CN102892897B

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Publication number: CN102892897B
Application number: CN201080064797.1A
Authority: CN
Inventors: 李健; 吴莹; 卢韶华; 朱虹光; 黄威; 任萍; 任一萍; 李兆勇
Original assignee: Fudan University
Current assignee: Shanghai Labway Clinical Laboratory Co ltd
Priority date: 2009-12-24
Filing date: 2010-12-24
Publication date: 2016-08-03
Anticipated expiration: 2030-12-24
Also published as: CN102892897A

Abstract

The present invention provides diagnostic kits for identifying molecular markers for one or more mammalian target cells exhibiting lung cancer, the kits comprising a plurality of nucleic acid molecules encoding miRNA sequences. One or more of the nucleic acid molecules encoding miRNA sequences are differentially expressed in target cells and in one or more control cells. The invention also provides methods of using the micrornas to identify one or more mammalian target cells exhibiting or having a predisposition to develop lung cancer, as well as methods and pharmaceutical compositions for preventing or treating lung cancer.

Description

Translated fromChinese

用于肺癌的微RNA表达谱分析的组合物和方法Compositions and methods for microRNA expression profiling of lung cancer

发明领域field of invention

本发明涉及用于肺癌(lungcancer)的微RNA(microRNA)表达谱分析的组合物和方法，特别是腺癌肺癌(adenocarcinomalungcancer)、鳞状细胞肺癌(squamouscelllungcancer)和小细胞肺癌(smallcelllungcancer)。The present invention relates to compositions and methods for microRNA (microRNA) expression profile analysis of lung cancer, especially adenocarcinoma lung cancer, squamous cell lung cancer and small cell lung cancer.

发明背景Background of the invention

肺癌依旧是世界范围内引起癌症相关死亡的最常见的原因之一。据估测2009年有140万例新发病例，平均年增长速度为2.51%(Frost&Sullivanestimates)，且绝大多数2009年被诊断为肺癌的患者将死于肺癌(Higgins,M.J.etal.(2009)ExpertRevAnticancerTher9,1365-1378)。尽管近几十年来手术方式和相关治疗手段取得了一定的进步，但在欧美，各期患者总体的五年生存率仅约15%。Lung cancer remains one of the most common causes of cancer-related death worldwide. It is estimated that there are 1.4 million new cases in 2009, with an average annual growth rate of 2.51% (Frost & Sullivanestimates), and the vast majority of patients diagnosed with lung cancer in 2009 will die of lung cancer (Higgins, M.J.etal. (2009) ExpertRevAnticancerTher9 , 1365-1378). Although surgical methods and related treatment methods have made some progress in recent decades, in Europe and the United States, the overall five-year survival rate of patients at each stage is only about 15%.

肺癌分为小细胞肺癌(smallcelllungcancer(SCLC))和非小细胞肺癌(non-smallcelllungcancer(NSCLC))。非小细胞肺癌是主要的组织学类型，占80%以上，包括腺癌和鳞状细胞癌。吸烟是肺癌最重要的危险因子，占全球男性病例的约80%，女性病例的50%。Lung cancer is divided into small cell lung cancer (small cell lung cancer (SCLC)) and non-small cell lung cancer (non-small cell lung cancer (NSCLC)). Non-small cell lung cancer is the main histological type, accounting for more than 80%, including adenocarcinoma and squamous cell carcinoma. Smoking is the most important risk factor for lung cancer, accounting for about 80% of cases in men and 50% in women worldwide.

针对肺癌不同亚型的治疗方式也各不相同。手术是早期NSCLC的治疗方式，总的五年生存率为40%。但大部分患者确诊时已处于中晚期，对这部分患者的一线治疗局限于多药化疗，估计生存时间也仅为8个月以下。近来靶向治疗的不断发展，要求对NSCLC进行更为精确的亚型分类。肿瘤血管生成抑制剂在鳞状细胞癌患者中极易引发副反应(Lebanoy，D.(2009)JClinOncol27,2030-2037)。小细胞肺癌是死亡率最高的肺癌类型，死亡率高达90%以上。尽管常可观察到高的最初反应率，局限期患者的中位生存率约为20个月。小细胞肺癌的治疗一般为化疗或者放化疗结合，而不选择手术治疗。There are different treatments for different subtypes of lung cancer. Surgery is the treatment of choice for early-stage NSCLC, with an overall five-year survival rate of 40%. However, most patients are already in the middle and advanced stages when they are diagnosed, and the first-line treatment for these patients is limited to multi-drug chemotherapy, and the estimated survival time is only less than 8 months. The recent continuous development of targeted therapy requires a more precise subtype classification of NSCLC. Tumor angiogenesis inhibitors are very likely to cause side effects in patients with squamous cell carcinoma (Lebanoy, D. (2009) JClinOncol27, 2030-2037). Small cell lung cancer is the type of lung cancer with the highest mortality rate, with a mortality rate of over 90%. Although high initial response rates are often observed, the median survival for patients with limited-stage disease is approximately 20 months. The treatment of small cell lung cancer is generally chemotherapy or a combination of radiotherapy and chemotherapy, rather than surgery.

除了针对不同亚型不同病因的肺癌患者采取的治疗手段各异外，观察者之间判断上的偏差和特异性标准化试验的缺乏，限制了当前对患者进行准确的分类和选择合适的治疗。未来对单个肺癌患者的治疗选择将取决于具体的肿瘤和宿主特性。因此寻找特异的分子标记物来区分不同的肺癌亚型十分必要。In addition to the different treatment methods for lung cancer patients with different subtypes and different etiologies, the judgment bias between observers and the lack of specific standardized tests limit the current accurate classification of patients and the selection of appropriate treatments. Future treatment options for individual lung cancer patients will depend on specific tumor and host properties. Therefore, it is necessary to find specific molecular markers to distinguish different lung cancer subtypes.

许多诊断试验由于只基于单一分子标记物，影响了结果的可靠性和精确性。此外，单一标记物往往不能具体预测潜伏期、肿瘤进展期等等。因此，依旧有必要寻找其他的分子标记物和试验方法来克服这些局限。Many diagnostic tests are based on a single molecular marker, compromising the reliability and precision of the results. In addition, a single marker often cannot specifically predict latency, tumor progression, and so on. Therefore, it is still necessary to find other molecular markers and assay methods to overcome these limitations.

一种解决这一问题的方法可以基于小调节RNA分子，特别是微RNA(miRNA)，它们是一类进化保守的内源表达的小的非编码RNA，大小为20-25个核苷酸(nt)，可以介导靶mRNA的表达。自从它们在大约10年前被发现就被认为在细胞发育、分化、增殖和凋亡中有重要功能(Bartel,D.P.(2004)Cell116,281-297,Ambros,V.(2004)Nature431,350-355;He,L.etal.(2004)NatRevGenet5,522-531)。此外，作为肿瘤标记物，miRNA优于mRNA，因其在体外更稳定，在体内半衰期更长(Lu,J.etal.,(2005)Nature435,834-838;Lim,L.P.etal.,(2005)Nature433,769-773)。One approach to address this issue could be based on small regulatory RNA molecules, specifically microRNAs (miRNAs), which are a class of evolutionarily conserved endogenously expressed small non-coding RNAs with a size of 20–25 nucleotides ( nt), which can mediate the expression of target mRNA. Since they were discovered about 10 years ago, they are considered to have important functions in cell development, differentiation, proliferation and apoptosis (Bartel, D.P. (2004) Cell 116, 281-297, Ambros, V. (2004) Nature 431, 350- 355; He, L. et al. (2004) Nat Rev Genet 5, 522-531). In addition, as a tumor marker, miRNA is superior to mRNA because it is more stable in vitro and has a longer half-life in vivo (Lu, J. et al., (2005) Nature 435, 834-838; Lim, L.P. et al., (2005) Nature 433, 769-773).

miRNA是从初级转录物产生的，初级转录物被RNaseIIIDrosha加工为茎-环结构前体(pre-miRNA)。在转运到细胞质后，另一种称为Dicer的RNaseIII切割pre-miRNA发夹的环以形成短双链(ds)RNA，其中一条链作为成熟miRNA掺入miRNA-蛋白质(miRNP)中。miRNA指导miRNP到达它们的靶mRNA，在此它们发挥功能(Bartel,D.P.(2004)Cell23,281-292；He,L.andHannon,G.J.(2004)Nat.Rev.Genet.5,522-531)。miRNAs are produced from primary transcripts that are processed by RNaseIII Drosha into stem-loop precursors (pre-miRNAs). After translocation into the cytoplasm, another RNaseIII called Dicer cleaves the loop of the pre-miRNA hairpin to form short double-stranded (ds) RNA, one strand of which is incorporated into miRNA-protein (miRNP) as mature miRNA. miRNAs direct miRNPs to their target mRNAs where they function (Bartel, D.P. (2004) Cell 23, 281-292; He, L. and Hannon, G.J. (2004) Nat. Rev. Genet. 5, 522-531).

根据miRNA与其靶之间的互补性程度，miRNA可以指导不同的调节过程。与miRNA高度互补的靶mRNA通过与RNA干扰(RNAi)相同的机制被特异切割。因此，在这种情况中，miRNA的功能是作为短干扰RNA(siRNA)。与miRNA互补性较低的靶mRNA要么被指引进入细胞降解途径要么被翻译性阻遏而不影响mRNA水平。但是，miRNA如何阻遏它们的靶mRNA的翻译的机制尚有争论。Depending on the degree of complementarity between the miRNA and its target, miRNAs can direct different regulatory processes. Target mRNAs that are highly complementary to miRNAs are specifically cleaved by the same mechanism as RNA interference (RNAi). Thus, in this case, the miRNA functions as short interfering RNA (siRNA). Target mRNAs with low complementarity to miRNAs are either directed into cellular degradation pathways or are translationally repressed without affecting mRNA levels. However, the mechanism of how miRNAs repress the translation of their target mRNAs is still debated.

高通量的miRNA定量技术，如miRNA微阵列、基于实时RT-PCR的TaqManmiRNA测定，为研究肿瘤全基因组中的总miRNA表达谱提供了有力的工具。可获得的现有数据表明miRNA表达的调节异常(dysregulation)可能与某些类型癌症的发生和/或发展相关。例如，已表明两种miRNA，miR-15及miR-16-1定位在慢性淋巴性白血病(CLL)中缺失的遗传基因座上，并且发现在大约70%的CLL患者中，两种miRNA基因被缺失或下调。另外，在结肠直肠瘤形成中观察到miR-143及miR-145的下调，而miRNAlet-7的表达在肺癌中经常降低(Michael,M.Z.etal.(2003)Mol.CancerRes.1,882-891；Mayr,C.etal.(2007)Science315,1576-1579)。事实上，基于miRNA表达中的癌症相关改变和miRNA通常位于参与癌症的基因组区域的观察推测miRNA可能既作为抑癌基因也作为癌基因(Esquela-Kerscher,A.andSlack,F.J(2006)Nat.Rev.Cancer6,259-269；Calin,G.A.andCroce,C.M.(2007)J.Clin.Invest.117,2059-2066；Blenkiron,C.andMiska,E.A.(2007)Hum.Mol.Genet.16,R106-R113)。miRNA在人类癌症中异常表达谱的发现提示其有望成为有效的诊断和预后标记物。High-throughput miRNA quantification technologies, such as miRNA microarrays and real-time RT-PCR-based TaqMan miRNA assays, provide powerful tools for studying total miRNA expression profiles in tumor genomes. Available existing data suggest that dysregulation of miRNA expression may be associated with the development and/or progression of certain types of cancer. For example, two miRNAs, miR-15 and miR-16-1, have been shown to localize at genetic loci that are missing in chronic lymphocytic leukemia (CLL), and both miRNA genes were found to be blocked in approximately 70% of CLL patients. missing or downregulated. In addition, downregulation of miR-143 and miR-145 was observed in colorectal neoplasia, whereas expression of miRNAlet-7 was often reduced in lung cancer (Michael, M.Z. et al. (2003) Mol. Cancer Res. 1, 882-891; Mayr, C. et al. (2007) Science 315, 1576-1579). In fact, miRNAs may function as both tumor suppressors and oncogenes based on cancer-associated changes in miRNA expression and the observation that miRNAs are often located in genomic regions involved in cancer (Esquela-Kerscher, A. and Slack, F.J (2006) Nat. Rev. .Cancer 6, 259-269; Calin, G.A. and Croce, C.M. (2007) J. Clin. Invest. 117, 2059-2066; Blenkiron, C. and Miska, E.A. (2007) Hum. Mol. Genet. 16, R106-R113) . The discovery of abnormal expression profiles of miRNAs in human cancers suggests that miRNAs are expected to be effective diagnostic and prognostic markers.

若干研究已报道了人类肺癌中的miRNA表达谱分析(Johnson,S.M.etal.(2005)Cell120,635-647;Liang,Y.etal.(2008)BMCMedGenomics1,61;Kumar,M.S.etal.(2008)ProcNatlAcadSciUSA105,3903-3908;Miko,E.etal.(2009)ExpLungRes35,646-664;Xie,Yetal.(2009)LungCancerMay13;Lebanony，D.etal.(2009)JClinOncol27,2030-2037;Kauppinen,S.etal.(2009)ClinCancerRes15,1177-1183;Mascaux,C.etal.(2009)EurRespirJ33,352-359)。同样地，这些研究均显示与非恶性肺组织相比，特异性miRNA在恶性组织中异常表达。研究还发现一些miRNA与预后有关(Yu,S.L.etal.(2008)CancerCell13,48-57;Raponi,M.etal(2009)CancerRes69,5776-5783)。因此，这种miRNA可提供关于涉及恶性转化和进展的细胞过程的认识。Several studies have reported miRNA expression profiling in human lung cancer (Johnson, S.M. et al. (2005) Cell 120, 635-647; Liang, Y. et al. (2008) BMC Med Genomics 1, 61; Kumar, M.S. et al. (2008) ProcNatlAcadSciUSA105 , 3903-3908; Miko, E.etal.(2009) ExpLungRes35, 646-664; Xie, Yetal.(2009) LungCancerMay13; Lebanony, D.etal.(2009) JClinOncol27, 2030-2037; Kauppinen, S.etal. (2009) Clin Cancer Res 15, 1177-1183; Mascaux, C. et al. (2009) EurRespir J 33, 352-359). Likewise, these studies all showed that specific miRNAs were abnormally expressed in malignant tissues compared with non-malignant lung tissues. Studies have also found that some miRNAs are associated with prognosis (Yu, S.L. et al. (2008) Cancer Cell 13, 48-57; Raponi, M. et al (2009) Cancer Res 69, 5776-5783). Thus, such miRNAs may provide insight into cellular processes involved in malignant transformation and progression.

因此，仍需要(一组)诊断标记物，特别是“表达特征(expressionsignature)”或“分子足迹(molecularfootprint)”形式，以使得可以快速、可靠和便宜地鉴定和/或治疗显示或具有发生不同类型肺癌倾向的细胞。另外，还持续需要相应的方法以鉴定和治疗展示这种癌表型的靶细胞。Therefore, there remains a need for (a set of) diagnostic markers, in particular in the form of "expression signatures" or "molecular footprints", so that rapid, reliable and inexpensive identification and/or treatment can be quickly, reliably and cheaply identified and/or treated. Type lung cancer prone cells. Additionally, there is a continuing need for corresponding methods to identify and treat target cells displaying this cancerous phenotype.

发明目的和发明概述Purpose of Invention and Summary of Invention

本发明的目的是提供通过确定多种核酸分子而诊断和/或治疗肺癌、和/或发生这种病症的倾向的新方法，每种所述核酸分子编码微RNA(miRNA)序列，其中与健康对照细胞相比，所述多种核酸分子的一或多种在所分析的靶细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表核酸表达特征，该核酸表达特征是存在肺癌或具有肺癌发生倾向的指征。It is an object of the present invention to provide new methods for diagnosing and/or treating lung cancer, and/or the predisposition to develop this condition, by identifying a plurality of nucleic acid molecules each encoding a microRNA (miRNA) sequence, which is associated with healthy One or more of the plurality of nucleic acid molecules is differentially expressed in the analyzed target cell compared to a control cell, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is Indications for the presence or predisposition to lung cancer.

更具体地，本发明的目的是提供核酸表达特征和/或组合物，用于鉴定展现出或具有发生肺癌倾向的一或多种哺乳动物靶细胞和/或区别不同类型的肺癌。肺癌的不同类型包括鳞状细胞癌、腺癌肺癌和小细胞肺癌。More specifically, it is an object of the present invention to provide nucleic acid expression signatures and/or compositions useful for identifying one or more mammalian target cells exhibiting or having a predisposition to develop lung cancer and/or distinguishing between different types of lung cancer. The different types of lung cancer include squamous cell carcinoma, adenocarcinoma lung cancer, and small cell lung cancer.

另外，本发明的目的是提供相应的方法，以用于鉴定展现出或具有发生肺癌倾向的一或多种哺乳动物靶细胞、区别不同类型的肺癌、以及用于预防或治疗这种病症。In addition, it is an object of the present invention to provide corresponding methods for identifying one or more mammalian target cells exhibiting or having a predisposition to develop lung cancer, distinguishing between different types of lung cancer, and for preventing or treating this condition.

这些及其它目的从以下描述将变得清楚，它们由独立权利要求的主题实现。本发明的一些优选实施方案由从属权利要求的主题限定。These and other objects will become apparent from the following description, which are achieved by the subject-matter of the independent claims. Some preferred embodiments of the invention are defined by the subject-matter of the dependent claims.

在第一方面，本发明涉及用于鉴定展现出或具有发展肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞中和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表核酸表达特征，该核酸表达特征是存在肺癌或具有发展肺癌倾向的指征。In a first aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop lung cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding MicroRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target cell and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature , the nucleic acid expression signature is indicative of the presence or predisposition to develop lung cancer.

本文限定的核酸表达特征可包括至少82种核酸分子，优选至少13种核酸分子，特别优选至少7种核酸分子。The nucleic acid expression signature defined herein may comprise at least 82 nucleic acid molecules, preferably at least 13 nucleic acid molecules, particularly preferably at least 7 nucleic acid molecules.

在优选的实施方案中，所述核酸表达特征包括至少一种编码微RNA序列的核酸分子，其表达在一或多种靶细胞中与一或多种对照细胞相比被上调；以及包括至少一种编码微RNA序列的核酸分子，其表达在一或多种靶细胞中与一或多种对照细胞相比被下调。In a preferred embodiment, the nucleic acid expression characteristics include at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in one or more target cells compared with one or more control cells; and includes at least one A nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in one or more target cells compared to one or more control cells.

在优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183、hsa-miR-451、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-145、hsa-miR-140-3p、hsa-miR-96、hsa-miR-144、has-miR-200c、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-218、hsa-miR-139-5p、has-miR-130b、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-425、hsa-miR-133b、hsa-miR-497、hsa-miR-182、hsa-miR-187^*、hsa-miR-30b、hsa-miR-93、hsa-miR-145^*、hsa-miR-429、hsa-miR-498、hsa-miR-200a、hsa-miR-625、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-98、hsa-miR-1、hsa-miR-106b、hsa-miR-150^*、hsa-miR-25、hsa-miR-134、hsa-miR-328、hsa-miR-638、hsa-miR-200b^*、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、has-miR-15b、hsa-miR-454、hsa-miR-301a、hsa-miR-135b、hsa-miR-100、hsa-miR-223、has-miR-196b、hsa-miR-196a、hsa-miR-1226^*、hsa-miR-424、hsa-miR-21^*、hsa-miR-572、hsa-miR-18b、hsa-miR-18a、hsa-miR-9^*、hsa-miR-301b、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、hsa-miR-30d、hsa-miR-29c、hsa-miR-199b-5p、hsa-miR-224、has-miR-125a-3p、hsa-miR-9、hsa-miR-200b、hsa-miR-195、hsa-miR-210、hsa-miR-7、has-miR-21、hsa-miR-17、hsa-miR-128、hsa-miR-141、hsa-miR-142-5p、hsa-miR-150、has-miR-205、hsa-miR-192、hsa-miR-215和hsa-miR-106a的核酸分子。In a preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183, hsa-miR-451, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-145 , hsa-miR-140-3p, hsa-miR-96, hsa-miR-144, has-miR-200c, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR-218, hsa -miR-139-5p, has-miR-130b, hsa-miR-30a^* , hsa-miR-144^* , hsa-miR-425, hsa-miR-133b, hsa-miR-497, hsa-miR-182 , hsa-miR-187^* , hsa-miR-30b, hsa-miR-93, hsa-miR-145^* , hsa-miR-429, hsa-miR-498, hsa-miR-200a, hsa-miR-625 , hsa-miR-130a, hsa-miR-30c-2^* , hsa-miR-98, hsa-miR-1, hsa-miR-106b, hsa-miR-150^* , hsa-miR-25, hsa-miR -134, hsa-miR-328, hsa-miR-638, hsa-miR-200b^* , hsa-miR-1225-5p, hsa-miR-1224-5p, hsa-miR-557, has-miR-15b, hsa-miR-454, hsa-miR-301a, hsa-miR-135b, hsa-miR-100, hsa-miR-223, has-miR-196b, hsa-miR-196a, hsa-miR-1226^* , hsa -miR-424, hsa-miR-21^* , hsa-miR-572, hsa-miR-18b, hsa-miR-18a, hsa-miR-9^* , hsa-miR-301b, hsa-miR-101, hsa - miR-143, hsa-miR-140-5p, hsa-miR-30d, hsa-miR-29c, hsa-miR-199b-5p, hsa-miR-224, has-miR-125a-3p, hsa-miR -9, hsa-miR-200b, hsa-miR-195, hsa-miR-210, hsa-miR-7, has-miR-21, hsa-miR-17, hsa-miR-128, hsa-miR-141 、h Nucleic acid molecules of sa-miR-142-5p, hsa-miR-150, has-miR-205, hsa-miR-192, hsa-miR-215 and hsa-miR-106a.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码has-miR-183、hsa-miR-96、hsa-miR-200c、hsa-miR-130b、hsa-miR-425、hsa-miR-182、has-miR-93、hsa-miR-429、hsa-miR-200a、hsa-miR-625、hsa-miR-98、hsa-miR-106b、has-miR-25、hsa-miR-200b^*、hsa-miR-15b、hsa-miR-454、hsa-miR-301a、hsa-miR-135b、hsa-miR-196b、hsa-miR-196a、hsa-miR-424、hsa-miR-21^*、hsa-miR-18b、hsa-miR-18a、hsa-miR-9^*、hsa-miR-301b、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-21、hsa-miR-17、hsa-miR-128、hsa-miR-141、hsa-miR-205、has-miR-192、hsa-miR-215、hsa-miR-106a的任意一种或多种核酸分子的表达被上调，而编码hsa-miR-451、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-145、hsa-miR-140-3p、hsa-miR-144、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-218、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-133b、hsa-miR-497、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-1、hsa-miR-150^*、hsa-miR-134、hsa-miR-328、has-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-100、hsa-miR-223、hsa-miR-1226^*、hsa-miR-572、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、has-miR-30d、hsa-miR-29c、hsa-miR-199b-5p、hsa-miR-125a-3p、hsa-miR-195、hsa-miR-142-5p和hsa-miR-150的任一或多种核酸分子的表达下调。Particularly preferably, has-miR-183, hsa-miR-96, hsa-miR-200c, hsa-miR-130b are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-425, hsa-miR-182, has-miR-93, hsa-miR-429, hsa-miR-200a, hsa-miR-625, hsa-miR-98, hsa-miR-106b, has -miR-25, hsa-miR-200b^* , hsa-miR-15b, hsa-miR-454, hsa-miR-301a, hsa-miR-135b, hsa-miR-196b, hsa-miR-196a, hsa- miR-424, hsa-miR-21^* , hsa-miR-18b, hsa-miR-18a, hsa-miR-9^* , hsa-miR-301b, hsa-miR-224, hsa-miR-9, hsa- miR-200b, hsa-miR-210, hsa-miR-7, hsa-miR-21, hsa-miR-17, hsa-miR-128, hsa-miR-141, hsa-miR-205, has-miR- 192. The expression of any one or more nucleic acid molecules of hsa-miR-215, hsa-miR-106a is up-regulated, while the encoding hsa-miR-451, hsa-miR-126, hsa-miR-126^* , hsa- miR-30a, hsa-miR-145, hsa-miR-140-3p, hsa-miR-144, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR-218, hsa-miR- 139-5p, hsa-miR-30a^* , hsa-miR-144^* , hsa-miR-133b, hsa-miR-497, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa-miR-498, hsa-miR-130a, hsa-miR-30c-2^* , hsa-miR-1, hsa-miR-150^* , hsa-miR-134, hsa-miR-328, has-miR- 638, hsa-miR-1225-5p, hsa-miR-1224-5p, hsa-miR-557, hsa-miR-100, hsa-miR-223, hsa-miR-1226^* , hsa-miR-572, hsa - miR-101, hsa - miR-143, hsa - miR-140-5p, has-miR-30d, hsa-miR-29c, hsa-miR-199b-5p, hsa-miR-125a-3p, hsa-miR-195, hsa-miR-142-5p and hsa - downregulation of expression of any one or more nucleic acid molecules of miR-150.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183、hsa-miR-451、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-145、hsa-miR-140-3p、hsa-miR-96、hsa-miR-144、has-miR-200c、hsa-miR-486-5p、hsa-miR-338-3p和hsa-miR-218的核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183, hsa-miR-451, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR- 145, hsa-miR-140-3p, hsa-miR-96, hsa-miR-144, has-miR-200c, hsa-miR-486-5p, hsa-miR-338-3p and hsa-miR-218 nucleic acid molecule.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-183、hsa-miR-96和hsa-miR-200c的任意一种或多种核酸分子的表达被上调，而编码hsa-miR-451、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-145、hsa-miR-140-3p、hsa-miR-144、hsa-miR-486-5p、hsa-miR-338-3p和hsa-miR-218的任一或多种核酸分子的表达下调。Particularly preferably, any one or more of hsa-miR-183, hsa-miR-96 and hsa-miR-200c is encoded in said one or more target cells compared to said one or more control cells The expression of a nucleic acid molecule is up-regulated, while encoding hsa-miR-451, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-145, hsa-miR-140-3p, hsa - Downregulation of expression of any one or more nucleic acid molecules of miR-144, hsa-miR-486-5p, hsa-miR-338-3p and hsa-miR-218.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183、hsa-miR-451、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-145和hsa-miR-140-3p的任意一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183, hsa-miR-451, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR- Any one or more nucleic acid molecules of 145 and hsa-miR-140-3p.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中hsa-miR-183的表达上调，而编码hsa-miR-451、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-145和hsa-miR-140-3p的任意一或多种核酸分子的表达下调。Particularly preferably, compared with the one or more control cells, the expression of hsa-miR-183 is up-regulated in the one or more target cells, and the expression of hsa-miR-451, hsa-miR-126, hsa - downregulation of expression of any one or more nucleic acid molecules of miR-126^* , hsa-miR-30a, hsa-miR-145 and hsa-miR-140-3p.

在第二方面，本发明涉及用于鉴定展现出或具有发展非小细胞肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述非小细胞肺癌包括鳞状细胞癌和腺癌肺癌。所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在非小细胞肺癌或有非小细胞肺癌发生倾向的指征，包括鳞状细胞癌和腺癌肺癌。In a second aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop non-small cell lung cancer, including squamous cell carcinoma and Adenocarcinoma lung cancer. The test kit comprises a plurality of nucleic acid molecules, each nucleic acid molecule encodes a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules is differentially expressed in the target cell and in one or more control cells, and wherein the One or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature indicative of the presence or predisposition to non-small cell lung cancer, including squamous cell carcinoma and adenocarcinoma lung cancer.

本文限定的核酸表达特征可包括至少70种核酸分子，优选地至少14种核酸分子，特别优选地至少9种核酸分子。The nucleic acid expression signature defined herein may comprise at least 70 nucleic acid molecules, preferably at least 14 nucleic acid molecules, particularly preferably at least 9 nucleic acid molecules.

在优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183、hsa-miR-451、has-miR-497、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-145、has-miR-140-3p、hsa-miR-96、hsa-miR-144、hsa-miR-200c、hsa-miR-486-5p、hsa-miR-139-5p、hsa-miR-5338-3p、hsa-miR-130b、hsa-miR-144^*、hsa-miR-425、hsa-miR-133b、has-miR-182、hsa-miR-187^*、hsa-miR-30b、hsa-miR-93、hsa-miR-145^*、hsa-miR-429、has-miR-498、hsa-miR-200a、hsa-miR-625、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-98、hsa-miR-1、hsa-miR-150^*、hsa-miR-25、hsa-miR-134、hsa-miR-328、hsa-miR-638、has-miR-200b^*、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-135b、has-miR-100、hsa-miR-424、hsa-miR-21^*、hsa-miR-30a^*、hsa-miR-494、hsa-miR-193b、has-miR-99a、hsa-miR-335、hsa-miR-106b、hsa-miR-140-5p、hsa-miR-101、hsa-miR-143、hsa-miR-30d、hsa-miR-29c、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-195、hsa-miR-210、hsa-miR-7、hsa-miR-21、hsa-miR-128、hsa-miR-141、hsa-miR-142-5p、hsa-miR-150、hsa-miR-205、hsa-miR-192和hsa-miR-215的任意一或多种核酸分子。In a preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183, hsa-miR-451, has-miR-497, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a , hsa-miR-218, hsa-miR-145, has-miR-140-3p, hsa-miR-96, hsa-miR-144, hsa-miR-200c, hsa-miR-486-5p, hsa-miR -139-5p, hsa-miR-5338-3p, hsa-miR-130b, hsa-miR-144^* , hsa-miR-425, hsa-miR-133b, has-miR-182, hsa-miR-187^* , hsa-miR-30b, hsa-miR-93, hsa-miR-145^* , hsa-miR-429, has-miR-498, hsa-miR-200a, hsa-miR-625, hsa-miR-130a, hsa-miR-30c-2^* , hsa-miR-98, hsa-miR-1, hsa-miR-150^* , hsa-miR-25, hsa-miR-134, hsa-miR-328, hsa-miR- 638, has-miR-200b^* , hsa-miR-1225-5p, hsa-miR-1224-5p, hsa-miR-557, hsa-miR-135b, has-miR-100, hsa-miR-424, hsa - miR-21^* , hsa-miR-30a^* , hsa-miR-494, hsa-miR-193b, has-miR-99a, hsa-miR-335, hsa-miR-106b, hsa-miR-140-5p , hsa-miR-101, hsa-miR-143, hsa-miR-30d, hsa-miR-29c, hsa-miR-224, hsa-miR-9, hsa-miR-200b, hsa-miR-195, hsa - miR-210, hsa-miR-7, hsa-miR-21, hsa-miR-128, hsa-miR-141, hsa-miR-142-5p, hsa-miR-150, hsa-miR-205, hsa - any one or more nucleic acid molecules of miR-192 and hsa-miR-215.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-183、hsa-miR-96、hsa-miR-200c、hsa-miR-130b、hsa-miR-425、hsa-miR-182、hsa-miR-93、hsa-miR-429、hsa-miR-200a、hsa-miR-625、hsa-miR-98、hsa-miR-25、hsa-miR-200b^*、hsa-miR-135b、hsa-miR-424、hsa-miR-21^*、hsa-miR-494、hsa-miR-193b、hsa-miR-106b、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-21、hsa-miR-128、hsa-miR-141、hsa-miR-205、hsa-miR-192、hsa-miR-215的任一或多种核酸分子表达上调，而编码hsa-miR-451、hsa-miR-497、hsa-miR-126、hsa-miR-126^*、has-miR-30a、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-144、hsa-miR-486-5p和hsa-miR-139-5p、hsa-miR-338-3p、hsa-miR-144^*、hsa-miR-133b、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-1、hsa-miR-150^*、hsa-miR-134、hsa-miR-328、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-100、hsa-miR-30a^*、hsa-miR-99a、hsa-miR-335、has-miR-140-5p、hsa-miR-101、hsa-miR-143、hsa-miR-30d、hsa-miR-29c、hsa-miR-195、hsa-miR-142-5p、hsa-miR-150的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-183, hsa-miR-96, hsa-miR-200c, hsa-miR-130b are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-425, hsa-miR-182, hsa-miR-93, hsa-miR-429, hsa-miR-200a, hsa-miR-625, hsa-miR-98, hsa-miR-25, hsa -miR-200b^* , hsa-miR-135b, hsa-miR-424, hsa-miR-21^* , hsa-miR-494, hsa-miR-193b, hsa-miR-106b, hsa-miR-224, hsa - miR-9, hsa-miR-200b, hsa-miR-210, hsa-miR-7, hsa-miR-21, hsa-miR-128, hsa-miR-141, hsa-miR-205, hsa-miR -192, hsa-miR-215 any one or more nucleic acid molecule expression is up-regulated, and coding hsa-miR-451, hsa-miR-497, hsa-miR-126, hsa-miR-126^* , has-miR- 30a, hsa-miR-218, hsa-miR-145, hsa-miR-140-3p, hsa-miR-144, hsa-miR-486-5p and hsa-miR-139-5p, hsa-miR-338- 3p, hsa-miR-144^* , hsa-miR-133b, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa-miR-498, hsa-miR-130a, hsa-miR -30c-2^* , hsa-miR-1, hsa-miR-150^* , hsa-miR-134, hsa-miR-328, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-1224 -5p, hsa-miR-557, hsa-miR-100, hsa-miR-30a^* , hsa-miR-99a, hsa-miR-335, has-miR-140-5p, hsa-miR-101, hsa- The expression of any one or more nucleic acid molecules of miR-143, hsa-miR-30d, hsa-miR-29c, hsa-miR-195, hsa-miR-142-5p, hsa-miR-150 is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183、hsa-miR-451、hsa-miR-497、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-96、hsa-miR-144、hsa-miR-200c、hsa-miR-486-5p和has-miR-139-5p的任意一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183, hsa-miR-451, hsa-miR-497, hsa-miR-126, hsa-miR-126^* , hsa-miR- 30a, hsa-miR-218, hsa-miR-145, hsa-miR-140-3p, hsa-miR-96, hsa-miR-144, hsa-miR-200c, hsa-miR-486-5p and has- Any one or more nucleic acid molecules of miR-139-5p.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-183、hsa-miR-96、hsa-miR-200c的任意一或多种核酸分子表达上调，而编码hsa-miR-451、hsa-miR-497、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-144、hsa-miR-486-5p和hsa-miR-139-5p的任意一或多种核酸分子的表达下调。Particularly preferably, compared with the one or more control cells, any one or more of hsa-miR-183, hsa-miR-96, hsa-miR-200c is encoded in the one or more target cells The expression of nucleic acid molecules is up-regulated, while the encoding hsa-miR-451, hsa-miR-497, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-218, hsa-miR-145, The expression of any one or more nucleic acid molecules of hsa-miR-140-3p, hsa-miR-144, hsa-miR-486-5p and hsa-miR-139-5p is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183、hsa-miR-451、hsa-miR-497、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p的任意一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183, hsa-miR-451, hsa-miR-497, hsa-miR-126, hsa-miR-126^* , hsa-miR- Any one or more nucleic acid molecules of 30a, hsa-miR-218, hsa-miR-145, hsa-miR-140-3p.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中hsa-miR-183的表达上调，而编码hsa-miR-451、hsa-miR-497、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p的任意一或多种核酸分子的表达下调。Particularly preferably, compared with the one or more control cells, the expression of hsa-miR-183 is up-regulated in the one or more target cells, while hsa-miR-451, hsa-miR-497, hsa - Down-regulation of expression of any one or more nucleic acid molecules of miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-218, hsa-miR-145, hsa-miR-140-3p.

在第三方面，本发明涉及用于鉴定展现出或具有发展腺癌肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在腺癌肺癌或有腺癌肺癌发展倾向的指征。In a third aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop adenocarcinoma lung cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid Molecularly encoded microRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a A nucleic acid expression signature that is indicative of the presence or predisposition to develop adenocarcinoma lung cancer.

本文限定的核酸表达特征可包括至少57种核酸分子，优选地至少13种核酸分子，特别优选地至少5种核酸分子。The nucleic acid expression signature defined herein may comprise at least 57 nucleic acid molecules, preferably at least 13 nucleic acid molecules, particularly preferably at least 5 nucleic acid molecules.

在优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-21、hsa-miR-183、hsa-miR-144、hsa-miR-451、hsa-miR-200c、hsa-miR-135b、hsa-miR-148a、hsa-miR-625、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-139-5p、hsa-miR-130b、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-425、hsa-miR-133b、hsa-miR-497、hsa-miR-182、hsa-miR-187^*、hsa-miR-30b、has-miR-145^*、hsa-miR-429、hsa-miR-498、hsa-miR-200a、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-134、hsa-miR-638、hsa-miR-200b^*、hsa-miR-1225-5p、hsa-miR-557、hsa-miR-100、hsa-miR-223、hsa-miR-424、hsa-miR-21^*、hsa-miR-99a、hsa-miR-34a、hsa-miR-542-3p、hsa-miR-200a^*、hsa-miR-375、hsa-miR-145、hsa-miR-140-3p、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、hsa-miR-224、hsa-miR-200b、hsa-miR-195、hsa-miR-210、hsa-miR-7、hsa-miR-141和hsa-miR-192的任意一或多种核酸分子。In a preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-96, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-218, hsa-miR-21 , hsa-miR-183, hsa-miR-144, hsa-miR-451, hsa-miR-200c, hsa-miR-135b, hsa-miR-148a, hsa-miR-625, hsa-miR-486-5p , hsa-miR-338-3p, hsa-miR-139-5p, hsa-miR-130b, hsa-miR-30a^* , hsa-miR-144^* , hsa-miR-425, hsa-miR-133b, hsa - miR-497, hsa-miR-182, hsa-miR-187^* , hsa-miR-30b, has-miR-145^* , hsa-miR-429, hsa-miR-498, hsa-miR-200a, hsa -miR-130a, hsa-miR-30c-2^* , hsa-miR-134, hsa-miR-638, hsa-miR-200b^* , hsa-miR-1225-5p, hsa-miR-557, hsa-miR -100, hsa-miR-223, hsa-miR-424, hsa-miR-21^* , hsa-miR-99a, hsa-miR-34a, hsa-miR-542-3p, hsa-miR-200a^* , hsa -miR-375, hsa-miR-145, hsa-miR-140-3p, hsa-miR-101, hsa-miR-143, hsa-miR-140-5p, hsa-miR-224, hsa-miR-200b Any one or more nucleic acid molecules of hsa-miR-195, hsa-miR-210, hsa-miR-7, hsa-miR-141 and hsa-miR-192.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-96、hsa-miR-21、hsa-miR-183、hsa-miR-200c、hsa-miR-135b、hsa-miR-148a、hsa-miR-625、hsa-miR-130b、hsa-miR-425、hsa-miR-182、has-miR-429、hsa-miR-200a、hsa-miR-200b^*、hsa-miR-424、hsa-miR-21^*、hsa-miR-34a、hsa-miR-542-3p、hsa-miR-200a^*、hsa-miR-375、hsa-miR-224、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-141、hsa-miR-192的任意一或多种核酸分子表达上调，而编码hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-144、hsa-miR-451、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-133b、hsa-miR-497、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、has-miR-134、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-557、hsa-miR-100、hsa-miR-223、hsa-miR-99a、hsa-miR-145、hsa-miR-140-3p、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、hsa-miR-195的任意一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-96, hsa-miR-21, hsa-miR-183, hsa-miR-200c are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-135b, hsa-miR-148a, hsa-miR-625, hsa-miR-130b, hsa-miR-425, hsa-miR-182, has-miR-429, hsa-miR-200a, hsa -miR-200b^* , hsa-miR-424, hsa-miR-21^* , hsa-miR-34a, hsa-miR-542-3p, hsa-miR-200a^* , hsa-miR-375, hsa-miR- 224. Expression of any one or more nucleic acid molecules of hsa-miR-200b, hsa-miR-210, hsa-miR-7, hsa-miR-141, hsa-miR-192 is up-regulated, while encoding hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-218, hsa-miR-144, hsa-miR-451, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR -139-5p, hsa-miR-30a^* , hsa-miR-144^* , hsa-miR-133b, hsa-miR-497, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa-miR-498, hsa-miR-130a, hsa-miR-30c-2^* , has-miR-134, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-557, hsa- miR-100, hsa-miR-223, hsa-miR-99a, hsa-miR-145, hsa-miR-140-3p, hsa-miR-101, hsa-miR-143, hsa-miR-140-5p, The expression of any one or more nucleic acid molecules of hsa-miR-195 is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-21、hsa-miR-183、hsa-miR-144、has-miR-451、hsa-miR-200c、hsa-miR-135b、hsa-miR-148a和hsa-miR-625的任意一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-96, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-218, hsa-miR- 21. Any one or more nucleic acids of hsa-miR-183, hsa-miR-144, has-miR-451, hsa-miR-200c, hsa-miR-135b, hsa-miR-148a and hsa-miR-625 molecular.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-96、hsa-miR-21、hsa-miR-183、hsa-miR-200c、hsa-miR-135b、hsa-miR-148a、hsa-miR-625的任一或多种核酸分子表达上，而编码hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-144、hsa-miR-451的任意一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-96, hsa-miR-21, hsa-miR-183, hsa-miR-200c are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-135b, hsa-miR-148a, hsa-miR-625 any one or more nucleic acid molecule expression, while encoding hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, The expression of any one or more nucleic acid molecules of hsa-miR-218, hsa-miR-144, and hsa-miR-451 is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218的任意一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include any one or more of hsa-miR-96, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-218 A nucleic acid molecule.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中hsa-miR-96的表达上调，而编码hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218的任意一或多种核酸分子的表达下调。Particularly preferably, compared with the one or more control cells, the expression of hsa-miR-96 is up-regulated in the one or more target cells, while hsa-miR-126, hsa-miR-126*, hsa-miR-126^* , The expression of any one or more nucleic acid molecules of hsa-miR-30a and hsa-miR-218 is down-regulated.

在第四方面，本发明涉及用于鉴定展现出或具有发展鳞状细胞肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在鳞状细胞肺癌或有鳞状细胞肺癌发展倾向的指征。In a fourth aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop squamous cell lung cancer, said kit comprising a plurality of nucleic acid molecules, each Nucleic acid molecules encode microRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a A nucleic acid expression signature that is indicative of the presence or predisposition to develop squamous cell lung cancer.

本文限定的核酸表达特征可包括至少112种核酸分子，优选地至少16种核酸分子，特别优选地至少9种核酸分子。The nucleic acid expression signature defined herein may comprise at least 112 nucleic acid molecules, preferably at least 16 nucleic acid molecules, particularly preferably at least 9 nucleic acid molecules.

在优选的实施方案中，所述核酸表达特征包括编码hsa-miR-144、hsa-miR-451、has-miR-200c、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-497、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-30b、hsa-miR-93、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-143、hsa-miR-103、hsa-miR-183、hsa-miR-96、hsa-miR-139-5p、hsa-miR-130b、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-425、hsa-miR-133b、hsa-miR-182、hsa-miR-187^*、hsa-miR-145^*、hsa-miR-429、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-98、hsa-miR-1、hsa-miR-106b、hsa-miR-150^*、hsa-miR-25、hsa-miR-134、has-miR-328、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-15b、hsa-miR-301a、hsa-miR-196b、hsa-miR-196a、hsa-miR-1226^*、hsa-miR-424、hsa-miR-21^*、hsa-miR-572、hsa-miR-18b、hsa-miR-18a、hsa-miR-9^*、hsa-miR-301b、has-miR-29c^*、hsa-miR-494、hsa-miR-193b、hsa-miR-375、hsa-miR-26a、hsa-miR-574-3p、hsa-miR-151-3p、hsa-miR-149、hsa-miR-768-5p、hsa-miR-181a、hsa-miR-20a、has-miR-181c、hsa-miR-29a、hsa-miR-29b、hsa-miR-505、hsa-miR-939、hsa-miR-769-5p、hsa-miR-30c、hsa-miR-590-5p、hsa-miR-590-5p、hsa-miR-671-5p、hsa-miR-625、has-miR-652、hsa-miR-139-3p、hsa-miR-28-5p、hsa-miR-623、hsa-miR-221、hsa-miR-19a、hsa-miR-944、hsa-miR-335、hsa-miR-125a-5p、hsa-miR-551b、hsa-let-7d^*、hsa-miR-31^*、hsa-miR-362-5p、hsa-miR-181a^*、hsa-miR-940、hsa-miR-30b^*、hsa-miR-601、has-miR-20b、hsa-miR-202、hsa-miR-101、hsa-miR-140-5p、hsa-miR-30d、hsa-miR-29c、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-195、hsa-miR-210、hsa-miR-7、has-miR-21、hsa-miR-17、hsa-miR-128、hsa-miR-141、hsa-miR-142-5p、hsa-miR-150和hsa-miR-205的任意一或多种核酸分子。In a preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-144, hsa-miR-451, has-miR-200c, hsa-miR-486-5p, hsa-miR-338-3p, hsa- miR-497, hsa-miR-218, hsa-miR-145, hsa-miR-140-3p, hsa-miR-30b, hsa-miR-93, hsa-miR-126, hsa-miR-126^* , hsa - miR-30a, hsa-miR-143, hsa-miR-103, hsa-miR-183, hsa-miR-96, hsa-miR-139-5p, hsa-miR-130b, hsa-miR-30a^* , hsa-miR-144^* , hsa-miR-425, hsa-miR-133b, hsa-miR-182, hsa-miR-187^* , hsa-miR-145^* , hsa-miR-429, hsa-miR-498 , hsa-miR-130a, hsa-miR-30c-2^* , hsa-miR-98, hsa-miR-1, hsa-miR-106b, hsa-miR-150^* , hsa-miR-25, hsa-miR -134, has-miR-328, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-1224-5p, hsa-miR-557, hsa-miR-15b, hsa-miR-301a, hsa - miR-196b, hsa-miR-196a, hsa-miR-1226^* , hsa-miR-424, hsa-miR-21^* , hsa-miR-572, hsa-miR-18b, hsa-miR-18a, hsa - miR-9^* , hsa-miR-301b, has-miR-29c^* , hsa-miR-494, hsa-miR-193b, hsa-miR-375, hsa-miR-26a, hsa-miR-574-3p , hsa-miR-151-3p, hsa-miR-149, hsa-miR-768-5p, hsa-miR-181a, hsa-miR-20a, has-miR-181c, hsa-miR-29a, hsa-miR -29b, hsa-miR-505, hsa-miR-939, hsa-miR-769-5p, hsa-miR-30c, hsa-miR-590-5p, hsa-miR-590-5p, hsa-miR-671 -5 p, hsa-miR-625, has-miR-652, hsa-miR-139-3p, hsa-miR-28-5p, hsa-miR-623, hsa-miR-221, hsa-miR-19a, hsa- miR-944, hsa-miR-335, hsa-miR-125a-5p, hsa-miR-551b, hsa-let-7d^* , hsa-miR-31^* , hsa-miR-362-5p, hsa-miR- 181a^* , hsa-miR-940, hsa-miR-30b^* , hsa-miR-601, has-miR-20b, hsa-miR-202, hsa-miR-101, hsa-miR-140-5p, hsa- miR-30d, hsa-miR-29c, hsa-miR-224, hsa-miR-9, hsa-miR-200b, hsa-miR-195, hsa-miR-210, hsa-miR-7, has-miR- 21. Any one or more nucleic acid molecules of hsa-miR-17, hsa-miR-128, hsa-miR-141, hsa-miR-142-5p, hsa-miR-150 and hsa-miR-205.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-200c、hsa-miR-93、hsa-miR-103、hsa-miR-183、hsa-miR-96、hsa-miR-130b、hsa-miR-425、hsa-miR-182、hsa-miR-429、hsa-miR-98、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-301a、hsa-miR-196b、hsa-miR-196a、has-miR-424、hsa-miR-21^*、hsa-miR-18b、hsa-miR-18a、hsa-miR-9^*、hsa-miR-301b、has-miR-494、hsa-miR-193b、hsa-miR-151-3p、hsa-miR-149、hsa-miR-20a、hsa-miR-505、hsa-miR-769-5p、hsa-miR-590-5p、hsa-miR-590-5p、hsa-miR-625、hsa-miR-28-5p、has-miR-221、hsa-miR-19a、hsa-miR-944、hsa-miR-31^*、hsa-miR-362-5p、hsa-miR-20b、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-21、has-miR-17、hsa-miR-128、hsa-miR-141、hsa-miR-205的任一或多种核酸分子表达上调，而编码hsa-miR-144、hsa-miR-451、hsa-miR-486-5p、hsa-miR-338-3p、hsa-5miR-497、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-30b、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-143、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-133b、hsa-miR-187^*、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-1、hsa-miR-150^*、has-miR-134、hsa-miR-328、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-1226^*、hsa-miR-572、hsa-miR-29c^*、hsa-miR-375、hsa-miR-26a、hsa-miR-574-3p、hsa-miR-768-5p、hsa-miR-181a、hsa-miR-181c、hsa-miR-29a、hsa-miR-29b、hsa-miR-939、hsa-miR-30c、hsa-miR-671-5p、hsa-miR-652、hsa-miR-139-3p、has-miR-623、hsa-miR-335、hsa-miR-125a-5p、hsa-miR-551b、hsa-let-7d^*、hsa-miR-181a^*、hsa-miR-940、hsa-miR-30b^*、hsa-miR-601、hsa-miR-202、hsa-miR-101、hsa-miR-140-5p、hsa-miR-30d、hsa-miR-29c、hsa-miR-195、hsa-miR-142-5p、hsa-miR-150的任意一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-200c, hsa-miR-93, hsa-miR-103, hsa-miR-183 are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-96, hsa-miR-130b, hsa-miR-425, hsa-miR-182, hsa-miR-429, hsa-miR-98, hsa-miR-106b, hsa-miR-25, hsa -miR-15b, hsa-miR-301a, hsa-miR-196b, hsa-miR-196a, has-miR-424, hsa-miR-21^* , hsa-miR-18b, hsa-miR-18a, hsa- miR-9^* , hsa-miR-301b, has-miR-494, hsa-miR-193b, hsa-miR-151-3p, hsa-miR-149, hsa-miR-20a, hsa-miR-505, hsa - miR-769-5p, hsa-miR-590-5p, hsa-miR-590-5p, hsa-miR-625, hsa-miR-28-5p, has-miR-221, hsa-miR-19a, hsa -miR-944, hsa-miR-31^* , hsa-miR-362-5p, hsa-miR-20b, hsa-miR-224, hsa-miR-9, hsa-miR-200b, hsa-miR-210, The expression of any or more nucleic acid molecules of hsa-miR-7, hsa-miR-21, has-miR-17, hsa-miR-128, hsa-miR-141, hsa-miR-205 is up-regulated, and the encoding hsa- miR-144, hsa-miR-451, hsa-miR-486-5p, hsa-miR-338-3p, hsa-5miR-497, hsa-miR-218, hsa-miR-145, hsa-miR-140- 3p, hsa-miR-30b, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-143, hsa-miR-139-5p, hsa-miR-30a^* , hsa- miR-144^* , hsa-miR-133b, hsa-miR-187^* , hsa-miR-145^* , hsa-miR-498, hsa-miR-130a, hsa-miR-30c-2^* , hsa-miR- 1. hsa-miR-150^* , has-miR-134, hsa-m iR-328, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-1224-5p, hsa-miR-557, hsa-miR-1226^* , hsa-miR-572, hsa-miR-29c^* , hsa-miR-375, hsa-miR-26a, hsa-miR-574-3p, hsa-miR-768-5p, hsa-miR-181a, hsa-miR-181c, hsa-miR-29a, hsa- miR-29b, hsa-miR-939, hsa-miR-30c, hsa-miR-671-5p, hsa-miR-652, hsa-miR-139-3p, has-miR-623, hsa-miR-335, hsa-miR-125a-5p, hsa-miR-551b, hsa-let-7d^* , hsa-miR-181a^* , hsa-miR-940, hsa-miR-30b^* , hsa-miR-601, hsa-miR Any of -202, hsa-miR-101, hsa-miR-140-5p, hsa-miR-30d, hsa-miR-29c, hsa-miR-195, hsa-miR-142-5p, hsa-miR-150 Expression of one or more nucleic acid molecules is downregulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-144、hsa-miR-451、hsa-miR-200c、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-497、hsa-miR-218、has-miR-145、hsa-miR-140-3p、hsa-miR-30b、hsa-miR-93、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-143和hsa-miR-103的任意一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-144, hsa-miR-451, hsa-miR-200c, hsa-miR-486-5p, hsa-miR-338-3p, hsa -miR-497, hsa-miR-218, has-miR-145, hsa-miR-140-3p, hsa-miR-30b, hsa-miR-93, hsa-miR-126, hsa-miR-126^* , Any one or more nucleic acid molecules of hsa-miR-30a, hsa-miR-143 and hsa-miR-103.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-200c、hsa-miR-93、hsa-miR-103的任意一或多种核酸分子表达上调，而编码hsa-miR-144、hsa-miR-451、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-497、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-30b、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、has-miR-143的任意一或多种核酸分子的表达下调。Particularly preferably, compared with the one or more control cells, any one or more of hsa-miR-200c, hsa-miR-93, hsa-miR-103 is encoded in the one or more target cells The expression of nucleic acid molecules is up-regulated, while encoding hsa-miR-144, hsa-miR-451, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR-497, hsa-miR-218, hsa-miR Any one or more nucleic acid molecules of -145, hsa-miR-140-3p, hsa-miR-30b, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, has-miR-143 Expression is downregulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-144、hsa-miR-451、hsa-miR-200c、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-497、hsa-miR-218、has-miR-145和hsa-miR-140-3p的任意一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-144, hsa-miR-451, hsa-miR-200c, hsa-miR-486-5p, hsa-miR-338-3p, hsa - any one or more nucleic acid molecules of miR-497, hsa-miR-218, has-miR-145 and hsa-miR-140-3p.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中hsa-miR-200c的表达上调，而编码hsa-miR-144、hsa-miR-451、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-497、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p的任意一或多种核酸分子的表达下调。Particularly preferably, compared with the one or more control cells, the expression of hsa-miR-200c is up-regulated in the one or more target cells, and the expression of hsa-miR-144, hsa-miR-451, hsa - expression of any one or more nucleic acid molecules of miR-486-5p, hsa-miR-338-3p, hsa-miR-497, hsa-miR-218, hsa-miR-145, hsa-miR-140-3p down.

在第五方面，本发明涉及用于鉴定展现或具有发展小细胞肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在小细胞肺癌或有小细胞肺癌发展倾向的指征。In a fifth aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop small cell lung cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule Encoding microRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid An expression signature, the nucleic acid expression signature is an indication of the presence of small cell lung cancer or the tendency to develop small cell lung cancer.

本文限定的核酸表达特征可包括至少116种核酸分子，优选地至少16种核酸分子，特别优选地至少11种核酸分子。The nucleic acid expression signature defined herein may comprise at least 116 nucleic acid molecules, preferably at least 16 nucleic acid molecules, particularly preferably at least 11 nucleic acid molecules.

在优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96、hsa-miR-144、has-miR-93、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-145、hsa-miR-375、has-miR-103、hsa-miR-20a、hsa-miR-107、hsa-miR-451、hsa-miR-130b、hsa-miR-454、has-miR-30a、hsa-miR-17、hsa-miR-183、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-425、hsa-miR-133b、hsa-miR-497、hsa-miR-182、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-429、hsa-miR-498、hsa-miR-200a、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-98、hsa-miR-1、hsa-miR-150^*、has-miR-134、hsa-miR-328、hsa-miR-638、hsa-miR-200b^*、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-152、hsa-miR-301a、hsa-miR-100、hsa-miR-223、has-miR-196b、hsa-miR-1226^*、hsa-miR-572、hsa-miR-18b、hsa-miR-9^*、hsa-miR-301b、hsa-miR-199b-3p、hsa-miR-34a、hsa-miR-200a^*、hsa-miR-26a、hsa-miR-574-3p、has-miR-768-5p、hsa-miR-29a、hsa-miR-29b、hsa-miR-505、hsa-miR-939、hsa-miR-769-5p、hsa-miR-625、hsa-miR-139-3p、hsa-miR-19a、hsa-miR-551b、hsa-miR-362-5p、has-miR-181a^*、hsa-miR-940、hsa-miR-601、hsa-miR-20b、hsa-miR-23a、hsa-miR-27a、has-miR-199a-5p、hsa-let-7i、hsa-miR-19b、hsa-miR-20a^*、hsa-miR-17^*、hsa-miR-22、has-miR-340^*、hsa-miR-214、hsa-miR-592、hsa-miR-335^*、hsa-miR-29c^*、hsa-miR-421、hsa-miR-886-3p、hsa-miR-16-2^*、hsa-miR-135a、hsa-miR-183^*、hsa-miR-7-1^*、has-miR-26b、hsa-miR-455-3p、hsa-miR-222、hsa-miR-95、hsa-miR-564、hsa-miR-636、has-miR-34b^*、hsa-miR-18a、hsa-miR-126、hsa-miR-126^*、hsa-miR-140-3p、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、hsa-miR-29c、hsa-miR-199b-5p、hsa-miR-125a-3p、has-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-128和hsa-miR-106a的任意一或多种核酸分子。In a preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-96, hsa-miR-144, has-miR-93, hsa-miR-106b, hsa-miR-25, hsa-miR-15b, hsa-miR-145, hsa-miR-375, has-miR-103, hsa-miR-20a, hsa-miR-107, hsa-miR-451, hsa-miR-130b, hsa-miR-454, has- miR-30a, hsa-miR-17, hsa-miR-183, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR-139-5p, hsa-miR-30a^* , hsa-miR -144^* , hsa-miR-425, hsa-miR-133b, hsa-miR-497, hsa-miR-182, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa- miR-429, hsa-miR-498, hsa-miR-200a, hsa-miR-130a, hsa-miR-30c-2^* , hsa-miR-98, hsa-miR-1, hsa-miR-150^* , has-miR-134, hsa-miR-328, hsa-miR-638, hsa-miR-200b^* , hsa-miR-1225-5p, hsa-miR-1224-5p, hsa-miR-557, hsa-miR -152, hsa-miR-301a, hsa-miR-100, hsa-miR-223, has-miR-196b, hsa-miR-1226^* , hsa-miR-572, hsa-miR-18b, hsa-miR- 9^* , hsa-miR-301b, hsa-miR-199b-3p, hsa-miR-34a, hsa-miR-200a^* , hsa-miR-26a, hsa-miR-574-3p, has-miR-768- 5p, hsa-miR-29a, hsa-miR-29b, hsa-miR-505, hsa-miR-939, hsa-miR-769-5p, hsa-miR-625, hsa-miR-139-3p, hsa- miR-19a, hsa-miR-551b, hsa-miR-362-5p, has-miR-181a^* , hsa-miR-940, hsa-miR-601, hsa-miR-20b, hsa-miR-23a, hsa -miR-27a, has-miR-199a-5p, hsa-let-7i, hsa-miR-19b, hsa-miR-20a^* , hsa-miR-17^* , hsa-miR-22, has-miR-340^* , hsa-miR-214, hsa-miR-592, hsa-miR-335^* , hsa-miR-29c^* , hsa-miR-421, hsa-miR-886-3p, hsa-miR-16-2^* , hsa-miR-135a, hsa-miR-183^* , hsa-miR-7-1^* , has-miR-26b, hsa-miR-455-3p, hsa-miR-222, hsa-miR-95, hsa -miR-564, hsa-miR-636, has-miR-34b^* , hsa-miR-18a, hsa-miR-126, hsa-miR-126^* , hsa-miR-140-3p, hsa-miR-101 , hsa-miR-143, hsa-miR-140-5p, hsa-miR-29c, hsa-miR-199b-5p, hsa-miR-125a-3p, has-miR-9, hsa-miR-200b, hsa - any one or more nucleic acid molecules of miR-210, hsa-miR-7, hsa-miR-128 and hsa-miR-106a.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-96、hsa-miR-93、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-375、hsa-miR-103、hsa-miR-20a、hsa-miR-107、hsa-miR-130b、hsa-miR-454、hsa-miR-17、hsa-miR-183、hsa-miR-425、hsa-miR-182、hsa-miR-429、hsa-miR-200a、hsa-miR-98、hsa-miR-200b^*、hsa-miR-301a、hsa-miR-196b、hsa-miR-505、hsa-miR-769-5p、hsa-miR-625、hsa-miR-19a、hsa-miR-362-5p、hsa-miR-20b、hsa-let-7i、has-miR-19b、hsa-miR-20a^*、hsa-miR-17^*、hsa-miR-340^*、hsa-miR-592、hsa-miR-335^*、hsa-miR-421、hsa-miR-16-2^*、hsa-miR-135a、hsa-miR-183^*、hsa-miR-7-1^*、hsa-miR-95、hsa-miR-18a、hsa-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-128和hsa-miR-106a的任一或多种核酸分子表达上调，而编码hsa-miR-144、hsa-miR-145、hsa-miR-451、hsa-miR-30a、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144、hsa-miR-133b、hsa-miR-497、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-498、has-miR-130a、hsa-miR-30c-2^*、hsa-miR-1、hsa-miR-150^*、hsa-miR-134、hsa-miR-328、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-152、hsa-miR-100、hsa-miR-223、hsa-miR-1226^*、hsa-miR-572、hsa-miR-18b、hsa-miR-9^*、hsa-miR-301b、hsa-miR-199b-3p、hsa-miR-34a、hsa-miR-200a^*、hsa-miR-26a、hsa-miR-574-3p、hsa-miR-768-5p、hsa-miR-29a、hsa-miR-29b、hsa-miR-939、hsa-miR-139-3p、hsa-miR-551b、hsa-miR-181a^*、hsa-miR-940、hsa-miR-601、hsa-miR-23a、hsa-miR-27a、hsa-miR-199a-5p、hsa-miR-22、hsa-miR-214、hsa-miR-29c^*、hsa-miR-886-3p、hsa-miR-26b、hsa-miR-455-3p、hsa-miR-222、hsa-miR-564、hsa-miR-636、hsa-miR-34b^*、hsa-miR-126、hsa-5miR-126^*、hsa-miR-140-3p、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、hsa-miR-29c、hsa-miR-199b-5p、hsa-miR-125a-3p的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-96, hsa-miR-93, hsa-miR-106b, hsa-miR-25 are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-15b, hsa-miR-375, hsa-miR-103, hsa-miR-20a, hsa-miR-107, hsa-miR-130b, hsa-miR-454, hsa-miR-17, hsa -miR-183, hsa-miR-425, hsa-miR-182, hsa-miR-429, hsa-miR-200a, hsa-miR-98, hsa-miR-200b^* , hsa-miR-301a, hsa- miR-196b, hsa-miR-505, hsa-miR-769-5p, hsa-miR-625, hsa-miR-19a, hsa-miR-362-5p, hsa-miR-20b, hsa-let-7i, has-miR-19b, hsa-miR-20a^* , hsa-miR-17^* , hsa-miR-340^* , hsa-miR-592, hsa-miR-335^* , hsa-miR-421, hsa-miR- 16-2^* , hsa-miR-135a, hsa-miR-183^* , hsa-miR-7-1^* , hsa-miR-95, hsa-miR-18a, hsa-miR-9, hsa-miR-200b , hsa-miR-210, hsa-miR-7, hsa-miR-128 and hsa-miR-106a any one or more nucleic acid molecule expression is up-regulated, while encoding hsa-miR-144, hsa-miR-145, hsa -miR-451, hsa-miR-30a, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR-139-5p, hsa-miR-30a^* , hsa-miR-144, hsa- miR-133b, hsa-miR-497, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa-miR-498, has-miR-130a, hsa-miR-30c-2^* , hsa-miR-1, hsa-miR-150^* , hsa-miR-134, hsa-miR-328, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-1224-5p, hsa- miR-557, hsa-miR-152, hsa-miR-100, hsa-miR-223, hsa-miR-1226^* , hsa-miR-572, hsa-miR-18b, hsa-miR-9^* , hsa-miR-301b, hsa-miR-199b-3p, hsa-miR- 34a, hsa-miR-200a^* , hsa-miR-26a, hsa-miR-574-3p, hsa-miR-768-5p, hsa-miR-29a, hsa-miR-29b, hsa-miR-939, hsa -miR-139-3p, hsa-miR-551b, hsa-miR-181a^* , hsa-miR-940, hsa-miR-601, hsa-miR-23a, hsa-miR-27a, hsa-miR-199a- 5p, hsa-miR-22, hsa-miR-214, hsa-miR-29c^* , hsa-miR-886-3p, hsa-miR-26b, hsa-miR-455-3p, hsa-miR-222, hsa - miR-564, hsa-miR-636, hsa-miR-34b^* , hsa-miR-126, hsa-5miR-126^* , hsa-miR-140-3p, hsa-miR-101, hsa-miR-143 The expression of any one or more nucleic acid molecules of hsa-miR-140-5p, hsa-miR-29c, hsa-miR-199b-5p, hsa-miR-125a-3p is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96、hsa-miR-144、hsa-miR-93、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-145、hsa-miR-375、hsa-miR-103、hsa-miR-20a、hsa-miR-107、hsa-miR-451、hsa-miR-130b、hsa-miR-454、hsa-miR-30a、hsa-miR-17的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-96, hsa-miR-144, hsa-miR-93, hsa-miR-106b, hsa-miR-25, hsa-miR-15b , hsa-miR-145, hsa-miR-375, hsa-miR-103, hsa-miR-20a, hsa-miR-107, hsa-miR-451, hsa-miR-130b, hsa-miR-454, hsa - any one or more nucleic acid molecules of miR-30a, hsa-miR-17.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-96、hsa-miR-93、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-375、hsa-miR-103、hsa-miR-20a、hsa-miR-107、hsa-miR-130b、hsa-miR-454、hsa-miR-17的任一或多种核酸分子表达上调，而编码hsa-miR-144、hsa-miR-145、hsa-miR-451、hsa-miR-30a的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-96, hsa-miR-93, hsa-miR-106b, hsa-miR-25 are encoded in said one or more target cells compared to said one or more control cells Any of hsa-miR-15b, hsa-miR-375, hsa-miR-103, hsa-miR-20a, hsa-miR-107, hsa-miR-130b, hsa-miR-454, hsa-miR-17 The expression of one or more nucleic acid molecules is up-regulated, while the expression of any one or more nucleic acid molecules encoding hsa-miR-144, hsa-miR-145, hsa-miR-451, hsa-miR-30a is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96、hsa-miR-144、hsa-miR-93、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-145、hsa-miR-375、hsa-miR-103、hsa-miR-20a和hsa-miR-107的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-96, hsa-miR-144, hsa-miR-93, hsa-miR-106b, hsa-miR-25, hsa-miR-15b Any one or more nucleic acid molecules of hsa-miR-145, hsa-miR-375, hsa-miR-103, hsa-miR-20a and hsa-miR-107.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-96、hsa-miR-93、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-375、hsa-miR-103、hsa-miR-20a和hsa-miR-107的任一或多种核酸分子表达上调，而编码hsa-miR-144、hsa-miR-145的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-96, hsa-miR-93, hsa-miR-106b, hsa-miR-25 are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-15b, hsa-miR-375, hsa-miR-103, hsa-miR-20a and hsa-miR-107 any one or more nucleic acid molecule expression up-regulation, while encoding hsa-miR-144, hsa - downregulation of expression of any one or more nucleic acid molecules of miR-145.

在第六方面，本发明涉及用于区别非小细胞肺癌和小细胞肺癌的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在非小细胞肺癌或小细胞肺癌的指征。In the sixth aspect, the present invention relates to a diagnostic kit for molecular markers for distinguishing non-small cell lung cancer and small cell lung cancer, said kit comprising a variety of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein said multiple One or more of the nucleic acid molecules are differentially expressed in the target cell and in the one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is present Indications for non-small cell lung cancer or small cell lung cancer.

本文限定的核酸表达特征可包括至少17种核酸分子，优选地至少8种核酸分子。A nucleic acid expression signature as defined herein may comprise at least 17 nucleic acid molecules, preferably at least 8 nucleic acid molecules.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-375、hsa-miR-335、hsa-miR-23a、hsa-miR-27a、hsa-miR-22、hsa-miR-592、hsa-miR-135a、hsa-miR-24、hsa-miR-454、hsa-miR-301a、hsa-miR-199b-5p、hsa-miR-9、hsa-miR-34a、hsa-miR-199a-5p、hsa-miR-335^*、hsa-miR-152和hsa-miR-216b的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-375, hsa-miR-335, hsa-miR-23a, hsa-miR-27a, hsa-miR-22, hsa-miR-592 , hsa-miR-135a, hsa-miR-24, hsa-miR-454, hsa-miR-301a, hsa-miR-199b-5p, hsa-miR-9, hsa-miR-34a, hsa-miR-199a - any one or more nucleic acid molecules of 5p, hsa-miR-335^* , hsa-miR-152 and hsa-miR-216b.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-23a、hsa-miR-27a、hsa-miR-22、hsa-miR-135a、hsa-miR-199b-5p、hsa-miR-34a、hsa-miR-199a-5p、hsa-miR-152的任一或多种核酸分子表达上调，而编码hsa-miR-375、hsa-miR-335、hsa-miR-592、hsa-miR-24、hsa-miR-454、hsa-miR-301a、hsa-miR-9、has-miR-335^*、hsa-miR-216b的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-23a, hsa-miR-27a, hsa-miR-22, hsa-miR-135a are encoded in said one or more target cells compared to said one or more control cells Any one or more nucleic acid molecules of hsa-miR-199b-5p, hsa-miR-34a, hsa-miR-199a-5p, hsa-miR-152 are up-regulated, while the encoding hsa-miR-375, hsa-miR Any or more of -335, hsa-miR-592, hsa-miR-24, hsa-miR-454, hsa-miR-301a, hsa-miR-9, has-miR-335^* , hsa-miR-216b The expression of a nucleic acid molecule is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-375、hsa-miR-335、hsa-miR-23a、hsa-miR-27a、hsa-miR-22、hsa-miR-592、hsa-miR-135a和hsa-miR-24的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-375, hsa-miR-335, hsa-miR-23a, hsa-miR-27a, hsa-miR-22, hsa-miR-592 Any one or more nucleic acid molecules of hsa-miR-135a, hsa-miR-24, hsa-miR-24.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-23a、hsa-miR-27a、hsa-miR-22、hsa-miR-135a的任一或多种核酸分子表达上调而编码hsa-miR-375、hsa-miR-335、hsa-miR-592、hsa-miR-24的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-23a, hsa-miR-27a, hsa-miR-22, hsa-miR-135a are encoded in said one or more target cells compared to said one or more control cells The expression of any one or more nucleic acid molecules encoding hsa-miR-375, hsa-miR-335, hsa-miR-592, hsa-miR-24 is down-regulated.

在第七方面，本发明涉及用于将腺癌肺癌、鳞状细胞肺癌或小细胞肺癌与其它两种区别开的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在腺癌肺癌、鳞状细胞肺癌或小细胞肺癌的指征。In a seventh aspect, the present invention relates to a diagnostic kit of molecular markers for distinguishing adenocarcinoma lung cancer, squamous cell lung cancer or small cell lung cancer from the other two, said kit comprising a plurality of nucleic acid molecules, each nucleic acid Molecularly encoded microRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a A nucleic acid expression signature that is indicative of the presence of adenocarcinoma lung cancer, squamous cell lung cancer, or small cell lung cancer.

本文限定的核酸表达特征可包括至少23种核酸分子，优选地至少7种核酸分子。A nucleic acid expression signature as defined herein may comprise at least 23 nucleic acid molecules, preferably at least 7 nucleic acid molecules.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-25、hsa-miR-205、hsa-miR-34a、hsa-miR-375、hsa-miR-29a、hsa-miR-27a、hsa-miR-29b、hsa-miR-93、hsa-miR-106b、hsa-miR-15b、hsa-miR-454、hsa-miR-301a、hsa-miR-145、hsa-miR-29c、hsa-miR-221、hsa-miR-335、hsa-miR-23a、hsa-miR-199a-5p、hsa-miR-22、hsa-miR-592、hsa-miR-95、hsa-miR-24和hsa-miR-92a的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-25, hsa-miR-205, hsa-miR-34a, hsa-miR-375, hsa-miR-29a, hsa-miR-27a , hsa-miR-29b, hsa-miR-93, hsa-miR-106b, hsa-miR-15b, hsa-miR-454, hsa-miR-301a, hsa-miR-145, hsa-miR-29c, hsa - miR-221, hsa-miR-335, hsa-miR-23a, hsa-miR-199a-5p, hsa-miR-22, hsa-miR-592, hsa-miR-95, hsa-miR-24 and hsa - any one or more nucleic acid molecules of miR-92a.

特别优选地，与鳞状细胞肺癌和小细胞肺癌相比，在腺癌肺癌中编码hsa-miR-34a、hsa-miR-29a、hsa-miR-29b、hsa-miR-145、hsa-miR-29c的任一或多种核酸分子表达上调，而编码hsa-miR-25、hsa-miR-93、hsa-miR-301a、hsa-miR-106b、hsa-miR-15b、has-miR-92a的任一或多种核酸分子的表达下调。在腺癌肺癌中编码hsa-miR-205、hsa-miR-27a、hsa-miR-221的任一或多种核酸分子，与小细胞肺癌相比表达上调，与鳞状细胞肺癌相比表达下调。在腺癌肺癌中编码hsa-miR-375的核酸分子，与鳞状细胞肺癌相比表达上调，与小细胞肺癌相比表达下调。与小细胞肺癌相比，在腺癌肺癌中编码hsa-miR-24、hsa-miR-23a、hsa-miR-22、hsa-miR-199a-5p的任一或多种核酸分子表达上调，而编码hsa-miR-592、hsa-miR-335、hsa-miR-95、hsa-miR-454的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-34a, hsa-miR-29a, hsa-miR-29b, hsa-miR-145, hsa-miR- The expression of any one or more nucleic acid molecules of 29c is up-regulated, while those encoding hsa-miR-25, hsa-miR-93, hsa-miR-301a, hsa-miR-106b, hsa-miR-15b, has-miR-92a Expression of any one or more nucleic acid molecules is downregulated. In adenocarcinoma lung cancer, any one or more nucleic acid molecules encoding hsa-miR-205, hsa-miR-27a, hsa-miR-221 are up-regulated compared with small cell lung cancer and down-regulated compared with squamous cell lung cancer . The nucleic acid molecule encoding hsa-miR-375 was upregulated in adenocarcinoma lung cancer compared with squamous cell lung cancer and downregulated compared with small cell lung cancer. Compared with small cell lung cancer, the expression of any one or more nucleic acid molecules encoding hsa-miR-24, hsa-miR-23a, hsa-miR-22, hsa-miR-199a-5p was up-regulated in adenocarcinoma lung cancer, while The expression of any one or more nucleic acid molecules encoding hsa-miR-592, hsa-miR-335, hsa-miR-95, hsa-miR-454 is down-regulated.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-25、hsa-miR-205、hsa-miR-34a、hsa-miR-375、hsa-miR-29a、hsa-miR-27a、hsa-miR-29b的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-25, hsa-miR-205, hsa-miR-34a, hsa-miR-375, hsa-miR-29a, hsa-miR-27a , any one or more nucleic acid molecules of hsa-miR-29b.

特别优选地，与鳞状细胞肺癌和小细胞肺癌相比，在腺癌肺癌中编码hsa-miR-34a、hsa-miR-29a、hsa-miR-29b的任一或多种核酸分子表达上调，而编码hsa-miR-25的核酸分子表达下调。在腺癌肺癌中编码hsa-miR-205、hsa-miR-27a的任一或多种核酸分子，与小细胞肺癌相比表达上调，与鳞状细胞肺癌相比表达下调。在腺癌肺癌中编码hsa-miR-375的核酸分子，与鳞状细胞肺癌相比表达上调，与小细胞肺癌相比表达下调。Particularly preferably, compared with squamous cell lung cancer and small cell lung cancer, the expression of any one or more nucleic acid molecules encoding hsa-miR-34a, hsa-miR-29a, hsa-miR-29b is up-regulated in adenocarcinoma lung cancer, The expression of nucleic acid molecules encoding hsa-miR-25 was down-regulated. In adenocarcinoma lung cancer, any one or more nucleic acid molecules encoding hsa-miR-205 and hsa-miR-27a are up-regulated compared with small cell lung cancer, and down-regulated compared with squamous cell lung cancer. The nucleic acid molecule encoding hsa-miR-375 was upregulated in adenocarcinoma lung cancer compared with squamous cell lung cancer and downregulated compared with small cell lung cancer.

在第八方面，本发明涉及用于区别腺癌肺癌和鳞状细胞肺癌的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在腺癌肺癌或鳞状细胞肺癌的指征。In an eighth aspect, the present invention relates to a molecular marker diagnostic kit for distinguishing adenocarcinoma lung cancer from squamous cell lung cancer, said kit comprising a variety of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein said multiple One or more of the nucleic acid molecules are differentially expressed in the target cell and in the one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is present Indications for adenocarcinoma lung or squamous cell lung cancer.

本文限定的核酸表达特征可包括至少14种核酸分子，优选地至少8种核酸分子。A nucleic acid expression signature as defined herein may comprise at least 14 nucleic acid molecules, preferably at least 8 nucleic acid molecules.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-497、hsa-miR-93、hsa-miR-25、hsa-miR-29c、hsa-miR-205、hsa-miR-375、hsa-miR-149、hsa-miR-29a、hsa-miR-1、hsa-miR-15b、hsa-miR-181c、hsa-miR-29b、hsa-miR-769-5p和hsa-miR-221的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-497, hsa-miR-93, hsa-miR-25, hsa-miR-29c, hsa-miR-205, hsa-miR-375 , hsa-miR-149, hsa-miR-29a, hsa-miR-1, hsa-miR-15b, hsa-miR-181c, hsa-miR-29b, hsa-miR-769-5p, and hsa-miR-221 any one or more nucleic acid molecules.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-497、hsa-miR-29c、hsa-miR-375、hsa-miR-29a、hsa-miR-1、hsa-miR-181c、hsa-miR-29b的任一或多种核酸分子表达上调，而编码hsa-miR-93、hsa-miR-25、hsa-miR-205、hsa-miR-149、hsa-miR-15b、hsa-miR-769-5p、hsa-miR-221的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-497, hsa-miR-29c, hsa-miR-375, hsa-miR-29a are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-1, hsa-miR-181c, hsa-miR-29b any one or more nucleic acid molecule expression is up-regulated, and coding hsa-miR-93, hsa-miR-25, hsa-miR-205, hsa - downregulation of expression of any one or more nucleic acid molecules of miR-149, hsa-miR-15b, hsa-miR-769-5p, hsa-miR-221.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-497、hsa-miR-93、hsa-miR-25、hsa-miR-29c、hsa-miR-205、hsa-miR-375、hsa-miR-149和hsa-miR-29a的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-497, hsa-miR-93, hsa-miR-25, hsa-miR-29c, hsa-miR-205, hsa-miR-375 Any one or more nucleic acid molecules of hsa-miR-149 and hsa-miR-29a.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-497、hsa-miR-29c、hsa-miR-375、hsa-miR-29a的任一或多种核酸分子表达上调，而编码hsa-miR-93、hsa-miR-25、hsa-miR-205、hsa-miR-149的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-497, hsa-miR-29c, hsa-miR-375, hsa-miR-29a are encoded in said one or more target cells compared to said one or more control cells The expression of any one or more nucleic acid molecules encoding hsa-miR-93, hsa-miR-25, hsa-miR-205, hsa-miR-149 is down-regulated.

在第九方面，本发明涉及一种鉴定显示或具有发生肺癌倾向的一或多种靶细胞的方法，所述方法包括：(a)在一或多种靶细胞中确定多种核酸分子的表达水平，每种核酸分子编码微RNA序列；(b)在一或多种健康对照细胞中确定所述多种核酸分子的表达水平；及(c)通过比较在步骤(a)和(b)中获得的各自表达水平而从所述多种核酸分子中鉴定在靶细胞和对照细胞中差异表达的一或多种核酸分子，其中所述一或多种差异表达的核酸分子一起代表了本文限定的核酸表达特征，该核酸表达特征是存在肺癌或有肺癌发生倾向的指征。In a ninth aspect, the present invention relates to a method of identifying one or more target cells exhibiting or having a predisposition to develop lung cancer, the method comprising: (a) determining the expression of a plurality of nucleic acid molecules in the one or more target cells Each nucleic acid molecule encodes a microRNA sequence; (b) determines the expression level of said plurality of nucleic acid molecules in one or more healthy control cells; and (c) by comparing in steps (a) and (b) One or more nucleic acid molecules that are differentially expressed in the target cell and the control cell are identified from the plurality of nucleic acid molecules based on the respective expression levels obtained, wherein the one or more differentially expressed nucleic acid molecules together represent the expression defined herein. A nucleic acid expression signature that is indicative of the presence or predisposition of lung cancer.

在本发明的更优选的实施方案中，所述方法进一步用于区别肺癌中的腺癌肺癌、鳞状细胞肺癌和小细胞肺癌。In a more preferred embodiment of the present invention, said method is further used to distinguish adenocarcinoma lung cancer, squamous cell lung cancer and small cell lung cancer among lung cancers.

在第十方面，本发明涉及一种预防或治疗肺癌的方法，所述方法包括：所述方法包括：(a)用本文限定的方法在一或多种靶细胞中鉴定核酸表达特征；和(b)在所述一或多种细胞中改变在所述核酸表达特征中所包含的编码微RNA序列的一或多种核酸分子的表达，所述改变以如下的方式进行，即其表达在所述一或多种靶细胞中被上调的核酸分子的表达被下调，而其表达在所述一或多种靶细胞中被下调的核酸分子的表达被上调。In a tenth aspect, the present invention relates to a method of preventing or treating lung cancer, said method comprising: said method comprising: (a) identifying nucleic acid expression signatures in one or more target cells using a method as defined herein; and ( b) in said one or more cells, alter the expression of one or more nucleic acid molecules encoding microRNA sequences contained in said nucleic acid expression signature, said alteration being carried out in such a way that it is expressed in said nucleic acid expression profile Expression of a nucleic acid molecule whose expression is downregulated in the one or more target cells is downregulated, and expression of a nucleic acid molecule whose expression is downregulated in the one or more target cells is upregulated.

在第十一方面，本发明涉及用于预防和/或治疗肺癌的药物组合物，所述组合物包含一或多种核酸分子，每种核酸分子编码序列，所述序列与本文限定的其表达在肺癌患者中被上调的核酸分子编码的微RNA序列至少部分互补，和/或所述序列对应于本文限定的其表达在肺癌患者中被下调的核酸分子编码的微RNA序列的序列。In an eleventh aspect, the present invention relates to a pharmaceutical composition for the prevention and/or treatment of lung cancer, said composition comprising one or more nucleic acid molecules, each nucleic acid molecule coding sequence, said sequence and its expression as defined herein The microRNA sequence encoded by the nucleic acid molecule upregulated in lung cancer patients is at least partially complementary and/or said sequence corresponds to the sequence defined herein of the microRNA sequence encoded by the nucleic acid molecule whose expression is downregulated in lung cancer patients.

最后，在第十二方面，本发明涉及所述药物组合物在制备用于预防和/或治疗肺癌的药物中的用途。Finally, in its twelfth aspect, the present invention relates to the use of said pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of lung cancer.

本发明的其它实施方案从以下详细描述中将变得明了。Other embodiments of the invention will become apparent from the following detailed description.

附图简述Brief description of the drawings

图1：描绘了流程图，示意性示出用于确定本发明的表达特征的关键方法步骤，所述表达特征用于鉴定显示或具有发生肺癌倾向的一或多种靶细胞。Figure 1 : depicts a flow diagram schematically illustrating the key method steps used to determine the expression signature of the present invention for identifying one or more target cells that exhibit or have a predisposition to develop lung cancer.

图2：示出在正常肺组织和不同类别肺癌中差异表达的miRNA的一个非监督层次分类。该分类将大部分正常样本归为一组，大部分肺癌样本归为另一组。肺癌的不同类别，包括小细胞肺癌、鳞状细胞肺癌、腺癌肺癌，也清楚地彼此区分开来。Figure 2: Shows an unsupervised hierarchical classification of miRNAs differentially expressed in normal lung tissue and different classes of lung cancer. This classification puts most normal samples into one group and most lung cancer samples into another group. The different classes of lung cancer, including small cell lung cancer, squamous cell lung cancer, and adenocarcinoma lung cancer, are also clearly distinguished from each other.

图3：示出本发明的第一方面中的前7个miRNA表达特征，用于鉴定显示肺癌的一或多种靶细胞。还示出该交叉验证分类的精确度，以及这些miRNA作为分类标记，与正常肺组织相比，在肺癌组织中的表达调节(即上调或下调)。Figure 3: Shows the top 7 miRNA expression signatures in the first aspect of the invention for identifying one or more target cells exhibiting lung cancer. Also shown is the accuracy of this cross-validated classification, as well as the expression regulation (ie, upregulation or downregulation) of these miRNAs as classification markers in lung cancer tissues compared to normal lung tissues.

图4：示出本发明的第二方面中的前9个miRNA表达特征，用于鉴定显示非小细胞肺癌的一或多种靶细胞，包括鳞状细胞肺癌和腺癌肺癌。还示出该交叉验证分类的精确度，以及这些miRNA作为分类标记，与正常肺组织相比，在非小细胞肺癌中的表达调节(即上调或下调)。Figure 4: shows the top 9 miRNA expression signatures in the second aspect of the invention for identifying one or more target cells exhibiting non-small cell lung cancer, including squamous cell lung cancer and adenocarcinoma lung cancer. Also shown is the accuracy of this cross-validated classification, as well as the expression modulation (ie, upregulation or downregulation) of these miRNAs as classification markers in non-small cell lung cancer compared to normal lung tissue.

图5：示出本发明的第三方面中的前5个miRNA表达特征，用于鉴定显示腺癌肺癌的一或多种靶细胞。还示出该交叉验证分类的精确度，以及这些miRNA作为分类标记，与正常肺组织相比，在腺癌肺癌中的表达调节(即上调或下调)。Figure 5: Shows the top 5 miRNA expression signatures for identifying one or more target cells exhibiting adenocarcinoma lung cancer in the third aspect of the invention. Also shown is the accuracy of this cross-validated classification, as well as the expression modulation (ie, upregulation or downregulation) of these miRNAs as classification markers in adenocarcinoma lung cancer compared to normal lung tissue.

图6：示出本发明的第四方面中的前9个miRNA表达特征，用于鉴定显示鳞状细胞肺癌的一或多种靶细胞。还示出该交叉验证分类的精确度，以及这些miRNA作为分类标记，与正常肺组织相比，在鳞状细胞肺癌中的表达调节(即上调或下调)。Figure 6: shows the top 9 miRNA expression signatures in the fourth aspect of the invention for identifying one or more target cells exhibiting squamous cell lung cancer. Also shown is the accuracy of this cross-validated classification, as well as the upregulated (ie upregulated or downregulated) expression of these miRNAs as classification markers in squamous cell lung cancer compared to normal lung tissue.

图7：示出本发明的第五方面中的前11个miRNA表达特征，用于鉴定显示小细胞肺癌的一或多种靶细胞。还示出该交叉验证分类的精确度，以及这些miRNA作为分类标记，与正常肺组织相比，在小细胞肺癌中的表达调节(即上调或下调)。Figure 7: shows the top 11 miRNA expression signatures in the fifth aspect of the invention for identifying one or more target cells exhibiting small cell lung cancer. Also shown is the accuracy of this cross-validated classification, as well as the expression modulation (ie, upregulation or downregulation) of these miRNAs as classification markers in small cell lung cancer compared to normal lung tissue.

图8：示出本发明的第六方面中的前8个miRNA表达特征，用于区别非小细胞肺癌(NSCLC)和小细胞肺癌(SCLC)。还示出该交叉验证分类的精确度，以及这些miRNA作为分类标记，与小细胞肺癌相比，在非小细胞肺癌中的表达调节(即上调或下调)。Figure 8: shows the top 8 miRNA expression signatures in the sixth aspect of the present invention for distinguishing non-small cell lung cancer (NSCLC) from small cell lung cancer (SCLC). Also shown is the accuracy of this cross-validated classification, as well as the expression modulation (ie, up- or down-regulation) of these miRNAs as classification markers in non-small cell lung cancer compared to small cell lung cancer.

图9：示出基于不同类别肺癌中差异表达的miRNA的一个非监督层次分类，显示小细胞肺癌、鳞状细胞肺癌和腺癌肺癌，清楚地彼此区分开来。Figure 9: shows an unsupervised hierarchical classification based on miRNAs differentially expressed in different classes of lung cancer, showing that small cell lung cancer, squamous cell lung cancer and adenocarcinoma lung cancer are clearly distinguished from each other.

图10：示出本发明的第八方面中的前8个miRNA表达特征，用于区别腺癌肺癌(AC)和鳞状细胞肺癌(SQ)。还示出该交叉验证分类的精确度，以及这些miRNA作为分类标记，与鳞状细胞肺癌相比，在腺癌肺癌中的表达调节(即上调或下调)。Figure 10: Shows the top 8 miRNA expression signatures in the eighth aspect of the invention for distinguishing adenocarcinoma lung cancer (AC) from squamous cell lung cancer (SQ). Also shown is the accuracy of this cross-validated classification, as well as the expression modulation (ie, upregulation or downregulation) of these miRNAs as classification markers in adenocarcinoma lung cancer compared to squamous cell lung cancer.

发明详细描述Detailed description of the invention

本发明基于如下出乎意料的发现，即基于特定的miRNA表达特征，可以高精确性和灵敏性可靠地鉴定肺癌，并区分不同类型的肺癌，其中所述表达特征如本文定义典型地包括被上调和下调的人类miRNA。更特别地，所述miRNA表达特征—通过分析整体miRNA表达模式和/或各个miRNA表达水平—使得能检测早期疾病状态下的肺癌以及区分不同类型的肺癌。The present invention is based on the unexpected discovery that lung cancers can be reliably identified with high precision and sensitivity and differentiated between different types of lung cancers based on specific miRNA expression signatures, as defined herein typically including being upregulated and downregulated human miRNAs. More particularly, the miRNA expression signature—by analyzing the overall miRNA expression pattern and/or individual miRNA expression levels—enables the detection of lung cancer in early disease states and the differentiation of different types of lung cancer.

本发明的以下例证可以在缺少本文中未具体揭示的任何一或多个元件、一或多种限制的条件下适当地实施。The following illustrations of the invention may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

本发明将根据特定的实施方案并参照附图加以描述，但是本发明不受其限制，仅受权利要求书限制。所描述的附图仅是示意性的，被认为是非限制性的。The present invention will be described with reference to particular embodiments and with reference to the accompanying drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are to be considered as non-limiting.

当术语“包含”被用于本发明说明书和权利要求书中时，其不排除其它元件或步骤。为本发明目的，术语“由…组成”被认为是术语“包含”的优选实施方案。如果在下文中组被限定为包含至少一定数目的实施方案，这也被理解为揭示了优选地仅由这些实施方案组成的组。When the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined as comprising at least a certain number of embodiments, this is also understood to reveal a group which preferably consists only of these embodiments.

当指代单数形式名词使用不定冠词或定冠词，例如“一个”或“一种”，“所述”时，包括该名词的复数形式，除非特别指出。When an indefinite or definite article is used when referring to a singular noun eg "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

术语“大约”在本发明中是指本领域技术人员理解仍能保证目的特征的技术效果的精确性区间。该术语通常表示偏离指示值的±10%，优选±5%。The term "about" in the present invention refers to the accuracy range understood by those skilled in the art that can still guarantee the technical effect of the objective feature. The term usually means ±10%, preferably ±5%, of the indicated value.

另外，术语第一、第二、第三、(a)、(b)、(c)等在说明书和权利要求书中用于区分类似的元件，不是描述顺序或时间次序必须的。应理解如此应用的术语在合适情况下可互换，并且本发明描述的实施方案能以不同于本文所述或例证的其它顺序操作。In addition, the terms first, second, third, (a), (b), (c), etc. are used in the description and claims to distinguish similar elements, and are not necessarily described in sequence or in time. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the described embodiments of the invention are capable of operation in other sequences than described or illustrated herein.

术语的进一步定义在以下使用术语时给出。Further definitions of terms are given below when terms are used.

以下术语或定义仅为了理解本发明而提供。这些定义不应被认为具有小于本领域技术人员理解的范围。The following terms or definitions are provided for the understanding of the present invention only. These definitions should not be considered to have less scope than understood by those skilled in the art.

本发明的目的之一是提供通过确定多种核酸分子而诊断和/或治疗肺癌和/或发生这种病症的倾向的新方法，每种核酸分子编码一个微RNA(miRNA)序列，其中与健康对照细胞相比，所述多种核酸分子的一或多种在所分析的靶细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在肺癌或有肺癌发生倾向的指征。One of the objects of the present invention is to provide new methods for diagnosing and/or treating lung cancer and/or the predisposition to develop this condition by identifying a plurality of nucleic acid molecules, each encoding a microRNA (miRNA) sequence, which is associated with healthy One or more of the plurality of nucleic acid molecules is differentially expressed in the target cell analyzed compared to the control cell, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that expresses Characterized by the presence or indication of a predisposition to lung cancer.

更具体地，本发明的目的之一是提供核酸表达特征和/或组合物，用于鉴定显示或具有发生肺癌倾向的一或多种哺乳动物靶细胞和/或区别肺癌的不同类型。肺癌的不同类型包括鳞状细胞癌、腺癌和小细胞肺癌。More specifically, it is an object of the present invention to provide nucleic acid expression signatures and/or compositions useful for identifying one or more mammalian target cells exhibiting or having a predisposition to develop lung cancer and/or distinguishing between different types of lung cancer. Different types of lung cancer include squamous cell carcinoma, adenocarcinoma, and small cell lung cancer.

本文所用术语“癌症”(也称为“癌(carcinoma)”)，通常指任何类型的恶性赘生物，即与未受影响的(健康)野生型对照细胞相比显示或具有发生癌特征倾向的靶细胞的任何形态学和/或生理学改变(基于遗传重编程(geneticre-programming))。这种改变的例子可涉及细胞大小和形状(变大或变小)、细胞增殖(细胞数增加)、细胞分化(生理学状态变化)、凋亡(程序性细胞死亡)或细胞存活。The term "cancer" (also referred to as "carcinoma"), as used herein, generally refers to any type of malignant neoplasm that exhibits or has a propensity to develop cancerous features compared to unaffected (healthy) wild-type control cells. Any morphological and/or physiological changes (based on geneticre-programming) of target cells. Examples of such changes may relate to cell size and shape (increase in size or size), cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death), or cell survival.

本文所用术语“肺癌”是指肺组织中失控的细胞生长，或肺的癌性生长。The term "lung cancer" as used herein refers to uncontrolled cell growth in lung tissue, or cancerous growth of the lung.

本文所用术语“肺癌的不同类型”包括腺癌肺癌、鳞状细胞肺癌和小细胞肺癌。The term "different types of lung cancer" as used herein includes adenocarcinoma lung cancer, squamous cell lung cancer and small cell lung cancer.

“腺癌肺癌”(“Adenocarcinomalungcancer”或“adenocarcinomalungcarcinoma”)是一种非小细胞肺癌。80%的肺癌是非小细胞肺癌，其中约50%是腺癌。肺的腺癌源起于肺的外周部位，可在确诊之前长时间内存在。最常见于女性的一种肺癌类型，且多见于非吸烟者。"Adenocarcinoma lung cancer" ("Adenocarcinomalungcancer" or "adenocarcinomalungcarcinoma") is a type of non-small cell lung cancer. 80% of lung cancers are non-small cell lung cancers, of which about 50% are adenocarcinomas. Adenocarcinomas of the lung arise in the peripheral parts of the lung and can persist long before diagnosis is made. The most common type of lung cancer in women and is more common in non-smokers.

“鳞状细胞肺癌”(“Squamouscelllungcancer”或“squamouscelllungcarcinoma”)是一种非小细胞肺癌。约30%的NSCLC是鳞状细胞肺癌。鳞状细胞肺癌通常源起于肺中央部位的支气管(大气道)。多数患者早期即出现症状，常见咯血(hemoptysis(coughingupblood))。"Squamous cell lung cancer" or "squamous cell lung cancer" is a type of non-small cell lung cancer. About 30% of NSCLC are squamous cell lung cancers. Squamous cell lung cancer usually starts in the bronchi (large airways) in the center of the lungs. Symptoms appear in most patients at an early stage, and hemoptysis (coughingupblood) is common.

“小细胞肺癌”(“Smallcelllungcancer”或“smallcelllungcarcinoma”)被认为起源于组成支气管(气道)上皮的一部分(内衬)的神经内分泌细胞。SCLC约占所有肺癌病例的18%。SCLC具有强侵袭性。生长迅速，可经由血流到达肝脏、肺、骨和脑。诊断时往往已可在这些器官发现肿瘤。"Small cell lung cancer" or "small cell lung cancer" is thought to arise from the neuroendocrine cells that make up part of the epithelium (lining) of the bronchi (airways). SCLC accounts for approximately 18% of all lung cancer cases. SCLC is highly aggressive. It grows rapidly and can reach the liver, lungs, bones, and brain via the bloodstream. Tumors are often found in these organs at the time of diagnosis.

本文所用术语“患者”是指至少被认为患有肺癌，或某种类型肺癌的人类。而术语“健康个体”或“健康对照”通常指代不具有该种癌性表型的健康人。但在一些应用中，例如比较肺癌的不同类别时，患有其他类别的肺癌的个体也通常被作为“对照”。The term "patient" as used herein refers to a human being who is at least believed to have lung cancer, or a certain type of lung cancer. Instead, the term "healthy individual" or "healthy control" generally refers to a healthy person who does not have the cancerous phenotype. However, in some applications, such as when comparing different types of lung cancer, individuals with other types of lung cancer are often also used as "controls".

在本发明的体外方法中用于检测的样品通常应以临床可接受的方式收集，优选以保护核酸(特别是RNA)或蛋白质的方式收集。待分析的样品典型地来源于组织。另外，血液及其它类型样品也可以使用。样品，特别在最初加工之后可以合并。但是也可以使用未合并的样品。The samples used for detection in the in vitro method of the present invention should generally be collected in a clinically acceptable manner, preferably in a manner that protects nucleic acid (especially RNA) or protein. The sample to be analyzed is typically derived from tissue. In addition, blood and other types of samples can also be used. Samples, especially after initial processing, can be pooled. But unpooled samples can also be used.

本文所用术语“微RNA”(或“miRNA”)是其在本领域的普通含义(Bartel,D.P.(2004)Cell23,281-292；He,L.andHannon,G.J.(2004)Nat.Rev.Genet.5,522-531)。因此，“微RNA”是指衍生自基因组基因座的RNA分子，其从可以形成局部RNA前体miRNA结构的转录物加工而来。成熟miRNA通常长度为20、21、22、23、24或25个核苷酸，其它数目的核苷酸也可存在，例如18、19、26或27个核苷酸。The term "microRNA" (or "miRNA") as used herein is its ordinary meaning in the art (Bartel, D.P. (2004) Cell 23, 281-292; He, L. and Hannon, G.J. (2004) Nat. Rev. Genet. 5, 522-531). Thus, "microRNA" refers to RNA molecules derived from genomic loci that are processed from transcripts that can form local RNA precursor miRNA structures. Mature miRNAs are typically 20, 21, 22, 23, 24 or 25 nucleotides in length, although other numbers of nucleotides may also be present, such as 18, 19, 26 or 27 nucleotides.

miRNA编码序列具有与侧翼基因组序列配对的潜力，将成熟miRNA置于非完全配对的RNA双链体之内(本文也称作茎-环或发夹结构或pre-miRNA)，所述双链体作为从更长的前体转录物进行miRNA加工的中间体。这一加工典型地通过分别称为Drosha和Dicer的两种特异的内切核酸酶的连续作用而发生。Drosha从初级转录物(本文也称作“pri-miRNA”)产生典型地折叠成发夹或茎-环结构的miRNA前体(本文也称作“pre-miRNA”)。从这个miRNA前体，通过Dicer切割miRNA双链体，其在发夹或茎-环结构的一条臂包含成熟miRNA，在另一条臂包含类似大小的节段(通常称为miRNA^*)。miRNA然后被导向其靶mRNA以发挥其功能，而miRNA^*被降解。另外，miRNA典型地衍生自与预测的蛋白质编码区不同的基因组节段。The miRNA coding sequence has the potential to pair with flanking genomic sequences, placing the mature miRNA within a non-perfectly paired RNA duplex (also referred to herein as a stem-loop or hairpin structure or pre-miRNA) that Serves as an intermediate in miRNA processing from longer precursor transcripts. This processing typically occurs through the sequential action of two specific endonucleases called Drosha and Dicer, respectively. Drosha produces miRNA precursors (also referred to herein as "pre-miRNAs") that typically fold into hairpin or stem-loop structures from primary transcripts (also referred to herein as "pri-miRNAs"). From this miRNA precursor, miRNA duplexes are cleaved by Dicer, which contain the mature miRNA in one arm of the hairpin or stem-loop structure and a similarly sized segment in the other arm (commonly referred to as miRNA^* ). miRNAs are then directed to their target mRNAs to carry out their functions, while miRNAs^* are degraded. In addition, miRNAs are typically derived from different genomic segments than predicted protein-coding regions.

本文所用术语“miRNA前体”(或“前体miRNA”或“pre-miRNA”)是指从其加工成熟miRNA的miRNA初级转录物的部分。典型地，pre-miRNA折叠成稳定的发夹(即双链体)或茎-环结构。发夹结构典型地长度为50-80个核苷酸，优选60-70个核苷酸(计数miRNA残基，与miRNA配对的残基，及任何间插节段，但排除更远端的序列)。The term "miRNA precursor" (or "pre-miRNA" or "pre-miRNA") as used herein refers to the portion of the primary transcript of the miRNA from which the mature miRNA is processed. Typically, pre-miRNAs fold into stable hairpin (ie, duplex) or stem-loop structures. Hairpin structures are typically 50-80 nucleotides in length, preferably 60-70 nucleotides (counting miRNA residues, residues paired with miRNAs, and any intervening segments, but excluding more distal sequences ).

本文所用术语“编码微RNA序列的核酸分子”是指编码微RNA(miRNA)的任何核酸分子。因此，该术语不仅指成熟miRNA，也指相应的如上所述的前体miRNA及初级miRNA转录物。另外，本发明不限于RNA分子，也包括相应的编码微RNA的DNA分子，例如通过逆转录miRNA序列产生的DNA分子。编码本发明的微RNA序列的核酸分子典型地编码单个miRNA序列(即个体miRNA)。但是，也可能这种核酸分子编码两个或多个miRNA序列(即两个或多个miRNA)，例如一个转录单位包含在常用调节序列如启动子或转录终止子控制下的两个或多个miRNA序列。The term "nucleic acid molecule encoding a microRNA sequence" as used herein refers to any nucleic acid molecule encoding a microRNA (miRNA). Thus, the term refers not only to mature miRNAs, but also to corresponding precursor miRNAs and primary miRNA transcripts as described above. In addition, the present invention is not limited to RNA molecules, but also includes corresponding DNA molecules encoding microRNAs, such as DNA molecules produced by reverse transcription of miRNA sequences. A nucleic acid molecule encoding a microRNA sequence of the invention typically encodes a single miRNA sequence (ie, an individual miRNA). However, it is also possible that such a nucleic acid molecule encodes two or more miRNA sequences (i.e. two or more miRNAs), for example a transcription unit comprising two or more miRNA sequences under the control of usual regulatory sequences such as promoters or transcription terminators. miRNA sequences.

本文所用术语“编码微RNA序列的核酸分子”也被理解为包括“有义核酸分子”(即核酸序列(5'→3')匹配或相应于所编码的miRNA(5'→3')序列的分子)及“反义核酸分子”(即核酸序列互补于所编码的miRNA(5'→3')序列或者换句话说匹配所编码的miRNA序列的反向互补序列(3'→5')的分子)。本文所用术语“互补”是指“反义”核酸分子序列与相应的“有义”核酸分子序列(具有互补于反义序列的序列)形成碱基对、优选Watson-Crick碱基对的能力。The term "nucleic acid molecule encoding a microRNA sequence" as used herein is also understood to include a "sense nucleic acid molecule" (i.e. a nucleic acid sequence (5'→3') matching or corresponding to the encoded miRNA (5'→3') sequence molecules) and "antisense nucleic acid molecules" (i.e., the nucleic acid sequence is complementary to the encoded miRNA (5'→3') sequence or in other words matches the reverse complementary sequence (3'→5') of the encoded miRNA sequence molecules). The term "complementary" as used herein refers to the ability of an "antisense" nucleic acid molecule sequence to form base pairs, preferably Watson-Crick base pairs, with a corresponding "sense" nucleic acid molecule sequence (having a sequence complementary to the antisense sequence).

在本发明范围内，两个核酸分子(即“有义”和“反义”分子)可以完全互补，即它们不含有任何碱基错配和/或额外的或缺失的核苷酸。或者，两个分子包含一或多个碱基错配或者在它们的核苷酸总数上不同(由于添加或缺失导致)。优选地，“互补”核酸分子包含与包含在相应的“有义”核酸分子中的序列显示完全互补性的至少10个连续核苷酸。Within the scope of the present invention, two nucleic acid molecules (ie "sense" and "antisense" molecules) may be fully complementary, ie they do not contain any base mismatches and/or additional or missing nucleotides. Alternatively, the two molecules contain one or more base mismatches or differ in their total number of nucleotides (due to additions or deletions). Preferably, a "complementary" nucleic acid molecule comprises at least 10 contiguous nucleotides showing complete complementarity to the sequence contained in the corresponding "sense" nucleic acid molecule.

因此，包含在本发明的诊断试剂盒中的编码miRNA序列的多种核酸分子可包括一或多种“有义核酸分子”和/或一或多种“反义核酸分子”。有时，诊断试剂盒包括一或多种“有义核酸分子”(即miRNA序列本身)，所述分子被认为组成了差异表达的miRNA(即分子标记)的全体或至少亚集合，所述差异表达的miRNA是存在或发生特定病症倾向的指征，本文是肺癌。另一方面，当诊断试剂盒包括一或多种“反义核酸分子”(即与miRNA序列互补的序列)时，所述分子可包含适合用于检测和/或定量给定样品中的一或多种特定(互补)miRNA序列的探针分子(用于进行杂交测定)和/或寡核苷酸引物(例如用于逆转录或PCR应用)。Accordingly, the plurality of nucleic acid molecules encoding miRNA sequences contained in the diagnostic kits of the present invention may include one or more "sense nucleic acid molecules" and/or one or more "antisense nucleic acid molecules". Sometimes, diagnostic kits include one or more "sense nucleic acid molecules" (i.e., miRNA sequences themselves) that are believed to make up the repertoire, or at least a subset, of differentially expressed miRNAs (i.e., molecular markers) that are differentially expressed miRNAs are indicative of the presence or predisposition to a particular condition, here lung cancer. On the other hand, when the diagnostic kit includes one or more "antisense nucleic acid molecules" (i.e., sequences complementary to miRNA sequences), the molecules may contain one or more molecules suitable for detecting and/or quantifying them in a given sample. Probe molecules (for performing hybridization assays) and/or oligonucleotide primers (eg for reverse transcription or PCR applications) of specific (complementary) miRNA sequences.

在本发明中定义的多种核酸分子可以包含至少2种、至少10种、至少50种、至少100种、至少200种、至少500种、至少1000种、至少10000种或至少100000种核酸分子，每种分子编码一个miRNA序列。A plurality of nucleic acid molecules as defined in the present invention may comprise at least 2, at least 10, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 10000 or at least 100000 nucleic acid molecules, Each molecule encodes a miRNA sequence.

本文所用术语“差异表达”是指特定miRNA在靶细胞中的表达水平相比于健康对照细胞是改变的，其可以是上调(即在靶细胞中miRNA浓度增加)或下调(即在靶细胞中miRNA浓度降低或消失)。换句话说，核酸分子在靶细胞中被激活至比在对照细胞中更高或更低的水平。The term "differential expression" as used herein means that the expression level of a particular miRNA in a target cell is altered compared to a healthy control cell, which can be up-regulated (i.e., the miRNA concentration in the target cell is increased) or down-regulated (i.e., in the target cell). miRNA concentration decreased or disappeared). In other words, the nucleic acid molecule is activated to a higher or lower level in target cells than in control cells.

在本发明范围内，核酸分子被认为是差异表达的，如果该核酸分子在靶细胞和对照细胞中的相应表达水平典型地相差至少5%或至少10%，优选地至少20%或至少25%，最优选地至少30%或至少50%。因此，后者的值相应于给定核酸分子在靶细胞中的表达水平相比于对照细胞分别上调至少1.3倍或至少1.5倍，或者反之在靶细胞中的表达水平下调至少0.7倍或至少0.5倍。Within the scope of the present invention, a nucleic acid molecule is considered differentially expressed if the corresponding expression levels of the nucleic acid molecule in the target cell and the control cell typically differ by at least 5% or at least 10%, preferably at least 20% or at least 25%. , most preferably at least 30% or at least 50%. Accordingly, the latter value corresponds to the expression level of the given nucleic acid molecule in the target cell being up-regulated by at least 1.3-fold or at least 1.5-fold, respectively, compared to the control cell, or conversely the expression level in the target cell is down-regulated by at least 0.7-fold or at least 0.5 times.

本文所用术语“表达水平”是指特定的miRNA序列从其基因组基因座被转录的程度，即miRNA在一或多种被分析细胞中的浓度。The term "expression level" as used herein refers to the degree to which a particular miRNA sequence is transcribed from its genomic locus, ie the concentration of the miRNA in one or more cells analyzed.

如上所述，术语“对照细胞”典型地是指收集自(健康)个体的不具有肺癌表型特征的细胞样本。但是，在一些应用中，例如比较肺癌的不同类别时，收集自其他类别的肺癌个体的细胞也通常被作为“对照细胞”。As noted above, the term "control cells" typically refers to a sample of cells collected from a (healthy) individual that does not have the phenotype characteristic of lung cancer. However, in some applications, such as when comparing different types of lung cancer, cells collected from individuals with lung cancer of other types are also often used as "control cells".

表达水平的确定典型地遵循本领域熟知的已建立的标准程序(Sambrook,J.etal.(1989)MolecularCloning:ALaboratoryManual.2ndEd.,ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY；Ausubel,F.M.etal.(2001)CurrentProtocolsinMolecularBiology.Wiley&Sons,Hoboken,NJ)。确定可以在RNA水平进行，例如使用miRNA特异性探针进行Northern印迹分析，或者在逆转录(及克隆)RNA群后例如通过定量PCR或实时PCR技术在DNA水平进行。本文所用术语“确定”包括分析编码上述微RNA序列的任何核酸分子。但是，由于pri-miRNA及pre-mRNA半寿期短，典型地仅测量成熟miRNA的浓度。Determination of expression levels typically follows established standard procedures well known in the art (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F. M. et al. (2001) Current Protocols in Molecular Biology. Wiley & Sons , Hoboken, NJ). Determination can be performed at the RNA level, for example by Northern blot analysis using miRNA-specific probes, or at the DNA level after reverse transcription (and cloning) of RNA populations, for example by quantitative PCR or real-time PCR techniques. The term "determining" as used herein includes analysis of any nucleic acid molecule encoding the aforementioned microRNA sequence. However, due to the short half-life of pri-miRNAs and pre-mRNAs, only the concentration of mature miRNAs is typically measured.

在特异的实施方案中，在给定样品的若干独立测量(例如两个、三个、五个或十个测量)和/或在一群靶细胞或对照细胞内的若干测量中获得的表达水平的标准值被用于分析。标准值可以用本领域已知的任何方法获得。例如，平均值±2SD(标准差)或平均值±3SD的范围可被用作标准值。In specific embodiments, the expression level obtained in several independent measurements (for example two, three, five or ten measurements) of a given sample and/or in several measurements within a population of target cells or control cells Standard values were used for analysis. Standard values can be obtained by any method known in the art. For example, a range of mean ± 2SD (standard deviation) or mean ± 3SD can be used as the standard value.

所获得的病变细胞和对照细胞的表达水平之间的差异可以归一化至进一步的对照核酸的表达水平，例如已知管家基因的表达水平不根据样本来源个体的疾病状态而不同。举例的管家基因包括β-肌动蛋白、甘油醛-3-磷酸脱氢酶和核糖体蛋白P1等。在优选的实施方案中，对照核酸是已知在样本来源个体的不同非癌和癌(前)状态中稳定表达的另一种miRNA。The difference between the obtained expression levels of diseased cells and control cells can be normalized to the expression level of a further control nucleic acid, for example the expression level of a known housekeeping gene does not vary according to the disease state of the individual from which the sample is derived. Exemplary housekeeping genes include β-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein P1, among others. In a preferred embodiment, the control nucleic acid is another miRNA known to be stably expressed in different non-cancerous and (pre-cancerous) states of the individual from which the sample is derived.

但是，代替在任何实验中确定一或多种对照细胞的表达水平，也可以基于实验证据和/或现有技术数据定义针对特定细胞表型(即疾病状态)的一或多个截断值。在这种情况中，一或多种靶细胞的相应表达水平可以用用于归一化的稳定表达的对照miRNA确定。如果计算的“归一化”的表达水平高于相应定义的截断值，则这一发现是基因表达上调的指征。反之，如果计算的“归一化”的表达水平低于相应定义的截断值，则这一发现是基因表达下调的指征。However, instead of determining the expression level of one or more control cells in any experiment, one or more cutoff values for a particular cellular phenotype (ie disease state) can also be defined based on experimental evidence and/or prior art data. In this case, the corresponding expression level of one or more target cells can be determined using a stably expressed control miRNA for normalization. If the calculated "normalized" expression level is above the corresponding defined cut-off value, this finding is indicative of up-regulation of gene expression. Conversely, if the calculated "normalized" expression level is below the corresponding defined cut-off value, this finding is indicative of down-regulation of gene expression.

在本发明中，术语“鉴定显示或具有发生肺癌倾向的一或多种哺乳动物靶细胞”也包括预测和可能性分析(“诊断”意义上)。本文公开的组合物和方法意在临床应用，以决定治疗形式，包括治疗性干预，诊断标准如疾病阶段，和疾病监控和疾病监视。根据本发明，可提供用于检查对象状态的中间结果。这种中间结果可与额外信息组合以帮助医生、护士或其它从业人员诊断出该对象患有该疾病。或者，本发明可用于从细胞样本中检测癌性改变，并提供有用信息给医生以进行诊断。另外，本发明还用于区别不同类型的肺癌。In the present invention, the term "identifying one or more mammalian target cells that show or have a predisposition to develop lung cancer" also includes prediction and likelihood analysis (in the sense of "diagnostic"). The compositions and methods disclosed herein are intended for clinical use to determine treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stage, and disease monitoring and surveillance. According to the present invention, intermediate results for checking the status of objects can be provided. Such intermediate results can be combined with additional information to assist a doctor, nurse or other practitioner in diagnosing the subject with the disease. Alternatively, the present invention can be used to detect cancerous changes in a sample of cells and provide useful information to a physician for diagnosis. In addition, the present invention is also useful for distinguishing between different types of lung cancer.

在本发明中，所鉴定的一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在肺癌的指征。本文所用术语“表达特征”是指一组核酸分子(例如miRNA)，其中各个核酸分子的表达水平在收集自肺癌患者的细胞和健康对照之间不同。本文中，核酸表达特征也指一组标记并代表最低数目的(不同)核酸分子，每种核酸分子编码能鉴定一个个体的表型状态的miRNA序列。In the present invention, one or more differentially expressed nucleic acid molecules identified together represent a nucleic acid expression signature that is indicative of the presence of lung cancer. The term "expression signature" as used herein refers to a set of nucleic acid molecules (eg, miRNAs), wherein the expression level of each nucleic acid molecule differs between cells collected from lung cancer patients and healthy controls. Herein, a nucleic acid expression signature also refers to a set of markers and represents a minimum number of (different) nucleic acid molecules each encoding a miRNA sequence capable of identifying the phenotypic state of an individual.

在第一方面，本发明涉及用于鉴定显示或具有发生肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在肺癌或有肺癌发生倾向的指征。In a first aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop lung cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a RNA sequences, wherein one or more of said plurality of nucleic acid molecules are differentially expressed in a target cell and in one or more control cells, and wherein said one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature , the nucleic acid expression feature is an indication of the presence of lung cancer or the tendency to develop lung cancer.

本文限定的核酸表达特征可包括至少82种核酸分子，优选地至少13种核酸分子，特别优选地至少7种核酸分子。The nucleic acid expression signature defined herein may comprise at least 82 nucleic acid molecules, preferably at least 13 nucleic acid molecules, particularly preferably at least 7 nucleic acid molecules.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183(SEQIDNO:1)、hsa-miR-451(SEQIDNO:4)、hsa-miR-126(SEQIDNO:54)、hsa-miR-126^*(SEQIDNO:55)、hsa-miR-30a(SEQIDNO:56)、hsa-miR-145(SEQIDNO:58)、hsa-miR-140-3p(SEQIDNO:59)、hsa-miR-96(SEQIDNO:2)、hsa-miR-144(SEQIDNO:3)、hsa-miR-200c(SEQIDNO:5)、hsa-miR-486-5p(SEQIDNO:6)、hsa-miR-338-3p(SEQIDNO:7)、hsa-miR-218(SEQIDNO:57)、hsa-miR-139-5p(SEQIDNO:8)、hsa-miR-130b(SEQIDNO:9)、hsa-miR-30a^*(SEQIDNO:10)、hsa-miR-144^*(SEQIDNO:11)、hsa-miR-425(SEQIDNO:12)、hsa-miR-133b(SEQIDNO:13)、hsa-miR-497(SEQIDNO:14)、hsa-miR-182(SEQIDNO:15)、hsa-miR-187^*(SEQIDNO:16)、hsa-miR-30b(SEQIDNO:17)、hsa-miR-93(SEQIDNO:18)、hsa-miR-145^*(SEQIDNO:19)、hsa-miR-429(SEQIDNO:20)、hsa-miR-498(SEQIDNO:21)、hsa-miR-200a(SEQIDNO:22)、hsa-miR-625(SEQIDNO:23)、hsa-miR-130a(SEQIDNO:24)、hsa-miR-30c-2^*(SEQIDNO:25)、hsa-miR-98(SEQIDNO:26)、hsa-miR-1(SEQIDNO:27)、hsa-miR-106b(SEQIDNO:28)、hsa-miR-150^*(SEQIDNO:29)、hsa-miR-25(SEQIDNO:30)、hsa-miR-134(SEQIDNO:31)、hsa-miR-328(SEQIDNO:32)、hsa-miR-638(SEQIDNO:33)、has-miR-200b^*(SEQIDNO:34)、hsa-miR-1225-5p(SEQIDNO:35)、hsa-miR-1224-5p(SEQIDNO:36)、hsa-miR-557(SEQIDNO:37)、hsa-miR-15b(SEQIDNO:38)、has-miR-454(SEQIDNO:39)、hsa-miR-301a(SEQIDNO:40)、hsa-miR-135b(SEQIDNO:41)、hsa-miR-100(SEQIDNO:42)、hsa-miR-223(SEQIDNO:43)、hsa-miR-196b(SEQIDNO:44)、hsa-miR-196a(SEQIDNO:45)、hsa-miR-1226^*(SEQIDNO:46)、hsa-miR-424(SEQIDNO:47)、hsa-miR-21^*(SEQIDNO:48)、hsa-miR-572(SEQIDNO:49)、hsa-miR-18b(SEQIDNO:50)、hsa-miR-18a(SEQIDNO:51)、hsa-miR-9^*(SEQIDNO:52)、hsa-miR-301b(SEQIDNO:53)、hsa-miR-101(SEQIDNO:63)、has-miR-143(SEQIDNO:61)、hsa-miR-140-5p(SEQIDNO:62)、hsa-miR-30d(SEQIDNO:63)、hsa-miR-29c(SEQIDNO:64)、hsa-miR-199b-5p(SEQIDNO:65)、hsa-miR-224(SEQIDNO:66)、hsa-miR-125a-3p(SEQIDNO:67)、hsa-miR-9(SEQIDNO:68)、hsa-5miR-200b(SEQIDNO:69)、hsa-miR-195(SEQIDNO:70)、hsa-miR-210(SEQIDNO:71)、hsa-miR-7(SEQIDNO:72)、hsa-miR-21(SEQIDNO:73)、hsa-miR-17(SEQIDNO:74)、hsa-miR-128(SEQIDNO:75)、hsa-miR-141(SEQIDNO:76)、hsa-miR-142-5p(SEQIDNO:77)、hsa-miR-150(SEQIDNO:78)、hsa-miR-205(SEQIDNO:79)、hsa-miR-192(SEQIDNO:80)、hsa-miR-215(SEQIDNO:81)和hsa-miR-106a(SEQIDNO:82)的核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183 (SEQ ID NO: 1), hsa-miR-451 (SEQ ID NO: 4), hsa-miR-126 (SEQ ID NO: 54), hsa- miR-126^* (SEQ ID NO:55), hsa-miR-30a (SEQ ID NO:56), hsa-miR-145 (SEQ ID NO:58), hsa-miR-140-3p (SEQ ID NO:59), hsa-miR-96 (SEQ ID NO: 2), hsa-miR-144 (SEQ ID NO: 3), hsa-miR-200c (SEQ ID NO: 5), hsa-miR-486-5p (SEQ ID NO: 6), hsa-miR-338-3p (SEQ ID NO: :7), hsa-miR-218 (SEQ ID NO: 57), hsa-miR-139-5p (SEQ ID NO: 8), hsa-miR-130b (SEQ ID NO: 9), hsa-miR-30a^* (SEQ ID NO: 10) , hsa-miR-144^* (SEQ ID NO: 11), hsa-miR-425 (SEQ ID NO: 12), hsa-miR-133b (SEQ ID NO: 13), hsa-miR-497 (SEQ ID NO: 14), hsa-miR- 182 (SEQ ID NO:15), hsa-miR-187^* (SEQ ID NO:16), hsa-miR-30b (SEQ ID NO:17), hsa-miR-93 (SEQ ID NO:18), hsa-miR-145^* (SEQ ID NO: 19), hsa-miR-429 (SEQ ID NO: 20), hsa-miR-498 (SEQ ID NO: 21), hsa-miR-200a (SEQ ID NO: 22), hsa-miR-625 (SEQ ID NO: 23), hsa-miR -130a (SEQ ID NO: 24), hsa-miR-30c-2^* (SEQ ID NO: 25), hsa-miR-98 (SEQ ID NO: 26), hsa-miR-1 (SEQ ID NO: 27), hsa-miR-106b ( SEQ ID NO: 28), hsa-miR-150^* (SEQ ID NO: 29), hsa-miR-25 (SEQ ID NO: 30), hsa-miR-134 (SEQ ID NO: 31), hsa-miR-328 (SEQ ID NO: 32), hsa-miR-638 (SEQ ID NO: 33), has-miR-200b^* (SEQ ID NO: 34), hsa-miR-1225-5p (SEQ ID NO: 35), hsa-miR-1224-5p (SEQ ID NO: 36), hsa-miR-557 (SEQ ID NO: 37), hsa-miR-15b (SEQ ID NO: :38), has-miR-454 (SEQ ID NO: 39), hsa-miR-301a (SEQ ID NO: 40), hsa-miR-135b (SEQ ID NO: 41), hsa-miR-100 (SEQ ID NO: 42), hsa- miR-223 (SEQ ID NO: 43), hsa-miR-196b (SEQ ID NO: 44), hsa-miR-196a (SEQ ID NO: 45), hsa-miR-1226^* (SEQ ID NO: 46), hsa-miR-424 (SEQ ID NO: :47), hsa-miR-21^* (SEQ ID NO: 48), hsa-miR-572 (SEQ ID NO: 49), hsa-miR-18b (SEQ ID NO: 50), hsa-miR-18a (SEQ ID NO: 51), hsa -miR-9^* (SEQ ID NO:52), hsa-miR-301b (SEQ ID NO:53), hsa-miR-101 (SEQ ID NO:63), has-miR-143 (SEQ ID NO:61), hsa-miR-140- 5p (SEQ ID NO:62), hsa-miR-30d (SEQ ID NO:63), hsa-miR-29c (SEQ ID NO:64), hsa-miR-199b-5p (SEQ ID NO:65), hsa-miR-224 (SEQ ID NO: 66), hsa-miR-125a-3p (SEQ ID NO: 67), hsa-miR-9 (SEQ ID NO: 68), hsa-5miR-200b (SEQ ID NO: 69), hsa-miR-195 (SEQ ID NO: 70), hsa - miR-210 (SEQ ID NO: 71), hsa-miR-7 (SEQ ID NO: 72), hsa-miR-21 (SEQ ID NO: 73), hsa-miR-17 (SEQ ID NO: 74), hsa-miR-128 (SEQ ID NO: : 75), hsa-miR-141 (SEQ ID NO: 76), hsa-miR-142-5p (SEQ ID NO: 77), hsa-miR-150 (SEQ ID NO: 78), hsa-miR-205 (SEQ ID NO: 79), hsa-miR-192 (SEQ ID NO: 8 O), nucleic acid molecules of hsa-miR-215 (SEQ ID NO: 81 ) and hsa-miR-106a (SEQ ID NO: 82).

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码has-miR-183、hsa-miR-96、hsa-miR-200c、hsa-miR-130b、hsa-miR-425、hsa-miR-182、has-miR-93、hsa-miR-429、hsa-miR-200a、hsa-miR-625、hsa-miR-98、hsa-miR-106b、has-miR-25、hsa-miR-200b^*、hsa-miR-15b、hsa-miR-454、hsa-miR-301a、hsa-miR-135b、hsa-miR-196b、hsa-miR-196a、hsa-miR-424、hsa-miR-21^*、hsa-miR-18b、hsa-miR-18a、hsa-miR-9^*、hsa-miR-301b、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-21、hsa-miR-17、hsa-miR-128、hsa-miR-141、hsa-miR-205、hsa-miR-192、hsa-miR-215、hsa-miR-106a的核酸分子表达上调，而编码hsa-miR-451、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-145、hsa-miR-140-3p、hsa-miR-144、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-218、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-133b、hsa-miR-497、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-1、hsa-miR-150^*、hsa-miR-134、hsa-miR-328、has-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-100、hsa-miR-223、hsa-miR-1226^*、hsa-miR-572、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、has-miR-30d、hsa-miR-29c、hsa-miR-199b-5p、hsa-miR-125a-3p、hsa-miR-195、hsa-miR-142-5p和hsa-miR-150的任一或多种核酸分子的表达下调。Particularly preferably, has-miR-183, hsa-miR-96, hsa-miR-200c, hsa-miR-130b are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-425, hsa-miR-182, has-miR-93, hsa-miR-429, hsa-miR-200a, hsa-miR-625, hsa-miR-98, hsa-miR-106b, has -miR-25, hsa-miR-200b^* , hsa-miR-15b, hsa-miR-454, hsa-miR-301a, hsa-miR-135b, hsa-miR-196b, hsa-miR-196a, hsa- miR-424, hsa-miR-21^* , hsa-miR-18b, hsa-miR-18a, hsa-miR-9^* , hsa-miR-301b, hsa-miR-224, hsa-miR-9, hsa- miR-200b, hsa-miR-210, hsa-miR-7, hsa-miR-21, hsa-miR-17, hsa-miR-128, hsa-miR-141, hsa-miR-205, hsa-miR- 192. The expression of nucleic acid molecules of hsa-miR-215 and hsa-miR-106a is up-regulated, while encoding hsa-miR-451, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR- 145. hsa-miR-140-3p, hsa-miR-144, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR-218, hsa-miR-139-5p, hsa-miR- 30a^* , hsa-miR-144^* , hsa-miR-133b, hsa-miR-497, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa-miR-498, hsa- miR-130a, hsa-miR-30c-2^* , hsa-miR-1, hsa-miR-150^* , hsa-miR-134, hsa-miR-328, has-miR-638, hsa-miR-1225- 5p, hsa-miR-1224-5p, hsa-miR-557, hsa-miR-100, hsa-miR-223, hsa-miR-1226^* , hsa-miR-572, hsa-miR-101, hsa-miR -143, hsa-miR-140- 5p, has-miR-30d, hsa-miR-29c, hsa-miR-199b-5p, hsa-miR-125a-3p, hsa-miR-195, hsa-miR-142-5p and hsa-miR-150 Expression of any one or more nucleic acid molecules is downregulated.

上述miRNA的核酸序列列于表1。The nucleic acid sequences of the above miRNAs are listed in Table 1.

表1Table 1

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-183 hsa-miR-183 uauggcacugguagaauucacu uauggcacugguagaauucacu hsa-miR-96 hsa-miR-96 uuuggcacuagcacauuuuugcu uuuggcacuagcacauuuuugcu hsa-miR-144 hsa-miR-144 uacaguauagaugauguacu uacaguauagaugauguacu hsa-miR-451 hsa-miR-451 aaaccguuaccauuacugaguu aaaccguuaccauuacugaguu hsa-miR-200c hsa-miR-200c uaauacugccggguaaugaugga uaauacugccggguaaugaugga hsa-miR-486-5p hsa-miR-486-5p uccuguacugagcugccccgag uccuguacugagcugccccgag hsa-miR-338-3p hsa-miR-338-3p uccagcaucagugauuuuguug uccagcaucagugauuuuguug hsa-miR-139-5p hsa-miR-139-5p ucuacagugcacgugucuccag ucuacagugcacgugucuccag hsa-miR-130b hsa-miR-130b cagugcaaugaugaaagggcau cagugcaaugaugaaagggcau hsa-miR-30a^*hsa-miR-30a^* cuuucagucggauguuugcagc cuuucagucggauguuugcagc hsa-miR-144^*hsa-miR-144^* ggauaucaucauauacuguaag ggauaucaucauauacuguaag hsa-miR-425 hsa-miR-425 aaugacacgaucacucccguuga aaugacacgaucacucccguuga hsa-miR-133b hsa-miR-133b uuugguccccuucaaccagcua uuugguccccuucaaccagcua hsa-miR-497 hsa-miR-497 cagcagcacacugugguuugu cagcagcacacuguguuugu hsa-miR-182 hsa-miR-182 uuuggcaaugguagaacucacacu uuuggcaaugguagaacucacacu hsa-miR-187^*hsa-miR-187^* ggcuacaacacaggacccgggc ggcuacaacacaggacccgggc hsa-miR-30b hsa-miR-30b uguaaacauccuacacucagcu uguaaacauccuacacucagcu hsa-miR-93 hsa-miR-93 caaagugcuguucgugcagguag caaagugcuguucgugcagguag hsa-miR-145^*hsa-miR-145^* ggauuccuggaaauacuguucu ggauuccuggaaauacuguucu hsa-miR-429 hsa-miR-429 uaauacugucugguaaaaccgu uaauacugucugguaaaaccgu hsa-miR-498 hsa-miR-498 uuucaagccagggggcguuuuuc uuucaagccaggggggcguuuuuc hsa-miR-200a hsa-miR-200a uaacacugucugguaacgaugu uaacacugucugguaacgaugu hsa-miR-625 hsa-miR-625 agggggaaaguucuauagucc19 --> agggggaaaguucuauagucc19 --> hsa-miR-130a hsa-miR-130a cagugcaauguuaaaagggcau cagugcaauguuaaaagggcau hsa-miR-30c-2^*hsa-miR-30c-2^* cugggagaaggcuguuuacuc cugggaaggcuguuuacuc hsa-miR-98 hsa-miR-98 ugagguaguaaguuguauuguu ugagguaguaaguuguauuguu hsa-miR-1 hsa-miR-1 uggaauguaaagaaguauguau uggaauguaaagaaguauguau hsa-miR-106b hsa-miR-106b uaaagugcugacagugcagau uaaagugcugacagugcagau hsa-miR-150^*hsa-miR-150^* cugguacaggccugggggacag cugguacaggccuggggggacag hsa-miR-25 hsa-miR-25 cauugcacuugucucggucuga cauugcacuugucucggucuga hsa-miR-134 hsa-miR-134 ugugacugguugaccagagggg ugugacugguugaccagggggg hsa-miR-328 hsa-miR-328 cuggcccucucugcccuuccgu cuggcccucucugcccuuccgu

hsa-miR-638 hsa-miR-638 agggaucgcgggcggguggcggccu agggaucgcgggcggguggcggccu hsa-miR-200b^*hsa-miR-200b^* caucuuacugggcagcauugga caucuuacugggcagcauugga hsa-miR-1225-5p hsa-miR-1225-5p guggguacggcccagugggggg guggguacggcccaggggggg hsa-miR-1224-5p hsa-miR-1224-5p gugaggacucgggaggugg gugaggacucgggagggg hsa-miR-557 hsa-miR-557 guuugcacgggugggccuugucu guuugcacgggggggccuugucu hsa-miR-15b hsa-miR-15b uagcagcacaucaugguuuaca uagcagcaacaucaugguuuaca hsa-miR-454 hsa-miR-454 uagugcaauauugcuuauagggu uagugcaauauugcuuauagggu hsa-miR-301a hsa-miR-301a cagugcaauaguauugucaaagc cagugcaauaguauugucaaagc hsa-miR-135b hsa-miR-135b uauggcuuuucauuccuauguga uauggcuuuucauuccuauguga hsa-miR-100 hsa-miR-100 aacccguagauccgaacuugug aacccguaagauccgaacuugug hsa-miR-223 hsa-miR-223 ugucaguuugucaaauacccca ugucaguuugucaaauaccccca hsa-miR-196b hsa-miR-196b uagguaguuuccuguuguuggg uagguaguuuccuguuguuggg hsa-miR-196a hsa-miR-196a uagguaguuucauguuguuggg uagguaguuucauguuguuggg hsa-miR-1226^*hsa-miR-1226^* gugagggcaugcaggccuggaugggg gugaggggcaugcaggccuggaugggggg hsa-miR-424 hsa-miR-424 cagcagcaauucauguuuugaa cagcagcaauucauguuuugaa hsa-miR-21^*hsa-miR-21^* caacaccagucgaugggcugu caacaccagucgaugggcugu hsa-miR-572 hsa-miR-572 guccgcucggcgguggccca guccgcucggcgguggccca hsa-miR-18b hsa-miR-18b uaaggugcaucuagugcaguuag uaaggugcaucuagugcaguuag hsa-miR-18a hsa-miR-18a uaaggugcaucuagugcagauag uaaggugcaucuagugcagauag hsa-miR-9^*hsa-miR-9^* auaaagcuagauaaccgaaagu auaaagcuagauaaccgaaagu hsa-miR-301b hsa-miR-301b cagugcaaugauauugucaaagc cagugcaaugauauugucaaagc hsa-miR-126 hsa-miR-126 ucguaccgugaguaauaaugcg ucguaccgugaguaauaaugcg hsa-miR-126^*hsa-miR-126^* cauuauuacuuuugguacgcg cauuauuacuuuugguacgcg hsa-miR-30a hsa-miR-30a uguaaacauccucgacuggaag uguaaacauccucgacuggaag hsa-miR-218 hsa-miR-218 uugugcuugaucuaaccaugu uugugcuugaucuaaccaugu hsa-miR-145 hsa-miR-145 guccaguuuucccaggaaucccu guccaguuuucccaggaaucccu hsa-miR-140-3p hsa-miR-140-3p uaccacaggguagaaccacgg uaccacaggguagaaccacgg hsa-miR-101 hsa-miR-101 uacaguacugugauaacugaa uacaguaacugugauaacugaa hsa-miR-143 hsa-miR-143 ugagaugaagcacuguagcuc20 --> ugagaugaagcacuguagcuc20 --> hsa-miR-140-5p hsa-miR-140-5p cagugguuuuacccuaugguag cagugguuuaccccuaugguag hsa-miR-30d hsa-miR-30d uguaaacauccccgacuggaag uguaaacauccccgacuggaag hsa-miR-29c hsa-miR-29c uagcaccauuugaaaucgguua uagcaccauuugaaaucgguua hsa-miR-199b-5p hsa-miR-199b-5p cccaguguuuagacuaucuguuc cccaguguuuagacuaucuguuc hsa-miR-224 hsa-miR-224 caagucacuagugguuccguu caagucacuaguguuccguu hsa-miR-125a-3p hsa-miR-125a-3p acaggugagguucuugggagcc acaggugagguucuuggagcc hsa-miR-9 hsa-miR-9 ucuuugguuaucuagcuguauga ucuuugguuaucuagcuguauga hsa-miR-200b hsa-miR-200b uaauacugccugguaaugauga uaauacugccuggaaugauga hsa-miR-195 hsa-miR-195 uagcagcacagaaauauuggc uagcagcacagaaauauuggc hsa-miR-210 hsa-miR-210 cugugcgugugacagcggcuga cugugcgugugacagcggcuga hsa-miR-7 hsa-miR-7 uggaagacuagugauuuuguugu uggaagacuagugauuuuguugu hsa-miR-21 hsa-miR-21 uagcuuaucagacugauguuga uagcuuaucagacugauguuga hsa-miR-17 hsa-miR-17 caaagugcuuacagugcagguag caaagugcuuacagugcagguag hsa-miR-128 hsa-miR-128 ucacagugaaccggucucuuu ucacagugaaccggucucuuu hsa-miR-141 hsa-miR-141 uaacacugucugguaaagaugg uaacacugucugguaaagaugg

hsa-miR-142-5p hsa-miR-142-5p cauaaaguagaaagcacuacu cauaaaguagaaagcacuacu hsa-miR-150 hsa-miR-150 ucucccaacccuuguaccagug ucucccaacccuuguaccagug hsa-miR-205 hsa-miR-205 uccuucauuccaccggagucug uccuucauuccaccggagugug hsa-miR-192 hsa-miR-192 cugaccuaugaauugacagcc cugaccuaugaauugacagcc hsa-miR-215 hsa-miR-215 augaccuaugaauugacagac augaccuaugaauugacagac hsa-miR-106a hsa-miR-106a aaaagugcuuacagugcagguag aaaagugcuuacagugcagguag

本文揭示的所有miRNA序列均已经保存在miRBase数据库中(http://microrna.sanger.ac.uk/；也参见Griffiths-JonesS.etal.(2008)Nucl.AcidsRes.36,D154-D158)。All miRNA sequences disclosed herein have been deposited in the miRBase database (http://microrna.sanger.ac.uk/; see also Griffiths-Jones S. et al. (2008) Nucl. Acids Res. 36, D154-D158).

本文所用术语“至少一种核酸分子”可涉及所述多种核酸分子的任何亚群，例如任一种、任两种、任三种、任四种、任五种、任六种、任七种、任八种、任九种、任十种等核酸分子，每种核酸分子均编码包含于所述核酸表达特征内的微RNA序列。The term "at least one nucleic acid molecule" as used herein may refer to any subgroup of said plurality of nucleic acid molecules, for example any one, any two, any three, any four, any five, any six, any seven Any eight kinds, any eight kinds, any nine kinds, any ten kinds of nucleic acid molecules, each kind of nucleic acid molecule all encodes the microRNA sequence included in the expression characteristics of the nucleic acid.

在第二方面，本发明涉及用于鉴定显示或具有发生非小细胞肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，包括鳞状细胞癌和腺癌。所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在非小细胞肺癌或有非小细胞肺癌发生倾向的指征，包括鳞状细胞癌和腺癌。In a second aspect, the invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop non-small cell lung cancer, including squamous cell carcinoma and adenocarcinoma. The test kit comprises a plurality of nucleic acid molecules, each nucleic acid molecule encodes a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules is differentially expressed in the target cell and in one or more control cells, and wherein the One or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature indicative of the presence or predisposition to non-small cell lung cancer, including squamous cell carcinoma and adenocarcinoma.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-183(SEQIDNO:1)、hsa-miR-451、(SEQIDNO:4)、hsa-miR-497(SEQIDNO:14)、hsa-miR-126(SEQIDNO:54)、hsa-miR-126^*(SEQIDNO:55)、hsa-miR-30a(SEQIDNO:56)、hsa-miR-218(SEQIDNO:57)、hsa-miR-145(SEQIDNO:58)、hsa-miR-140-3p(SEQIDNO:59)、hsa-miR-96(SEQIDNO:2)、hsa-miR-144(SEQIDNO:3)、hsa-miR-200c(SEQIDNO:5)、hsa-miR-486-5p(SEQIDNO:6)、hsa-miR-139-5p(SEQIDNO:8)、hsa-miR-338-3p(SEQIDNO:7)、hsa-miR-130b(SEQIDNO:9)、hsa-miR-144^*(SEQIDNO:11)、hsa-miR-425(SEQIDNO:12)、hsa-miR-133b(SEQIDNO:13)、hsa-miR-182(SEQIDNO:15)、hsa-miR-187^*(SEQIDNO:16)、hsa-miR-30b(SEQIDNO:17)、hsa-miR-93(SEQIDNO:18)、hsa-miR-145^*(SEQIDNO:19)、hsa-miR-429(SEQIDNO:20)、hsa-miR-498(SEQIDNO:21)、hsa-miR-200a(SEQIDNO:22)、hsa-miR-625(SEQIDNO:23)、hsa-miR-130a(SEQIDNO:24)、hsa-miR-30c-2^*(SEQIDNO:25)、hsa-miR-98(SEQIDNO:26)、hsa-miR-1(SEQIDNO:27)、hsa-miR-150^*(SEQIDNO:29)、hsa-miR-25(SEQIDNO:30)、hsa-miR-134(SEQIDNO:31)、hsa-miR-328(SEQIDNO:32)、hsa-miR-638(SEQIDNO:33)、hsa-miR-200b^*(SEQIDNO:34)、hsa-miR-1225-5p(SEQIDNO:35)、hsa-miR-1224-5p(SEQIDNO:36)、hsa-miR-557(SEQIDNO:37)、hsa-miR-135b(SEQIDNO:41)、hsa-miR-100(SEQIDNO:42)、has-miR-424(SEQIDNO:47)、hsa-miR-21^*(SEQIDNO:48)、hsa-miR-30a^*(SEQIDNO:10)、hsa-miR-494(SEQIDNO:83)、hsa-miR-193b(SEQIDNO:84)、hsa-miR-99a(SEQIDNO:85)、hsa-miR-335(SEQIDNO:86)、hsa-miR-106b(SEQIDNO:28)、has-miR-140-5p(SEQIDNO:59)、hsa-miR-101(SEQIDNO:60)、hsa-miR-143(SEQIDNO:61)、hsa-miR-30d(SEQIDNO:63)、hsa-miR-29c(SEQIDNO:64)、hsa-miR-224(SEQIDNO:66)、hsa-miR-9(SEQIDNO:68)、hsa-miR-200b(SEQIDNO:69)、has-miR-195(SEQIDNO:70)、hsa-miR-210(SEQIDNO:71)、hsa-miR-7(SEQIDNO:72)、hsa-miR-21(SEQIDNO:83)、hsa-miR-128(SEQIDNO:75)、hsa-miR-141(SEQIDNO:76)、hsa-miR-142-5p(SEQIDNO:77)、hsa-miR-150(SEQIDNO:78)、hsa-miR-205(SEQIDNO:79)、hsa-miR-192(SEQIDNO:80)和hsa-miR-215(SEQIDNO:215).的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-183 (SEQ ID NO: 1), hsa-miR-451, (SEQ ID NO: 4), hsa-miR-497 (SEQ ID NO: 14), hsa - miR-126 (SEQ ID NO: 54), hsa-miR-126^* (SEQ ID NO: 55), hsa-miR-30a (SEQ ID NO: 56), hsa-miR-218 (SEQ ID NO: 57), hsa-miR-145 ( SEQ ID NO:58), hsa-miR-140-3p (SEQ ID NO:59), hsa-miR-96 (SEQ ID NO:2), hsa-miR-144 (SEQ ID NO:3), hsa-miR-200c (SEQ ID NO:5) , hsa-miR-486-5p (SEQ ID NO: 6), hsa-miR-139-5p (SEQ ID NO: 8), hsa-miR-338-3p (SEQ ID NO: 7), hsa-miR-130b (SEQ ID NO: 9) , hsa-miR-144^* (SEQ ID NO: 11), hsa-miR-425 (SEQ ID NO: 12), hsa-miR-133b (SEQ ID NO: 13), hsa-miR-182 (SEQ ID NO: 15), hsa-miR- 187^* (SEQ ID NO:16), hsa-miR-30b (SEQ ID NO:17), hsa-miR-93 (SEQ ID NO:18), hsa-miR-145^* (SEQ ID NO:19), hsa-miR-429 (SEQ ID NO: 20), hsa-miR-498 (SEQ ID NO: 21), hsa-miR-200a (SEQ ID NO: 22), hsa-miR-625 (SEQ ID NO: 23), hsa-miR-130a (SEQ ID NO: 24), hsa-miR -30c-2^* (SEQ ID NO:25), hsa-miR-98 (SEQ ID NO:26), hsa-miR-1 (SEQ ID NO:27), hsa-miR-150^* (SEQ ID NO:29), hsa-miR-25 (SEQ ID NO:30), hsa-miR-134 (SEQ ID NO:31), hsa-miR-328 (SEQ ID NO:32), hsa-miR-638 (SEQ ID NO:33), hsa-miR-200b^* (SEQ ID NO:34) , hsa-miR-1225-5p (SEQ ID NO: 35), hsa-miR-12 24-5p (SEQ ID NO:36), hsa-miR-557 (SEQ ID NO:37), hsa-miR-135b (SEQ ID NO:41), hsa-miR-100 (SEQ ID NO:42), has-miR-424 (SEQ ID NO: 47), hsa-miR-21^* (SEQ ID NO:48), hsa-miR-30a^* (SEQ ID NO:10), hsa-miR-494 (SEQ ID NO:83), hsa-miR-193b (SEQ ID NO:84), hsa - miR-99a (SEQ ID NO: 85), hsa-miR-335 (SEQ ID NO: 86), hsa-miR-106b (SEQ ID NO: 28), has-miR-140-5p (SEQ ID NO: 59), hsa-miR-101 (SEQ ID NO:60), hsa-miR-143 (SEQ ID NO:61), hsa-miR-30d (SEQ ID NO:63), hsa-miR-29c (SEQ ID NO:64), hsa-miR-224 (SEQ ID NO:66), hsa-miR-9 (SEQ ID NO: 68), hsa-miR-200b (SEQ ID NO: 69), has-miR-195 (SEQ ID NO: 70), hsa-miR-210 (SEQ ID NO: 71), hsa-miR-7 ( SEQ ID NO:72), hsa-miR-21 (SEQ ID NO:83), hsa-miR-128 (SEQ ID NO:75), hsa-miR-141 (SEQ ID NO:76), hsa-miR-142-5p (SEQ ID NO:77) Any one or more of hsa-miR-150 (SEQ ID NO:78), hsa-miR-205 (SEQ ID NO:79), hsa-miR-192 (SEQ ID NO:80) and hsa-miR-215 (SEQ ID NO:215). A nucleic acid molecule.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-183、hsa-miR-96、hsa-miR-200c、hsa-miR-130b、hsa-miR-425、hsa-miR-182、hsa-miR-93、hsa-miR-429、hsa-miR-200a、hsa-miR-625、hsa-miR-98、hsa-miR-25、hsa-miR-200b^*、hsa-miR-135b、hsa-miR-424、hsa-miR-21^*、hsa-miR-494、hsa-miR-193b、hsa-miR-106b、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-21、hsa-miR-128、hsa-miR-141、hsa-miR-205、hsa-miR-192、hsa-miR-215的任一或多种核酸分子表达上调，而编码hsa-miR-451、hsa-miR-497、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-144、hsa-miR-486-5p和hsa-miR-139-5p、hsa-miR-338-3p、hsa-miR-144^*、hsa-miR-133b、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-1、hsa-miR-150^*、hsa-miR-134、hsa-miR-328、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-100、hsa-miR-30a^*、hsa-miR-99a、hsa-miR-335、has-miR-140-5p、hsa-miR-101、hsa-miR-143、hsa-miR-30d、hsa-miR-29c、hsa-miR-195、hsa-miR-142-5p、hsa-miR-150的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-183, hsa-miR-96, hsa-miR-200c, hsa-miR-130b are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-425, hsa-miR-182, hsa-miR-93, hsa-miR-429, hsa-miR-200a, hsa-miR-625, hsa-miR-98, hsa-miR-25, hsa -miR-200b^* , hsa-miR-135b, hsa-miR-424, hsa-miR-21^* , hsa-miR-494, hsa-miR-193b, hsa-miR-106b, hsa-miR-224, hsa - miR-9, hsa-miR-200b, hsa-miR-210, hsa-miR-7, hsa-miR-21, hsa-miR-128, hsa-miR-141, hsa-miR-205, hsa-miR -192, hsa-miR-215 any one or more nucleic acid molecule expression up-regulated, and encoding hsa-miR-451, hsa-miR-497, hsa-miR-126, hsa-miR-126^* , hsa-miR- 30a, hsa-miR-218, hsa-miR-145, hsa-miR-140-3p, hsa-miR-144, hsa-miR-486-5p and hsa-miR-139-5p, hsa-miR-338- 3p, hsa-miR-144^* , hsa-miR-133b, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa-miR-498, hsa-miR-130a, hsa-miR -30c-2^* , hsa-miR-1, hsa-miR-150^* , hsa-miR-134, hsa-miR-328, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-1224 -5p, hsa-miR-557, hsa-miR-100, hsa-miR-30a^* , hsa-miR-99a, hsa-miR-335, has-miR-140-5p, hsa-miR-101, hsa- The expression of any one or more nucleic acid molecules of miR-143, hsa-miR-30d, hsa-miR-29c, hsa-miR-195, hsa-miR-142-5p, hsa-miR-150 is down-regulated.

上述miRNA的核酸序列列于表2。The nucleic acid sequences of the above miRNAs are listed in Table 2.

表2Table 2

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-183 hsa-miR-183 uauggcacugguagaauucacu uauggcacugguagaauucacu hsa-miR-96 hsa-miR-96 uuuggcacuagcacauuuuugcu uuuggcacuagcacauuuuugcu hsa-miR-144 hsa-miR-144 uacaguauagaugauguacu uacaguauagaugauguacu hsa-miR-451 hsa-miR-451 aaaccguuaccauuacugaguu aaaccguuaccauuacugaguu hsa-miR-200c hsa-miR-200c uaauacugccggguaaugaugga uaauacugccggguaaugaugga hsa-miR-486-5p hsa-miR-486-5p uccuguacugagcugccccgag uccuguacugagcugccccgag hsa-miR-139-5p hsa-miR-139-5p ucuacagugcacgugucuccag ucuacagugcacgugucuccag hsa-miR-497 hsa-miR-497 cagcagcacacugugguuugu cagcagcacacuguguuugu hsa-miR-338-3p hsa-miR-338-3p uccagcaucagugauuuuguug uccagcaucagugauuuuguug hsa-miR-130b hsa-miR-130b cagugcaaugaugaaagggcau cagugcaaugaugaaagggcau hsa-miR-144^*hsa-miR-144^* ggauaucaucauauacuguaag ggauaucaucauauacuguaag hsa-miR-425 hsa-miR-425 aaugacacgaucacucccguuga aaugacacgaucacucccguuga hsa-miR-133b hsa-miR-133b uuugguccccuucaaccagcua uuugguccccuucaaccagcua hsa-miR-182 hsa-miR-182 uuuggcaaugguagaacucacacu uuuggcaaugguagaacucacacu hsa-miR-187^*hsa-miR-187^* ggcuacaacacaggacccgggc ggcuacaacacaggacccgggc hsa-miR-30b hsa-miR-30b uguaaacauccuacacucagcu uguaaacauccuacacucagcu hsa-miR-93 hsa-miR-93 caaagugcuguucgugcagguag caaagugcuguucgugcagguag hsa-miR-145^*hsa-miR-145^* ggauuccuggaaauacuguucu ggauuccuggaaauacuguucu hsa-miR-429 hsa-miR-429 uaauacugucugguaaaaccgu uaauacugucugguaaaaccgu hsa-miR-498 hsa-miR-498 uuucaagccagggggcguuuuuc uuucaagccaggggggcguuuuuc hsa-miR-200a hsa-miR-200a uaacacugucugguaacgaugu uaacacugucugguaacgaugu hsa-miR-572 hsa-miR-572 guccgcucggcgguggccca guccgcucggcgguggccca hsa-miR-130a hsa-miR-130a cagugcaauguuaaaagggcau cagugcaauguuaaaagggcau hsa-miR-30c-2^*hsa-miR-30c-2^* cugggagaaggcuguuuacuc cugggaaggcuguuuacuc hsa-miR-98 hsa-miR-98 ugagguaguaaguuguauuguu ugagguaguaaguuguauuguu hsa-miR-1 hsa-miR-1 uggaauguaaagaaguauguau uggaauguaaagaaguauguau hsa-miR-150^*hsa-miR-150^* cugguacaggccugggggacag cugguacaggccuggggggacag hsa-miR-25 hsa-miR-25 cauugcacuugucucggucuga cauugcacuugucucggucuga hsa-miR-134 hsa-miR-134 ugugacugguugaccagagggg ugugacugguugaccagggggg

hsa-miR-328 hsa-miR-328 cuggcccucucugcccuuccgu cuggcccucucugcccuuccgu hsa-miR-638 hsa-miR-638 agggaucgcgggcggguggcggccu agggaucgcgggcggguggcggccu hsa-miR-200b^*hsa-miR-200b^* caucuuacugggcagcauugga caucuuacugggcagcauugga hsa-miR-1225-5p hsa-miR-1225-5p guggguacggcccagugggggg guggguacggcccaggggggg hsa-miR-1224-5p hsa-miR-1224-5p gugaggacucgggaggugg gugaggacucgggagggg hsa-miR-557 hsa-miR-557 guuugcacgggugggccuugucu guuugcacgggggggccuugucu hsa-miR-135b hsa-miR-135b uauggcuuuucauuccuauguga23 --> uauggcuuuucauuccuauguga23 --> hsa-miR-100 hsa-miR-100 aacccguagauccgaacuugug aacccguaagauccgaacuugug hsa-miR-424 hsa-miR-424 cagcagcaauucauguuuugaa cagcagcaauucauguuuugaa hsa-miR-21^*hsa-miR-21^* caacaccagucgaugggcugu caacaccagucgaugggcugu hsa-miR-30a^*hsa-miR-30a^* cuuucagucggauguuugcagc cuuucagucggauguuugcagc hsa-miR-494 hsa-miR-494 ugaaacauacacgggaaaccuc ugaaacauacacgggaaaccuc hsa-miR-193b hsa-miR-193b aacuggcccucaaagucccgcu aacuggcccucaaagucccgcu hsa-miR-99a hsa-miR-99a aacccguagauccgaucuugug aacccguagauccgaucuugug hsa-miR-335 hsa-miR-335 ucaagagcaauaacgaaaaaugu ucaagagcaauaacgaaaaaugu hsa-miR-126 hsa-miR-126 ucguaccgugaguaauaaugcg ucguaccgugaguaauaaugcg hsa-miR-126^*hsa-miR-126^* cauuauuacuuuugguacgcg cauuauuacuuuugguacgcg hsa-miR-30a hsa-miR-30a uguaaacauccucgacuggaag uguaaacauccucgacuggaag hsa-miR-218 hsa-miR-218 uugugcuugaucuaaccaugu uugugcuugaucuaaccaugu hsa-miR-145 hsa-miR-145 guccaguuuucccaggaaucccu guccaguuuucccaggaaucccu hsa-miR-140-3p hsa-miR-140-3p uaccacaggguagaaccacgg uaccacaggguagaaccacgg hsa-miR-106b hsa-miR-106b uaaagugcugacagugcagau uaaagugcugacagugcagau hsa-miR-140-5p hsa-miR-140-5p cagugguuuuacccuaugguag cagugguuuaccccuaugguag hsa-miR-101 hsa-miR-101 uacaguacugugauaacugaa uacaguaacugugauaacugaa hsa-miR-143 hsa-miR-143 ugagaugaagcacuguagcuc ugagaugaagcacuguagcuc hsa-miR-30d hsa-miR-30d uguaaacauccccgacuggaag uguaaacauccccgacuggaag hsa-miR-29c hsa-miR-29c uagcaccauuugaaaucgguua uagcaccauuugaaaucgguua hsa-miR-224 hsa-miR-224 caagucacuagugguuccguu caagucacuaguguuccguu hsa-miR-9 hsa-miR-9 ucuuugguuaucuagcuguauga ucuuugguuaucuagcuguauga hsa-miR-200b hsa-miR-200b uaauacugccugguaaugauga uaauacugccuggaaugauga hsa-miR-195 hsa-miR-195 uagcagcacagaaauauuggc uagcagcacagaaauauuggc hsa-miR-210 hsa-miR-210 cugugcgugugacagcggcuga cugugcgugugacagcggcuga hsa-miR-7 hsa-miR-7 uggaagacuagugauuuuguugu uggaagacuagugauuuuguugu hsa-miR-21 hsa-miR-21 uagcuuaucagacugauguuga uagcuuaucagacugauguuga hsa-miR-128 hsa-miR-128 ucacagugaaccggucucuuu ucacagugaaccggucucuuu hsa-miR-141 hsa-miR-141 uaacacugucugguaaagaugg uaacacugucugguaaagaugg hsa-miR-142-5p hsa-miR-142-5p cauaaaguagaaagcacuacu cauaaaguagaaagcacuacu hsa-miR-150 hsa-miR-150 ucucccaacccuuguaccagug ucucccaacccuuguaccagug hsa-miR-205 hsa-miR-205 uccuucauuccaccggagucug uccuucauuccaccggagugug hsa-miR-192 hsa-miR-192 cugaccuaugaauugacagcc cugaccuaugaauugacagcc hsa-miR-215 hsa-miR-215 augaccuaugaauugacagac augaccuaugaauugacagac

在第三方面，本发明涉及用于鉴定显示或具有发生腺癌肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在腺癌肺癌或有腺癌肺癌发生倾向的指征。In a third aspect, the invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop adenocarcinoma lung cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule Encoding microRNA sequences, wherein one or more of said plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein said one or more differentially expressed nucleic acid molecules together represent a nucleic acid An expression feature, the nucleic acid expression feature is an indication of the presence of adenocarcinoma lung cancer or the tendency to develop adenocarcinoma lung cancer.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96(SEQIDNO:2)、hsa-miR-126(SEQIDNO:54)、hsa-miR-126^*(SEQIDNO:55)、hsa-miR-30a(SEQIDNO:56)、hsa-miR-218(SEQIDNO:57)、hsa-miR-21(SEQIDNO:73)、hsa-miR-183(SEQIDNO:1)、hsa-miR-144(SEQIDNO:3)、hsa-miR-451(SEQIDNO:4)、hsa-miR-200c(SEQIDNO:5)、hsa-miR-135b(SEQIDNO:41)、hsa-miR-148a(SEQIDNO:86)、hsa-miR-625(SEQIDNO:108)、hsa-miR-486-5p(SEQIDNO:6)、hsa-miR-338-3p(SEQIDNO:7)、hsa-miR-139-5p(SEQIDNO:8)、hsa-miR-130b(SEQIDNO:9)、has-miR-30a^*(SEQIDNO:10)、hsa-miR-144^*(SEQIDNO:11)、hsa-miR-425(SEQIDNO:12)、hsa-miR-133b(SEQIDNO:13)、hsa-miR-497(SEQIDNO:14)、hsa-miR-182(SEQIDNO:15)、hsa-miR-187^*(SEQIDNO:16)、hsa-miR-30b(SEQIDNO:17)、has-miR-145^*(SEQIDNO:19)、hsa-miR-429(SEQIDNO:20)、hsa-miR-498(SEQIDNO:21)、hsa-miR-200a(SEQIDNO:22)、hsa-miR-130a(SEQIDNO:24)、hsa-miR-30c-2^*(SEQIDNO:25)、hsa-miR-134(SEQIDNO:31)、hsa-miR-638(SEQIDNO:33)、hsa-miR-200b^*(SEQIDNO:34)、hsa-miR-1225-5p(SEQIDNO:35)、hsa-miR-557(SEQIDNO:37)、hsa-miR-100(SEQIDNO:42)、hsa-miR-223(SEQIDNO:43)、hsa-miR-424(SEQIDNO:47)、hsa-miR-21^*(SEQIDNO:48)、hsa-miR-99a(SEQIDNO:85)、hsa-miR-34a(SEQIDNO:87)、hsa-miR-542-3p(SEQIDNO:88)、hsa-miR-200a^*(SEQIDNO:89)、hsa-miR-375(SEQIDNO:90)、hsa-miR-145(SEQIDNO:58)、hsa-miR-140-3p(SEQIDNO:59)、hsa-miR-101(SEQIDNO:60)、has-miR-143(SEQIDNO:61)、hsa-miR-140-5p(SEQIDNO:62)、hsa-miR-224(SEQIDNO:66)、hsa-miR-200b(SEQIDNO:69)、hsa-miR-195(SEQIDNO:70)、hsa-miR-210(SEQIDNO:71)、hsa-miR-7(SEQIDNO:72)、hsa-miR-141(SEQIDNO:76)和has-miR-192(SEQIDNO:80).的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-96 (SEQ ID NO: 2), hsa-miR-126 (SEQ ID NO: 54), hsa-miR-126^* (SEQ ID NO: 55), hsa - miR-30a (SEQ ID NO: 56), hsa-miR-218 (SEQ ID NO: 57), hsa-miR-21 (SEQ ID NO: 73), hsa-miR-183 (SEQ ID NO: 1), hsa-miR-144 (SEQ ID NO: :3), hsa-miR-451 (SEQ ID NO: 4), hsa-miR-200c (SEQ ID NO: 5), hsa-miR-135b (SEQ ID NO: 41), hsa-miR-148a (SEQ ID NO: 86), hsa- miR-625 (SEQ ID NO: 108), hsa-miR-486-5p (SEQ ID NO: 6), hsa-miR-338-3p (SEQ ID NO: 7), hsa-miR-139-5p (SEQ ID NO: 8), hsa- miR-130b (SEQ ID NO:9), has-miR-30a^* (SEQ ID NO:10), hsa-miR-144^* (SEQ ID NO:11), hsa-miR-425 (SEQ ID NO:12), hsa-miR-133b ( SEQ ID NO: 13), hsa-miR-497 (SEQ ID NO: 14), hsa-miR-182 (SEQ ID NO: 15), hsa-miR-187^* (SEQ ID NO: 16), hsa-miR-30b (SEQ ID NO: 17), has-miR-145^* (SEQ ID NO:19), hsa-miR-429 (SEQ ID NO:20), hsa-miR-498 (SEQ ID NO:21), hsa-miR-200a (SEQ ID NO:22), hsa-miR-130a (SEQ ID NO:24), hsa-miR-30c-2^* (SEQ ID NO:25), hsa-miR-134 (SEQ ID NO:31), hsa-miR-638 (SEQ ID NO:33), hsa-miR-200b^* (SEQ ID NO :34), hsa-miR-1225-5p (SEQ ID NO: 35), hsa-miR-557 (SEQ ID NO: 37), hsa-miR-100 (SEQ ID NO: 42), hsa-miR-223 (SEQ ID NO: 43), hsa-miR-424 (SEQ ID NO: 47), hsa-miR -21^* (SEQ ID NO:48), hsa-miR-99a (SEQ ID NO:85), hsa-miR-34a (SEQ ID NO:87), hsa-miR-542-3p (SEQ ID NO:88), hsa-miR-200a^* (SEQ ID NO:89), hsa-miR-375 (SEQ ID NO:90), hsa-miR-145 (SEQ ID NO:58), hsa-miR-140-3p (SEQ ID NO:59), hsa-miR-101 (SEQ ID NO:60 ), has-miR-143 (SEQ ID NO: 61), hsa-miR-140-5p (SEQ ID NO: 62), hsa-miR-224 (SEQ ID NO: 66), hsa-miR-200b (SEQ ID NO: 69), hsa- miR-195 (SEQ ID NO:70), hsa-miR-210 (SEQ ID NO:71), hsa-miR-7 (SEQ ID NO:72), hsa-miR-141 (SEQ ID NO:76) and has-miR-192 (SEQ ID NO: 80). Any one or more nucleic acid molecules.

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-96、hsa-miR-21、hsa-miR-183、hsa-miR-200c、hsa-miR-135b、hsa-miR-148a、hsa-miR-625、hsa-miR-130b、hsa-miR-425、hsa-miR-182、has-miR-429、hsa-miR-200a、hsa-miR-200b^*、hsa-miR-424、hsa-miR-21^*、hsa-miR-34a、hsa-miR-542-3p、hsa-miR-200a^*、hsa-miR-375、hsa-miR-224、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-141、hsa-miR-192的任一或多种核酸分子表达上调，而编码hsa-miR-126、hsa-miR-126^*、has-miR-30a、hsa-miR-218、hsa-miR-144、hsa-miR-451、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-133b、hsa-miR-497、hsa-miR-187^*、hsa-miR-30b、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、has-miR-134、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-557、hsa-miR-100、hsa-miR-223、hsa-miR-99a、hsa-miR-145、hsa-miR-140-3p、hsa-miR-101、hsa-miR-143、hsa-miR-140-5p、hsa-miR-195的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-96, hsa-miR-21, hsa-miR-183, hsa-miR-200c are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-135b, hsa-miR-148a, hsa-miR-625, hsa-miR-130b, hsa-miR-425, hsa-miR-182, has-miR-429, hsa-miR-200a, hsa -miR-200b^* , hsa-miR-424, hsa-miR-21^* , hsa-miR-34a, hsa-miR-542-3p, hsa-miR-200a^* , hsa-miR-375, hsa-miR- 224. The expression of any one or more nucleic acid molecules of hsa-miR-200b, hsa-miR-210, hsa-miR-7, hsa-miR-141, hsa-miR-192 is up-regulated, while encoding hsa-miR-126, hsa-miR-126^* , has-miR-30a, hsa-miR-218, hsa-miR-144, hsa-miR-451, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR -139-5p, hsa-miR-30a^* , hsa-miR-144^* , hsa-miR-133b, hsa-miR-497, hsa-miR-187^* , hsa-miR-30b, hsa-miR-145^* , hsa-miR-498, hsa-miR-130a, hsa-miR-30c-2^* , has-miR-134, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-557, hsa- miR-100, hsa-miR-223, hsa-miR-99a, hsa-miR-145, hsa-miR-140-3p, hsa-miR-101, hsa-miR-143, hsa-miR-140-5p, The expression of any one or more nucleic acid molecules of hsa-miR-195 is down-regulated.

上述miRNA的核酸序列列于表3。The nucleic acid sequences of the above miRNAs are listed in Table 3.

表3table 3

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-183 hsa-miR-183 uauggcacugguagaauucacu uauggcacugguagaauucacu hsa-miR-96 hsa-miR-96 uuuggcacuagcacauuuuugcu uuuggcacuagcacauuuuugcu hsa-miR-144 hsa-miR-144 uacaguauagaugauguacu uacaguauagaugauguacu hsa-miR-451 hsa-miR-451 aaaccguuaccauuacugaguu aaaccguuaccauuacugaguu hsa-miR-200c hsa-miR-200c uaauacugccggguaaugaugga uaauacugccggguaaugaugga hsa-miR-135b hsa-miR-135b uauggcuuuucauuccuauguga uauggcuuuucauuccuauguga hsa-miR-486-5p hsa-miR-486-5p uccuguacugagcugccccgag uccuguacugagcugccccgag

hsa-miR-338-3p hsa-miR-338-3p uccagcaucagugauuuuguug uccagcaucagugauuuuguug hsa-miR-139-5p hsa-miR-139-5p ucuacagugcacgugucuccag ucuacagugcacgugucuccag hsa-miR-130b hsa-miR-130b cagugcaaugaugaaagggcau cagugcaaugaugaaagggcau hsa-miR-30a^*hsa-miR-30a^* cuuucagucggauguuugcagc cuuucagucggauguuugcagc hsa-miR-144^*hsa-miR-144^* ggauaucaucauauacuguaag ggauaucaucauauacuguaag hsa-miR-425 hsa-miR-425 aaugacacgaucacucccguuga aaugacacgaucacucccguuga hsa-miR-133b hsa-miR-133b uuugguccccuucaaccagcua uuugguccccuucaaccagcua hsa-miR-497 hsa-miR-497 cagcagcacacugugguuugu cagcagcacacuguguuugu hsa-miR-182 hsa-miR-182 uuuggcaaugguagaacucacacu uuuggcaaugguagaacucacacu hsa-miR-187^*hsa-miR-187^* ggcuacaacacaggacccgggc ggcuacaacacaggacccgggc hsa-miR-30b hsa-miR-30b uguaaacauccuacacucagcu uguaaacauccuacacucagcu hsa-miR-145^*hsa-miR-145^* ggauuccuggaaauacuguucu ggauuccuggaaauacuguucu hsa-miR-429 hsa-miR-429 uaauacugucugguaaaaccgu uaauacugucugguaaaaccgu hsa-miR-498 hsa-miR-498 uuucaagccagggggcguuuuuc uuucaagccaggggggcguuuuuc hsa-miR-200a hsa-miR-200a uaacacugucugguaacgaugu uaacacugucugguaacgaugu hsa-miR-625 hsa-miR-625 agggggaaaguucuauagucc agggggaaaguucuauagucc hsa-miR-130a hsa-miR-130a cagugcaauguuaaaagggcau cagugcaauguuaaaagggcau hsa-miR-30c-2^*hsa-miR-30c-2^* cugggagaaggcuguuuacuc cugggaaggcuguuuacuc hsa-miR-134 hsa-miR-134 ugugacugguugaccagagggg ugugacugguugaccagggggg hsa-miR-638 hsa-miR-638 agggaucgcgggcggguggcggccu agggaucgcgggcggguggcggccu hsa-miR-200b^*hsa-miR-200b^* caucuuacugggcagcauugga caucuuacugggcagcauugga hsa-miR-1225-5p hsa-miR-1225-5p guggguacggcccagugggggg guggguacggcccaggggggg hsa-miR-557 hsa-miR-557 guuugcacgggugggccuugucu guuugcacgggggggccuugucu hsa-miR-100 hsa-miR-100 aacccguagauccgaacuugug aacccguaagauccgaacuugug hsa-miR-223 hsa-miR-223 ugucaguuugucaaauacccca26 --> ugucaguuugucaaauacccca26 --> hsa-miR-424 hsa-miR-424 cagcagcaauucauguuuugaa cagcagcaauucauguuuugaa hsa-miR-21^*hsa-miR-21^* caacaccagucgaugggcugu caacaccagucgaugggcugu hsa-miR-99a hsa-miR-99a aacccguagauccgaucuugug aacccguagauccgaucuugug hsa-miR-148a hsa-miR-148a ucagugcacuacagaacuuugu ucagugcacuacagaacuuugu hsa-miR-34a hsa-miR-34a uggcagugucuuagcugguugu uggcagugucuuagcugguugu hsa-miR-542-3p hsa-miR-542-3p ugugacagauugauaacugaaa ugugacagauugauaacugaaa hsa-miR-200a^*hsa-miR-200a^* caucuuaccggacagugcugga caucuuaccggacagugcugga hsa-miR-375 hsa-miR-375 uuuguucguucggcucgcguga uuuguucguucggcucgcguga hsa-miR-126 hsa-miR-126 ucguaccgugaguaauaaugcg ucguaccgugaguaauaaugcg hsa-miR-126^*hsa-miR-126^* cauuauuacuuuugguacgcg cauuauuacuuuugguacgcg hsa-miR-30a hsa-miR-30a uguaaacauccucgacuggaag uguaaacauccucgacuggaag hsa-miR-218 hsa-miR-218 uugugcuugaucuaaccaugu uugugcuugaucuaaccaugu hsa-miR-21 hsa-miR-21 uagcuuaucagacugauguuga uagcuuaucagacugauguuga hsa-miR-145 hsa-miR-145 guccaguuuucccaggaaucccu guccaguuuucccaggaaucccu hsa-miR-140-3p hsa-miR-140-3p uaccacaggguagaaccacgg uaccacaggguagaaccacgg hsa-miR-101 hsa-miR-101 uacaguacugugauaacugaa uacaguaacugugauaacugaa hsa-miR-143 hsa-miR-143 ugagaugaagcacuguagcuc ugagaugaagcacuguagcuc hsa-miR-140-5p hsa-miR-140-5p cagugguuuuacccuaugguag cagugguuuaccccuaugguag hsa-miR-224 hsa-miR-224 caagucacuagugguuccguu caagucacuaguguuccguu

hsa-miR-200b hsa-miR-200b uaauacugccugguaaugauga uaauacugccuggaaugauga hsa-miR-195 hsa-miR-195 uagcagcacagaaauauuggc uagcagcacagaaauauuggc hsa-miR-210 hsa-miR-210 cugugcgugugacagcggcuga cugugcgugugacagcggcuga hsa-miR-7 hsa-miR-7 uggaagacuagugauuuuguugu uggaagacuagugauuuuguugu hsa-miR-141 hsa-miR-141 uaacacugucugguaaagaugg uaacacugucugguaaagaugg hsa-miR-192 hsa-miR-192 cugaccuaugaauugacagcc cugaccuaugaauugacagcc

在第四方面，本发明涉及用于鉴定显示或具有发生鳞状细胞肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在鳞状细胞肺癌或有鳞状细胞肺癌发生倾向的指征。In a fourth aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop squamous cell lung cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid Molecularly encoded microRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a A nucleic acid expression signature that is indicative of the presence or predisposition to squamous cell lung cancer.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-144(SEQIDNO:3)、hsa-miR-451(SEQIDNO:4)、hsa-miR-200c(SEQIDNO:5)、hsa-miR-486-5p(SEQIDNO:6)、hsa-miR-338-3p(SEQIDNO:7)、hsa-miR-497(SEQIDNO:14)、hsa-miR-218(SEQIDNO:57)、hsa-miR-145(SEQIDNO:58)、hsa-miR-140-3p(SEQIDNO:59)、hsa-miR-30b(SEQIDNO:17)、hsa-miR-93(SEQIDNO:18)、hsa-miR-126(SEQIDNO:54)、hsa-miR-126^*(SEQIDNO:55)、hsa-miR-30a(SEQIDNO:56)、hsa-miR-143(SEQIDNO:61)、hsa-miR-103(SEQIDNO:91)、hsa-miR-183(SEQIDNO:1)、has-miR-96(SEQIDNO:2)、hsa-miR-139-5p(SEQIDNO:8)、hsa-miR-130b(SEQIDNO:9)、hsa-miR-30a^*(SEQIDNO:10)、hsa-miR-144^*(SEQIDNO:11)、hsa-miR-425(SEQIDNO:12)、hsa-miR-133b(SEQIDNO:13)、hsa-miR-182(SEQIDNO:15)、has-miR-187^*(SEQIDNO:16)、hsa-miR-145^*(SEQIDNO:19)、hsa-miR-429(SEQIDNO:20)、hsa-miR-498(SEQIDNO:21)、hsa-miR-130a(SEQIDNO:24)、hsa-miR-30c-2^*(SEQIDNO:25)、hsa-miR-98(SEQIDNO:26)、hsa-miR-1(SEQIDNO:27)、has-miR-106b(SEQIDNO:28)、hsa-miR-150^*(SEQIDNO:29)、hsa-miR-25(SEQIDNO:30)、hsa-miR-134(SEQIDNO:31)、hsa-miR-328(SEQIDNO:32)、hsa-miR-638(SEQIDNO:33)、hsa-miR-1225-5p(SEQIDNO:35)、hsa-miR-1224-5p(SEQIDNO:36)、hsa-miR-557(SEQIDNO:37)、hsa-miR-15b(SEQIDNO:38)、hsa-miR-301a(SEQIDNO:40)、hsa-miR-196b(SEQIDNO:44)、hsa-miR-196a(SEQIDNO:45)、hsa-miR-1226^*(SEQIDNO:46)、hsa-miR-424(SEQIDNO:47)、hsa-miR-21^*(SEQIDNO:48)、hsa-miR-572(SEQIDNO:49)、hsa-miR-18b(SEQIDNO:50)、hsa-miR-18a(SEQIDNO:51)、hsa-miR-9^*(SEQIDNO:52)、hsa-miR-301b(SEQIDNO:53)、hsa-miR-29c^*(SEQIDNO:64)、hsa-miR-494(SEQIDNO:83)、hsa-miR-193b(SEQIDNO:84)、hsa-miR-375(SEQIDNO:90)、hsa-miR-26a(SEQIDNO:92)、hsa-miR-574-3p(SEQIDNO:93)、hsa-miR-151-3p(SEQIDNO:94)、hsa-miR-149(SEQIDNO:95)、hsa-miR-768-5p(SEQIDNO:96)、hsa-miR-181a(SEQIDNO:97)、hsa-miR-20a(SEQIDNO:98)、hsa-miR-181c(SEQIDNO:99)、hsa-miR-29a(SEQIDNO:100)、hsa-miR-29b(SEQIDNO:101)、hsa-miR-505(SEQIDNO:102)、hsa-miR-939(SEQIDNO:103)、hsa-miR-769-5p(SEQIDNO:104)、hsa-miR-30c(SEQIDNO:105)、has-miR-590-5p(SEQIDNO:106)、hsa-miR-671-5p(SEQIDNO:107)、hsa-miR-625(SEQIDNO:108)、hsa-miR-652(SEQIDNO:109)、hsa-miR-139-3p(SEQIDNO:110)、has-miR-28-5p(SEQIDNO:111)、hsa-miR-623(SEQIDNO:112)、hsa-miR-221(SEQIDNO:113)、hsa-miR-19a(SEQIDNO:114)、hsa-miR-944(SEQIDNO:115)、hsa-miR-335(SEQIDNO:116)、hsa-miR-125a-5p(SEQIDNO:117)、hsa-miR-551b(SEQIDNO:118)、hsa-let-7d^*(SEQIDNO:119)、hsa-miR-31^*(SEQIDNO:120)、hsa-miR-362-5p(SEQIDNO:121)、hsa-miR-181a^*(SEQIDNO:122)、hsa-miR-940(SEQIDNO:123)、hsa-miR-30b^*(SEQIDNO:124)、hsa-miR-601(SEQIDNO:125)、hsa-miR-20b(SEQIDNO:126)、hsa-miR-202(SEQIDNO:127)、hsa-miR-101(SEQIDNO:60)、hsa-miR-140-5p(SEQIDNO:62)、hsa-miR-30d(SEQIDNO:6)3、hsa-miR-29c(SEQIDNO:64)、hsa-miR-224(SEQIDNO:66)、hsa-miR-9(SEQIDNO:68)、hsa-miR-200b(SEQIDNO:69)、hsa-miR-195(SEQIDNO:70)、hsa-miR-210(SEQIDNO:71)、has-miR-7(SEQIDNO:72)、hsa-miR-21(SEQIDNO:73)、hsa-miR-17(SEQIDNO:74)、hsa-miR-128(SEQIDNO:75)、hsa-miR-141(SEQIDNO:76)、hsa-miR-142-5p(SEQIDNO:77)、hsa-miR-150(SEQIDNO:78)和hsa-miR-205(SEQIDNO:79)的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression features include encoding hsa-miR-144 (SEQ ID NO: 3), hsa-miR-451 (SEQ ID NO: 4), hsa-miR-200c (SEQ ID NO: 5), hsa- miR-486-5p (SEQ ID NO: 6), hsa-miR-338-3p (SEQ ID NO: 7), hsa-miR-497 (SEQ ID NO: 14), hsa-miR-218 (SEQ ID NO: 57), hsa-miR- 145 (SEQ ID NO:58), hsa-miR-140-3p (SEQ ID NO:59), hsa-miR-30b (SEQ ID NO:17), hsa-miR-93 (SEQ ID NO:18), hsa-miR-126 (SEQ ID NO: 54), hsa-miR-126^* (SEQ ID NO: 55), hsa-miR-30a (SEQ ID NO: 56), hsa-miR-143 (SEQ ID NO: 61), hsa-miR-103 (SEQ ID NO: 91), hsa- miR-183 (SEQ ID NO: 1), has-miR-96 (SEQ ID NO: 2), hsa-miR-139-5p (SEQ ID NO: 8), hsa-miR-130b (SEQ ID NO: 9), hsa-miR-30a^* (SEQ ID NO: 10), hsa-miR-144^* (SEQ ID NO: 11), hsa-miR-425 (SEQ ID NO: 12), hsa-miR-133b (SEQ ID NO: 13), hsa-miR-182 (SEQ ID NO: 15) , has-miR-187^* (SEQ ID NO: 16), hsa-miR-145^* (SEQ ID NO: 19), hsa-miR-429 (SEQ ID NO: 20), hsa-miR-498 (SEQ ID NO: 21), hsa-miR -130a (SEQ ID NO: 24), hsa-miR-30c-2^* (SEQ ID NO: 25), hsa-miR-98 (SEQ ID NO: 26), hsa-miR-1 (SEQ ID NO: 27), has-miR-106b ( SEQ ID NO: 28), hsa-miR-150^* (SEQ ID NO: 29), hsa-miR-25 (SEQ ID NO: 30), hsa-miR-134 (SEQ ID NO: 31), hsa-miR-328 (SEQ ID NO: 32), hsa-miR-638 (SEQ ID NO: 33), hsa-miR-1225-5p ( SEQ ID NO:35), hsa-miR-1224-5p (SEQ ID NO:36), hsa-miR-557 (SEQ ID NO:37), hsa-miR-15b (SEQ ID NO:38), hsa-miR-301a (SEQ ID NO:40) , hsa-miR-196b (SEQ ID NO: 44), hsa-miR-196a (SEQ ID NO: 45), hsa-miR-1226^* (SEQ ID NO: 46), hsa-miR-424 (SEQ ID NO: 47), hsa-miR- 21^* (SEQ ID NO:48), hsa-miR-572 (SEQ ID NO:49), hsa-miR-18b (SEQ ID NO:50), hsa-miR-18a (SEQ ID NO:51), hsa-miR-9^* (SEQ ID NO: 52), hsa-miR-301b (SEQ ID NO: 53), hsa-miR-29c^* (SEQ ID NO: 64), hsa-miR-494 (SEQ ID NO: 83), hsa-miR-193b (SEQ ID NO: 84), hsa- miR-375 (SEQ ID NO: 90), hsa-miR-26a (SEQ ID NO: 92), hsa-miR-574-3p (SEQ ID NO: 93), hsa-miR-151-3p (SEQ ID NO: 94), hsa-miR- 149 (SEQ ID NO:95), hsa-miR-768-5p (SEQ ID NO:96), hsa-miR-181a (SEQ ID NO:97), hsa-miR-20a (SEQ ID NO:98), hsa-miR-181c (SEQ ID NO: 99), hsa-miR-29a (SEQ ID NO: 100), hsa-miR-29b (SEQ ID NO: 101), hsa-miR-505 (SEQ ID NO: 102), hsa-miR-939 (SEQ ID NO: 103), hsa-miR -769-5p (SEQ ID NO: 104), hsa-miR-30c (SEQ ID NO: 105), has-miR-590-5p (SEQ ID NO: 106), hsa-miR-671-5p (SEQ ID NO: 107), hsa-miR -625 (SEQ ID NO: 108), hsa-miR-652 (SEQ ID NO: 109), hsa-miR-139-3p (SEQ ID NO: 110), has-miR-28-5p (SEQ ID NO: 111), hsa-miR-623 (SEQ ID NO: 112), hsa-miR-221 (SEQ ID NO: 113), hsa-miR-19a (SEQ ID NO: 114), hsa-miR-944 (SEQ ID NO: 115), hsa-miR-335 (SEQ ID NO: 116), hsa-miR -125a-5p (SEQ ID NO:117), hsa-miR-551b (SEQ ID NO:118), hsa-let-7d^* (SEQ ID NO:119), hsa-miR-31^* (SEQ ID NO:120), hsa-miR-362 -5p (SEQ ID NO:121), hsa-miR-181a^* (SEQ ID NO:122), hsa-miR-940 (SEQ ID NO:123), hsa-miR-30b^* (SEQ ID NO:124), hsa-miR-601 (SEQ ID NO:124) : 125), hsa-miR-20b (SEQ ID NO: 126), hsa-miR-202 (SEQ ID NO: 127), hsa-miR-101 (SEQ ID NO: 60), hsa-miR-140-5p (SEQ ID NO: 62), hsa-miR-30d (SEQ ID NO: 6) 3, hsa-miR-29c (SEQ ID NO: 64), hsa-miR-224 (SEQ ID NO: 66), hsa-miR-9 (SEQ ID NO: 68), hsa-miR-200b (SEQ ID NO: 69), hsa-miR-195 (SEQ ID NO: 70), hsa-miR-210 (SEQ ID NO: 71), has-miR-7 (SEQ ID NO: 72), hsa-miR-21 (SEQ ID NO: 73), hsa-miR-17 (SEQ ID NO: 74), hsa-miR-128 (SEQ ID NO: 75), hsa-miR-141 (SEQ ID NO: 76), hsa-miR-142-5p (SEQ ID NO: 77), hsa-miR- Any one or more nucleic acid molecules of 150 (SEQ ID NO: 78) and hsa-miR-205 (SEQ ID NO: 79).

特别优选地，与所述一或多种对照细胞相比，在所述一或多种靶细胞中编码hsa-miR-200c、hsa-miR-93、hsa-miR-103、hsa-miR-183、hsa-miR-96、hsa-miR-130b、hsa-miR-425、hsa-miR-182、hsa-miR-429、hsa-miR-98、hsa-miR-106b、hsa-miR-25、hsa-miR-15b、hsa-miR-301a、hsa-miR-196b、hsa-miR-196a、hsa-miR-424、hsa-miR-21^*、hsa-miR-18b、hsa-miR-18a、hsa-miR-9^*、hsa-miR-301b、has-miR-494、hsa-miR-193b、hsa-miR-151-3p、hsa-miR-149、hsa-miR-20a、hsa-miR-505、hsa-miR-769-5p、hsa-miR-590-5p、hsa-miR-590-5p、hsa-miR-625、hsa-miR-28-5p、has-miR-221、hsa-miR-19a、hsa-miR-944、hsa-miR-31^*、hsa-miR-362-5p、hsa-miR-20b、hsa-miR-224、hsa-miR-9、hsa-miR-200b、hsa-miR-210、hsa-miR-7、hsa-miR-21、has-miR-17、hsa-miR-128、hsa-miR-141、hsa-miR-205的任一或多种核酸分子表达上调，而编码hsa-miR-144、hsa-miR-451、hsa-miR-486-5p、hsa-miR-338-3p、hsa-miR-497、hsa-miR-218、hsa-miR-145、hsa-miR-140-3p、hsa-miR-30b、hsa-miR-126、hsa-miR-126^*、hsa-miR-30a、hsa-miR-143、hsa-miR-139-5p、hsa-miR-30a^*、hsa-miR-144^*、hsa-miR-133b、hsa-miR-187^*、hsa-miR-145^*、hsa-miR-498、hsa-miR-130a、hsa-miR-30c-2^*、hsa-miR-1、hsa-miR-150^*、has-miR-134、hsa-miR-328、hsa-miR-638、hsa-miR-1225-5p、hsa-miR-1224-5p、hsa-miR-557、hsa-miR-1226^*、hsa-miR-572、hsa-miR-29c^*、hsa-miR-375、hsa-miR-26a、hsa-miR-574-3p、hsa-miR-768-5p、hsa-miR-181a、hsa-miR-181c、hsa-miR-29a、hsa-miR-29b、hsa-miR-939、hsa-miR-530c、hsa-miR-671-5p、hsa-miR-652、hsa-miR-139-3p、hsa-miR-623、hsa-miR-335、hsa-miR-125a-5p、hsa-miR-551b、hsa-let-7d^*、hsa-miR-181a^*、hsa-miR-940、hsa-miR-30b^*、hsa-miR-601、hsa-miR-202、hsa-miR-101、hsa-miR-140-5p、hsa-miR-30d、hsa-miR-29c、hsa-miR-195、hsa-miR-142-5p、hsa-miR-150的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-200c, hsa-miR-93, hsa-miR-103, hsa-miR-183 are encoded in said one or more target cells compared to said one or more control cells , hsa-miR-96, hsa-miR-130b, hsa-miR-425, hsa-miR-182, hsa-miR-429, hsa-miR-98, hsa-miR-106b, hsa-miR-25, hsa -miR-15b, hsa-miR-301a, hsa-miR-196b, hsa-miR-196a, hsa-miR-424, hsa-miR-21^* , hsa-miR-18b, hsa-miR-18a, hsa- miR-9^* , hsa-miR-301b, has-miR-494, hsa-miR-193b, hsa-miR-151-3p, hsa-miR-149, hsa-miR-20a, hsa-miR-505, hsa - miR-769-5p, hsa-miR-590-5p, hsa-miR-590-5p, hsa-miR-625, hsa-miR-28-5p, has-miR-221, hsa-miR-19a, hsa -miR-944, hsa-miR-31^* , hsa-miR-362-5p, hsa-miR-20b, hsa-miR-224, hsa-miR-9, hsa-miR-200b, hsa-miR-210, The expression of any or more nucleic acid molecules of hsa-miR-7, hsa-miR-21, has-miR-17, hsa-miR-128, hsa-miR-141, hsa-miR-205 is up-regulated, and the encoding hsa- miR-144, hsa-miR-451, hsa-miR-486-5p, hsa-miR-338-3p, hsa-miR-497, hsa-miR-218, hsa-miR-145, hsa-miR-140- 3p, hsa-miR-30b, hsa-miR-126, hsa-miR-126^* , hsa-miR-30a, hsa-miR-143, hsa-miR-139-5p, hsa-miR-30a^* , hsa- miR-144^* , hsa-miR-133b, hsa-miR-187^* , hsa-miR-145^* , hsa-miR-498, hsa-miR-130a, hsa-miR-30c-2^* , hsa-miR- 1. hsa-miR-150^* , has-miR-134, hsa-mi R-328, hsa-miR-638, hsa-miR-1225-5p, hsa-miR-1224-5p, hsa-miR-557, hsa-miR-1226^* , hsa-miR-572, hsa-miR-29c^* , hsa-miR-375, hsa-miR-26a, hsa-miR-574-3p, hsa-miR-768-5p, hsa-miR-181a, hsa-miR-181c, hsa-miR-29a, hsa- miR-29b, hsa-miR-939, hsa-miR-530c, hsa-miR-671-5p, hsa-miR-652, hsa-miR-139-3p, hsa-miR-623, hsa-miR-335, hsa-miR-125a-5p, hsa-miR-551b, hsa-let-7d^* , hsa-miR-181a^* , hsa-miR-940, hsa-miR-30b^* , hsa-miR-601, hsa-miR Any of -202, hsa-miR-101, hsa-miR-140-5p, hsa-miR-30d, hsa-miR-29c, hsa-miR-195, hsa-miR-142-5p, hsa-miR-150 Expression of one or more nucleic acid molecules is downregulated.

上述miRNA的核酸序列列于表4。The nucleic acid sequences of the above miRNAs are listed in Table 4.

表4Table 4

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-144 hsa-miR-144 uacaguauagaugauguacu uacaguauagaugauguacu hsa-miR-451 hsa-miR-451 aaaccguuaccauuacugaguu aaaccguuaccauuacugaguu hsa-miR-200c hsa-miR-200c uaauacugccggguaaugaugga uaauacugccggguaaugaugga hsa-miR-486-5p hsa-miR-486-5p uccuguacugagcugccccgag uccuguacugagcugccccgag hsa-miR-338-3p hsa-miR-338-3p uccagcaucagugauuuuguug uccagcaucagugauuuuguug hsa-miR-497 hsa-miR-497 cagcagcacacugugguuugu cagcagcacacuguguuugu hsa-miR-30b hsa-miR-30b uguaaacauccuacacucagcu uguaaacauccuacacucagcu hsa-miR-93 hsa-miR-93 caaagugcuguucgugcagguag caaagugcuguucgugcagguag hsa-miR-103 hsa-miR-103 agcagcauuguacagggcuauga agcagcauuguacagggcuauga hsa-miR-183 hsa-miR-183 uauggcacugguagaauucacu uauggcacugguagaauucacu hsa-miR-96 hsa-miR-96 uuuggcacuagcacauuuuugcu uuuggcacuagcacauuuuugcu hsa-miR-139-5p hsa-miR-139-5p ucuacagugcacgugucuccag29 --> ucuacagugcacgugucuccag29 --> hsa-miR-130b hsa-miR-130b cagugcaaugaugaaagggcau cagugcaaugaugaaagggcau hsa-miR-30a^*hsa-miR-30a^* cuuucagucggauguuugcagc cuuucagucggauguuugcagc hsa-miR-144^*hsa-miR-144^* ggauaucaucauauacuguaag ggauaucaucauauacuguaag hsa-miR-425 hsa-miR-425 aaugacacgaucacucccguuga aaugacacgaucacucccguuga hsa-miR-133b hsa-miR-133b uuugguccccuucaaccagcua uuugguccccuucaaccagcua hsa-miR-182 hsa-miR-182 uuuggcaaugguagaacucacacu uuuggcaaugguagaacucacacu hsa-miR-187^*hsa-miR-187^* ggcuacaacacaggacccgggc ggcuacaacacaggacccgggc hsa-miR-145^*hsa-miR-145^* ggauuccuggaaauacuguucu ggauuccuggaaauacuguucu hsa-miR-429 hsa-miR-429 uaauacugucugguaaaaccgu uaauacugucugguaaaaccgu hsa-miR-498 hsa-miR-498 uuucaagccagggggcguuuuuc uuucaagccaggggggcguuuuuc hsa-miR-130a hsa-miR-130a cagugcaauguuaaaagggcau cagugcaauguuaaaagggcau hsa-miR-30c-2^*hsa-miR-30c-2^* cugggagaaggcuguuuacuc cugggaaggcuguuuacuc hsa-miR-98 hsa-miR-98 ugagguaguaaguuguauuguu ugagguaguaaguuguauuguu hsa-miR-1 hsa-miR-1 uggaauguaaagaaguauguau uggaauguaaagaaguauguau hsa-miR-106b hsa-miR-106b uaaagugcugacagugcagau uaaagugcugacagugcagau hsa-miR-150^*hsa-miR-150^* cugguacaggccugggggacag cugguacaggccuggggggacag hsa-miR-25 hsa-miR-25 cauugcacuugucucggucuga cauugcacuugucucggucuga hsa-miR-134 hsa-miR-134 ugugacugguugaccagagggg ugugacugguugaccagggggg hsa-miR-328 hsa-miR-328 cuggcccucucugcccuuccgu cuggcccucucugcccuuccgu hsa-miR-638 hsa-miR-638 agggaucgcgggcggguggcggccu agggaucgcgggcggguggcggccu hsa-miR-1225-5p hsa-miR-1225-5p guggguacggcccagugggggg guggguacggcccaggggggg hsa-miR-1224-5p hsa-miR-1224-5p gugaggacucgggaggugg gugaggacucgggagggg hsa-miR-557 hsa-miR-557 guuugcacgggugggccuugucu guuugcacgggggggccuugucu

hsa-miR-15b hsa-miR-15b uagcagcacaucaugguuuaca uagcagcaacaucaugguuuaca hsa-miR-301a hsa-miR-301a cagugcaauaguauugucaaagc cagugcaauaguauugucaaagc hsa-miR-196b hsa-miR-196b uagguaguuuccuguuguuggg uagguaguuuccuguuguuggg hsa-miR-196a hsa-miR-196a uagguaguuucauguuguuggg uagguaguuucauguuguuggg hsa-miR-1226^*hsa-miR-1226^* gugagggcaugcaggccuggaugggg gugaggggcaugcaggccuggaugggggg hsa-miR-424 hsa-miR-424 cagcagcaauucauguuuugaa cagcagcaauucauguuuugaa hsa-miR-21^*hsa-miR-21^* caacaccagucgaugggcugu caacaccagucgaugggcugu hsa-miR-572 hsa-miR-572 guccgcucggcgguggccca guccgcucggcgguggccca hsa-miR-18b hsa-miR-18b uaaggugcaucuagugcaguuag uaaggugcaucuagugcaguuag hsa-miR-18a hsa-miR-18a uaaggugcaucuagugcagauag uaaggugcaucuagugcagauag hsa-miR-9^*hsa-miR-9^* auaaagcuagauaaccgaaagu auaaagcuagauaaccgaaagu hsa-miR-301b hsa-miR-301b cagugcaaugauauugucaaagc cagugcaaugauauugucaaagc hsa-miR-29c hsa-miR-29c uagcaccauuugaaaucgguua uagcaccauuugaaaucgguua hsa-miR-494 hsa-miR-494 ugaaacauacacgggaaaccuc ugaaacauacacgggaaaccuc hsa-miR-193b hsa-miR-193b aacuggcccucaaagucccgcu30 --> aacuggcccucaaagucccgcu30 --> hsa-miR-375 hsa-miR-375 uuuguucguucggcucgcguga uuuguucguucggcucgcguga hsa-miR-26a hsa-miR-26a uucaaguaauccaggauaggcu uucaaguaauccaggauaggcu hsa-miR-574-3p hsa-miR-574-3p cacgcucaugcacacacccaca cacgcucaugcacacacccaca hsa-miR-151-3p hsa-miR-151-3p cuagacugaagcuccuugagg cuagacugaagcuccuugagg hsa-miR-149 hsa-miR-149 ucuggcuccgugucuucacuccc ucuggcuccgugucuucacuccc hsa-miR-768-5p hsa-miR-768-5p Bad entry! Bad entry! hsa-miR-181a hsa-miR-181a aacauucaacgcugucggugagu aacauucaacgcugucggugagu hsa-miR-20a hsa-miR-20a uaaagugcuuauagugcagguag uaaagugcuuauagugcagguag hsa-miR-181c hsa-miR-181c aacauucaaccugucggugagu aacauucaaccugucggugagu hsa-miR-29a hsa-miR-29a uagcaccaucugaaaucgguua uagcaccaucugaaaucgguua hsa-miR-29b hsa-miR-29b uagcaccauuugaaaucaguguu uagcaccauuugaaaucaguguu hsa-miR-505 hsa-miR-505 cgucaacacuugcugguuuccu cgucaacacuugcugguuuccu hsa-miR-939 hsa-miR-939 uggggagcugaggcucugggggug uggggagcugaggcucugggggggg hsa-miR-769-5p hsa-miR-769-5p ugagaccucuggguucugagcu ugagaccucuggguucugagcu hsa-miR-30c hsa-miR-30c uguaaacauccuacacucucagc uguaaacauccuacacucucacagc hsa-miR-590-5p hsa-miR-590-5p gagcuuauucauaaaagugcag gagcuuauucauaaaagugcag hsa-miR-671-5p hsa-miR-671-5p aggaagcccuggaggggcuggag aggaagccccuggaggggcuggag hsa-miR-625 hsa-miR-625 agggggaaaguucuauagucc agggggaaaguucuauagucc hsa-miR-652 hsa-miR-652 aauggcgccacuaggguugug aauggcgccacuaggguugug hsa-miR-139-3p hsa-miR-139-3p ggagacgcggcccuguuggagu ggagacgcggcccuguuggagu hsa-miR-28-5p hsa-miR-28-5p aaggagcucacagucuauugag aaggagcucacagucuauugag hsa-miR-623 hsa-miR-623 aucccuugcaggggcuguugggu aucccuugcaggggcuguugggu hsa-miR-221 hsa-miR-221 agcuacauugucugcuggguuuc agcuacauugucugcuggguuuc hsa-miR-19a hsa-miR-19a ugugcaaaucuaugcaaaacuga ugugcaaaucuaugcaaaacuga hsa-miR-944 hsa-miR-944 aaauuauuguacaucggaugag aaauuauuguacaucggaugg hsa-miR-335 hsa-miR-335 ucaagagcaauaacgaaaaaugu ucaagagcaauaacgaaaaaugu hsa-miR-125a-5p hsa-miR-125a-5p ucccugagacccuuuaaccuguga ucccugagacccuuuaaccuguga hsa-miR-551b hsa-miR-551b gcgacccauacuugguuucag gcgacccauacuugguucag hsa-let-7d^*hsa-let-7d^* cuauacgaccugcugccuuucu cuauacgaccugcugccuuuucu

hsa-miR-31^*hsa-miR-31^* ugcuaugccaacauauugccau ugcuaugccaacauauugccaau hsa-miR-362-5p hsa-miR-362-5p aauccuuggaaccuaggugugagu aauccuuggaaccuaggugugagu hsa-miR-181a^*hsa-miR-181a^* accaucgaccguugauuguacc accaucgaccguugauuguacc hsa-miR-940 hsa-miR-940 aaggcagggcccccgcucccc aaggcagggcccccgcucccc hsa-miR-30b^*hsa-miR-30b^* cugggagguggauguuuacuuc cuggggagggaguguuuacuuc hsa-miR-601 hsa-miR-601 uggucuaggauuguuggaggag uggucuaggauuguuggagggag hsa-miR-20b hsa-miR-20b caaagugcucauagugcagguag caaagugcucauagugcagguag hsa-miR-202 hsa-miR-202 agagguauagggcaugggaa agagguauagggcaugggaa hsa-miR-218 hsa-miR-218 uugugcuugaucuaaccaugu31 --> uugugcuugaucuaaccaugu31 --> hsa-miR-145 hsa-miR-145 guccaguuuucccaggaaucccu guccaguuuucccaggaaucccu hsa-miR-140-3p hsa-miR-140-3p uaccacaggguagaaccacgg uaccacaggguagaaccacgg hsa-miR-126 hsa-miR-126 ucguaccgugaguaauaaugcg ucguaccgugaguaauaaugcg hsa-miR-126^*hsa-miR-126^* cauuauuacuuuugguacgcg cauuauuacuuuugguacgcg hsa-miR-30a hsa-miR-30a uguaaacauccucgacuggaag uguaaacauccucgacuggaag hsa-miR-143 hsa-miR-143 ugagaugaagcacuguagcuc ugagaugaagcacuguagcuc hsa-miR-101 hsa-miR-101 uacaguacugugauaacugaa uacaguaacugugauaacugaa hsa-miR-140-5p hsa-miR-140-5p cagugguuuuacccuaugguag cagugguuuaccccuaugguag hsa-miR-30d hsa-miR-30d uguaaacauccccgacuggaag uguaaacauccccgacuggaag hsa-miR-224 hsa-miR-224 caagucacuagugguuccguu caagucacuaguguuccguu hsa-miR-9 hsa-miR-9 ucuuugguuaucuagcuguauga ucuuugguuaucuagcuguauga hsa-miR-200b hsa-miR-200b uaauacugccugguaaugauga uaauacugccuggaaugauga hsa-miR-195 hsa-miR-195 uagcagcacagaaauauuggc uagcagcacagaaauauuggc hsa-miR-210 hsa-miR-210 cugugcgugugacagcggcuga cugugcgugugacagcggcuga hsa-miR-7 hsa-miR-7 uggaagacuagugauuuuguugu uggaagacuagugauuuuguugu hsa-miR-21 hsa-miR-21 uagcuuaucagacugauguuga uagcuuaucagacugauguuga hsa-miR-17 hsa-miR-17 caaagugcuuacagugcagguag caaagugcuuacagugcagguag hsa-miR-128 hsa-miR-128 ucacagugaaccggucucuuu ucacagugaaccggucucuuu hsa-miR-141 hsa-miR-141 uaacacugucugguaaagaugg uaacacugucugguaaagaugg hsa-miR-142-5p hsa-miR-142-5p cauaaaguagaaagcacuacu cauaaaguagaaagcacuacu hsa-miR-150 hsa-miR-150 ucucccaacccuuguaccagug ucucccaacccuuguaccagug hsa-miR-205 hsa-miR-205 uccuucauuccaccggagucug uccuucauuccaccggagugug

在第五方面，本发明涉及用于鉴定显示或具有发生小细胞肺癌倾向的一或多种哺乳动物靶细胞的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在小细胞肺癌或有小细胞肺癌发生倾向的指征。In a fifth aspect, the present invention relates to a diagnostic kit for identifying molecular markers of one or more mammalian target cells exhibiting or having a predisposition to develop small cell lung cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule Encoding microRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid An expression feature, the nucleic acid expression feature is an indication of the presence of small cell lung cancer or the tendency to develop small cell lung cancer.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-96(SEQIDNO:2)、hsa-miR-144(SEQIDNO:3)、hsa-miR-93(SEQIDNO:18)、hsa-miR-106b(SEQIDNO:28)、hsa-miR-25(SEQIDNO:30)、hsa-miR-15b(SEQIDNO:38)、hsa-miR-145(SEQIDNO:58)、hsa-miR-375(SEQIDNO:90)、hsa-miR-103(SEQIDNO:91)、hsa-miR-20a(SEQIDNO:98)、hsa-miR-107(SEQIDNO:129)、hsa-miR-451(SEQIDNO:4)、hsa-miR-130b(SEQIDNO:7)、hsa-miR-454(SEQIDNO:39)、hsa-miR-30a(SEQIDNO:56)、hsa-miR-17(SEQIDNO:74)、hsa-miR-183(SEQIDNO:1)、hsa-miR-486-5p(SEQIDNO:6)、hsa-miR-338-3p(SEQIDNO:7)、hsa-miR-139-5p(SEQIDNO:8)、hsa-miR-30a^*(SEQIDNO:10)、hsa-miR-144^*(SEQIDNO:11)、hsa-miR-425(SEQIDNO:12)、hsa-miR-133b(SEQIDNO:13)、hsa-miR-497(SEQIDNO:14)、hsa-miR-182(SEQIDNO:15)、hsa-miR-187^*(SEQIDNO:16)、hsa-miR-30b(SEQIDNO:17)、hsa-miR-145^*(SEQIDNO:19)、hsa-miR-429(SEQIDNO:20)、hsa-miR-498(SEQIDNO:21)、hsa-miR-200a(SEQIDNO:22)、hsa-miR-130a(SEQIDNO:24)、hsa-miR-30c-2^*(SEQIDNO:25)、hsa-miR-98(SEQIDNO:26)、hsa-miR-1(SEQIDNO:27)、hsa-miR-150^*(SEQIDNO:29)、hsa-miR-134(SEQIDNO:31)、hsa-miR-328(SEQIDNO:32)、hsa-miR-638(SEQIDNO:33)、hsa-miR-200b^*(SEQIDNO:34)、hsa-miR-1225-5p(SEQIDNO:35)、hsa-miR-1224-5p(SEQIDNO:36)、hsa-miR-557(SEQIDNO:37)、hsa-miR-152(SEQIDNO:38)、hsa-miR-301a、(SEQIDNO:40)、hsa-miR-100(SEQIDNO:42)、hsa-miR-223(SEQIDNO:43)、hsa-miR-196b(SEQIDNO:44)、hsa-miR-1226^*(SEQIDNO:46)、hsa-miR-572(SEQIDNO:49)、hsa-miR-18b(SEQIDNO:50)、hsa-miR-9^*(SEQIDNO:52)、hsa-miR-301b(SEQIDNO:53)、hsa-miR-199b-3p(SEQIDNO:65)、hsa-miR-34a(SEQIDNO:87)、hsa-miR-200a^*(SEQIDNO:89)、hsa-miR-26a(SEQIDNO:92)、hsa-miR-574-3p(SEQIDNO:93)、hsa-miR-768-5p(SEQIDNO:96)、hsa-miR-29a(SEQIDNO:100)、hsa-miR-29b(SEQIDNO:101)、hsa-miR-505(SEQIDNO:102)、hsa-miR-939(SEQIDNO:103)、hsa-miR-769-5p(SEQIDNO:104)、hsa-miR-625(SEQIDNO:108)、hsa-miR-139-3p(SEQIDNO:110)、hsa-miR-19a(SEQIDNO:114)、hsa-miR-551b(SEQIDNO:118)、has-miR-362-5p(SEQIDNO:121)、hsa-miR-181a^*(SEQIDNO:122)、hsa-miR-940(SEQIDNO:123)、hsa-miR-601(SEQIDNO:125)、hsa-miR-20b(SEQIDNO:126)、has-miR-23a(SEQIDNO:130)、hsa-miR-27a(SEQIDNO:131)、hsa-miR-199a-5p(SEQIDNO:132)、hsa-let-7i(SEQIDNO:133)、hsa-miR-19b(SEQIDNO:134)、hsa-miR-20a^*(SEQIDNO:135)、hsa-miR-17^*(SEQIDNO:136)、hsa-miR-22(SEQIDNO:137)、hsa-miR-340^*(SEQIDNO:138)、hsa-miR-214(SEQIDNO:139)、hsa-miR-592(SEQIDNO:140)、hsa-miR-335^*(SEQIDNO:141)、hsa-miR-29c^*(SEQIDNO:142)、hsa-miR-421(SEQIDNO:143)、hsa-miR-886-3p(SEQIDNO:144)、hsa-miR-16-2^*(SEQIDNO:145)、hsa-miR-135a(SEQIDNO:146)、hsa-miR-183^*(SEQIDNO:147)、hsa-miR-7-1^*(SEQIDNO:148)、hsa-miR-26b(SEQIDNO:149)、has-miR-455-3p(SEQIDNO:150)、hsa-miR-222(SEQIDNO:151)、hsa-miR-95(SEQIDNO:152)、hsa-miR-564(SEQIDNO:153)、hsa-miR-636(SEQIDNO:154)、hsa-miR-34b^*(SEQIDNO:155)、hsa-miR-18a(SEQIDNO:51)、hsa-miR-126(SEQIDNO:54)、hsa-miR-126^*(SEQIDNO:55)、hsa-miR-140-3p(SEQIDNO:59)、hsa-miR-101(SEQIDNO:60)、hsa-miR-143(SEQIDNO:61)、hsa-miR-140-5p(SEQIDNO:62)、hsa-miR-29c(SEQIDNO:64)、hsa-miR-199b-5p(SEQIDNO:65)、hsa-miR-125a-3p(SEQIDNO:67)、hsa-miR-9(SEQIDNO:68)、hsa-miR-200b(SEQIDNO:69)、hsa-miR-210(SEQIDNO:71)、hsa-miR-7(SEQIDNO:72)、hsa-miR-128(SEQIDNO:75)和hsa-miR-106a(SEQIDNO:82)的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-96 (SEQ ID NO: 2), hsa-miR-144 (SEQ ID NO: 3), hsa-miR-93 (SEQ ID NO: 18), hsa- miR-106b (SEQ ID NO:28), hsa-miR-25 (SEQ ID NO:30), hsa-miR-15b (SEQ ID NO:38), hsa-miR-145 (SEQ ID NO:58), hsa-miR-375 (SEQ ID NO: 90), hsa-miR-103 (SEQ ID NO: 91), hsa-miR-20a (SEQ ID NO: 98), hsa-miR-107 (SEQ ID NO: 129), hsa-miR-451 (SEQ ID NO: 4), hsa-miR -130b (SEQ ID NO: 7), hsa-miR-454 (SEQ ID NO: 39), hsa-miR-30a (SEQ ID NO: 56), hsa-miR-17 (SEQ ID NO: 74), hsa-miR-183 (SEQ ID NO: 1 ), hsa-miR-486-5p (SEQ ID NO:6), hsa-miR-338-3p (SEQ ID NO:7), hsa-miR-139-5p (SEQ ID NO:8), hsa-miR-30a^* (SEQ ID NO: 10), hsa-miR-144^* (SEQ ID NO: 11), hsa-miR-425 (SEQ ID NO: 12), hsa-miR-133b (SEQ ID NO: 13), hsa-miR-497 (SEQ ID NO: 14), hsa- miR-182 (SEQ ID NO: 15), hsa-miR-187^* (SEQ ID NO: 16), hsa-miR-30b (SEQ ID NO: 17), hsa-miR-145^* (SEQ ID NO: 19), hsa-miR-429 ( SEQ ID NO:20), hsa-miR-498 (SEQ ID NO:21), hsa-miR-200a (SEQ ID NO:22), hsa-miR-130a (SEQ ID NO:24), hsa-miR-30c-2^* (SEQ ID NO:25 ), hsa-miR-98 (SEQ ID NO:26), hsa-miR-1 (SEQ ID NO:27), hsa-miR-150^* (SEQ ID NO:29), hsa-miR-134 (SEQ ID NO:31), hsa-miR -328 (SEQ ID NO: 32), hsa-miR-638 (SEQ ID NO: 3 3), hsa-miR-200b^* (SEQ ID NO: 34), hsa-miR-1225-5p (SEQ ID NO: 35), hsa-miR-1224-5p (SEQ ID NO: 36), hsa-miR-557 (SEQ ID NO: 37 ), hsa-miR-152 (SEQ ID NO:38), hsa-miR-301a, (SEQ ID NO:40), hsa-miR-100 (SEQ ID NO:42), hsa-miR-223 (SEQ ID NO:43), hsa-miR -196b (SEQ ID NO:44), hsa-miR-1226^* (SEQ ID NO:46), hsa-miR-572 (SEQ ID NO:49), hsa-miR-18b (SEQ ID NO:50), hsa-miR-9^* (SEQ ID NO :52), hsa-miR-301b (SEQ ID NO: 53), hsa-miR-199b-3p (SEQ ID NO: 65), hsa-miR-34a (SEQ ID NO: 87), hsa-miR-200a^* (SEQ ID NO: 89) , hsa-miR-26a (SEQ ID NO: 92), hsa-miR-574-3p (SEQ ID NO: 93), hsa-miR-768-5p (SEQ ID NO: 96), hsa-miR-29a (SEQ ID NO: 100), hsa - miR-29b (SEQ ID NO: 101), hsa-miR-505 (SEQ ID NO: 102), hsa-miR-939 (SEQ ID NO: 103), hsa-miR-769-5p (SEQ ID NO: 104), hsa-miR-625 (SEQ ID NO: 108), hsa-miR-139-3p (SEQ ID NO: 110), hsa-miR-19a (SEQ ID NO: 114), hsa-miR-551b (SEQ ID NO: 118), has-miR-362-5p (SEQ ID NO: : 121), hsa-miR-181a^* (SEQ ID NO: 122), hsa-miR-940 (SEQ ID NO: 123), hsa-miR-601 (SEQ ID NO: 125), hsa-miR-20b (SEQ ID NO: 126), has - miR-23a (SEQ ID NO: 130), hsa-miR-27a (SEQ ID NO: 131), hsa-miR-199a-5p (SEQ ID NO: 132), hsa-let-7i (SEQ ID NO: 133), hsa-miR-19b (SEQ ID NO:134), hsa-miR-20a^* (SEQ ID NO:135), hsa-miR-17^* (SEQ ID NO:136), hsa-miR-22 (SEQ ID NO:137), hsa-miR-340^* (SEQ ID NO:138 ), hsa-miR-214 (SEQ ID NO: 139), hsa-miR-592 (SEQ ID NO: 140), hsa-miR-335^* (SEQ ID NO: 141), hsa-miR-29c^* (SEQ ID NO: 142), hsa- miR-421 (SEQ ID NO: 143), hsa-miR-886-3p (SEQ ID NO: 144), hsa-miR-16-2^* (SEQ ID NO: 145), hsa-miR-135a (SEQ ID NO: 146), hsa-miR -183^* (SEQ ID NO:147), hsa-miR-7-1^* (SEQ ID NO:148), hsa-miR-26b (SEQ ID NO:149), has-miR-455-3p (SEQ ID NO:150), hsa-miR -222 (SEQ ID NO: 151), hsa-miR-95 (SEQ ID NO: 152), hsa-miR-564 (SEQ ID NO: 153), hsa-miR-636 (SEQ ID NO: 154), hsa-miR-34b^* (SEQ ID NO: 155), hsa-miR-18a (SEQ ID NO:51), hsa-miR-126 (SEQ ID NO:54), hsa-miR-126^* (SEQ ID NO:55), hsa-miR-140-3p (SEQ ID NO:59), hsa-miR-101 (SEQ ID NO:60), hsa-miR-143 (SEQ ID NO:61), hsa-miR-140-5p (SEQ ID NO:62), hsa-miR-29c (SEQ ID NO:64), hsa-miR- 199b-5p (SEQ ID NO: 65), hsa-miR-125a-3p (SEQ ID NO: 67), hsa-miR-9 (SEQ ID NO: 68), hsa-miR-200b (SEQ ID NO: 69), hsa-miR-210 ( Any one or more nucleic acid molecules of SEQ ID NO:71), hsa-miR-7 (SEQ ID NO:72), hsa-miR-128 (SEQ ID NO:75) and hsa-miR-106a (SEQ ID NO:82).

上述miRNA的核酸序列列于表5。The nucleic acid sequences of the above miRNAs are listed in Table 5.

表5table 5

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-96 hsa-miR-96 uuuggcacuagcacauuuuugcu uuuggcacuagcacauuuuugcu hsa-miR-144 hsa-miR-144 uacaguauagaugauguacu uacaguauagaugauguacu hsa-miR-451 hsa-miR-451 aaaccguuaccauuacugaguu aaaccguuaccauuacugaguu hsa-miR-130b hsa-miR-130b cagugcaaugaugaaagggcau cagugcaaugaugaaagggcau hsa-miR-93 hsa-miR-93 caaagugcuguucgugcagguag caaagugcuguucgugcagguag hsa-miR-25 hsa-miR-25 cauugcacuugucucggucuga cauugcacuugucucggucuga

hsa-miR-15b hsa-miR-15b uagcagcacaucaugguuuaca uagcagcaacaucaugguuuaca hsa-miR-454 hsa-miR-454 uagugcaauauugcuuauagggu uagugcaauauugcuuauagggu hsa-miR-375 hsa-miR-375 uuuguucguucggcucgcguga uuuguucguucggcucgcguga hsa-miR-103 hsa-miR-103 agcagcauuguacagggcuauga agcagcauuguacagggcuauga hsa-miR-107 hsa-miR-107 agcagcauuguacagggcuauca agcagcauuguacagggcuauca hsa-miR-183 hsa-miR-183 uauggcacugguagaauucacu34 --> uauggcacugguagaauucacu34 --> hsa-miR-486-5p hsa-miR-486-5p uccuguacugagcugccccgag uccuguacugagcugccccgag hsa-miR-338-3p hsa-miR-338-3p uccagcaucagugauuuuguug uccagcaucagugauuuuguug hsa-miR-139-5p hsa-miR-139-5p ucuacagugcacgugucuccag ucuacagugcacgugucuccag hsa-miR-30a^*hsa-miR-30a^* cuuucagucggauguuugcagc cuuucagucggauguuugcagc hsa-miR-144^*hsa-miR-144^* ggauaucaucauauacuguaag ggauaucaucauauacuguaag hsa-miR-425 hsa-miR-425 aaugacacgaucacucccguuga aaugacacgaucacucccguuga hsa-miR-133b hsa-miR-133b uuugguccccuucaaccagcua uuugguccccuucaaccagcua hsa-miR-497 hsa-miR-497 cagcagcacacugugguuugu cagcagcacacuguguuugu hsa-miR-182 hsa-miR-182 u uuggcaaugguagaacucacacu u uuggcaaugguagaacucacacu hsa-miR-187^*hsa-miR-187^* ggcuacaacacaggacccgggc ggcuacaacacaggacccgggc hsa-miR-30b hsa-miR-30b uguaaacauccuacacucagcu uguaaacauccuacacucagcu hsa-miR-145^*hsa-miR-145^* ggauuccuggaaauacuguucu ggauuccuggaaauacuguucu hsa-miR-429 hsa-miR-429 uaauacugucugguaaaaccgu uaauacugucugguaaaaccgu hsa-miR-498 hsa-miR-498 uuucaagccagggggcguuuuuc uuucaagccaggggggcguuuuuc hsa-miR-200a hsa-miR-200a uaacacugucugguaacgaugu uaacacugucugguaacgaugu hsa-miR-130a hsa-miR-130a cagugcaauguuaaaagggcau cagugcaauguuaaaagggcau hsa-miR-30c-2^*hsa-miR-30c-2^* cugggagaaggcuguuuacuc cugggaaggcuguuuacuc hsa-miR-98 hsa-miR-98 ugagguaguaaguuguauuguu ugagguaguaaguuguauuguu hsa-miR-1 hsa-miR-1 uggaauguaaagaaguauguau uggaauguaaagaaguauguau hsa-miR-150^*hsa-miR-150^* cugguacaggccugggggacag cugguacaggccuggggggacag hsa-miR-134 hsa-miR-134 ugugacugguugaccagagggg ugugacugguugaccagggggg hsa-miR-328 hsa-miR-328 cuggcccucucugcccuuccgu cuggcccucucugcccuuccgu hsa-miR-638 hsa-miR-638 agggaucgcgggcggguggcggccu agggaucgcgggcggguggcggccu hsa-miR-200b^*hsa-miR-200b^* caucuuacugggcagcauugga caucuuacugggcagcauugga hsa-miR-1225-5p hsa-miR-1225-5p guggguacggcccagugggggg guggguacggcccaggggggg hsa-miR-1224-5p hsa-miR-1224-5p gugaggacucgggaggugg gugaggacucgggagggg hsa-miR-557 hsa-miR-557 guuugcacgggugggccuugucu guuugcacgggggggccuugucu hsa-miR-152 hsa-miR-152 ucagugcaugacagaacuugg ucagugcaugacagaacuugg hsa-miR-301a hsa-miR-301a cagugcaauaguauugucaaagc cagugcaauaguauugucaaagc hsa-miR-100 hsa-miR-100 aacccguagauccgaacuugug aacccguaagauccgaacuugug hsa-miR-223 hsa-miR-223 ugucaguuugucaaauacccca ugucaguuugucaaauaccccca hsa-miR-196b hsa-miR-196b uagguaguuuccuguuguuggg uagguaguuuccuguuguuggg hsa-miR-1226^*hsa-miR-1226^* gugagggcaugcaggccuggaugggg gugaggggcaugcaggccuggaugggggg hsa-miR-572 hsa-miR-572 guccgcucggcgguggccca guccgcucggcgguggccca hsa-miR-18b hsa-miR-18b uaaggugcaucuagugcaguuag uaaggugcaucuagugcaguuag hsa-miR-9^*hsa-miR-9^* auaaagcuagauaaccgaaagu auaaagcuagauaaccgaaagu hsa-miR-301b hsa-miR-301b cagugcaaugauauugucaaagc cagugcaaugauauugucaaagc hsa-miR-199b-3p hsa-miR-199b-3p cccaguguuuagacuaucuguuc cccaguguuuagacuaucuguuc

hsa-miR-34a hsa-miR-34a uggcagugucuuagcugguugu uggcagugucuuagcugguugu hsa-miR-200a^*hsa-miR-200a^* caucuuaccggacagugcugga caucuuaccggacagugcugga hsa-miR-26a hsa-miR-26a uucaaguaauccaggauaggcu uucaaguaauccaggauaggcu hsa-miR-574-3p hsa-miR-574-3p cacgcucaugcacacacccaca cacgcucaugcacacacccaca hsa-miR-768-5p hsa-miR-768-5p Bad entry bad entry hsa-miR-29a hsa-miR-29a uagcaccaucugaaaucgguua uagcaccaucugaaaucgguua hsa-miR-29b hsa-miR-29b uagcaccauuugaaaucaguguu uagcaccauuugaaaucaguguu hsa-miR-505 hsa-miR-505 cgucaacacuugcugguuuccu cgucaacacuugcugguuuccu hsa-miR-939 hsa-miR-939 uggggagcugaggcucugggggug uggggagcugaggcucugggggggg hsa-miR-769-5p hsa-miR-769-5p ugagaccucuggguucugagcu ugagaccucuggguucugagcu hsa-miR-625 hsa-miR-625 agggggaaaguucuauagucc agggggaaaguucuauagucc hsa-miR-139-3p hsa-miR-139-3p ggagacgcggcccuguuggagu ggagacgcggcccuguuggagu hsa-miR-19a hsa-miR-19a ugugcaaaucuaugcaaaacuga ugugcaaaucuaugcaaaacuga hsa-miR-551b hsa-miR-551b gcgacccauacuugguuucag gcgacccauacuugguucag hsa-miR-362-5p hsa-miR-362-5p aauccuuggaaccuaggugugagu aauccuuggaaccuaggugugagu hsa-miR-181a^*hsa-miR-181a^* accaucgaccguugauuguacc accaucgaccguugauuguacc hsa-miR-940 hsa-miR-940 aaggcagggcccccgcucccc aaggcagggcccccgcucccc hsa-miR-601 hsa-miR-601 uggucuaggauuguuggaggag uggucuaggauuguuggagggag hsa-miR-20b hsa-miR-20b caaagugcucauagugcagguag caaagugcucauagugcagguag hsa-miR-23a hsa-miR-23a aucacauugccagggau uucc aucacauugccagggauuucc hsa-miR-27a hsa-miR-27a uucacaguggcuaaguuccgc uucacaguggcuaaguuccgc hsa-miR-199a-5p hsa-miR-199a-5p cccaguguucagacuaccuguuc cccaguguucagacuaccuguuc hsa-let-7i hsa-let-7i ugagguaguaguuugugcuguu ugagguaguaguuugugcuguu hsa-miR-19b hsa-miR-19b ugugcaaauccaugcaaaacuga ugugcaaauccaugcaaaacuga hsa-miR-20a^*hsa-miR-20a^* acugcauuaugagcacuuaaag acugcauuaugagcacuuaaag hsa-miR-17^*hsa-miR-17^* acugcagugaaggcacuuguag acugcagugaaggcacuuguag hsa-miR-22 hsa-miR-22 aagcugccaguugaagaacugu aagcugccaguugaagaacugu hsa-miR-340^*hsa-miR-340^* uccgucucaguuacuuuauagc uccgucucaguuacuuuauagc hsa-miR-214 hsa-miR-214 acagcaggcacagacaggcagu acagcaggcacagacaggcagu hsa-miR-592 hsa-miR-592 uugugucaauaugcgaugaugu uugugucaauaugcgaugaugu hsa-miR-335^*hsa-miR-335^* uuuuucauuauugcuccugacc uuuuucauuauugcuccugacc hsa-miR-29c^*hsa-miR-29c^* ugaccgauuucuccugguguuc ugaccgauuucucccugguguuc hsa-miR-421 hsa-miR-421 aucaacagacauuaauugggcgc aucaacagacauuaauugggcgc hsa-miR-886-3p hsa-miR-886-3p cgcgggugcuuacugacccuu cgcgggugcuuacugaccccuu hsa-miR-16-2^*hsa-miR-16-2^* ccaauauuacugugcugcuuua ccaauauuacugugcugcuuua hsa-miR-135a hsa-miR-135a uauggcuuuuuauuccuauguga uauggcuuuuuauuccuauguga hsa-miR-183^*hsa-miR-183^* gugaauuaccgaagggccauaa gugaauuaccgaagggccauaa hsa-miR-7-1^*hsa-miR-7-1^* caacaaaucacagucugccaua36 --> caacaaaucacagucugccaua36 --> hsa-miR-26b hsa-miR-26b uucaaguaauucaggauaggu uucaaguaauucaggauaggu hsa-miR-455-3p hsa-miR-455-3p gcaguccaugggcauauacac gcaguccaugggcauauacac hsa-miR-222 hsa-miR-222 agcuacaucuggcuacugggu agcuacaucuggcuacugggu hsa-miR-95 hsa-miR-95 uucaacggguauuuauugagca uucaacggguauuuauugagca hsa-miR-564 hsa-miR-564 aggcacggugucagcaggc aggcacggugucagcaggc hsa-miR-636 hsa-miR-636 ugugcuugcucgucccgcccgca ugugcuugcucgucccgcccgca

hsa-miR-34b^*hsa-miR-34b^* uaggcagugucauuagcugauug uaggcagugucauuagcugauug hsa-miR-106b hsa-miR-106b uaaagugcugacagugcagau uaaagugcugacagugcagau hsa-miR-30a hsa-miR-30a uguaaacauccucgacuggaag uguaaacauccucgacuggaag hsa-miR-145 hsa-miR-145 guccaguuuucccaggaaucccu guccaguuuucccaggaaucccu hsa-miR-17 hsa-miR-17 caaagugcuuacagugcagguag caaagugcuuacagugcagguag hsa-miR-20a hsa-miR-20a uaaagugcuuauagugcagguag uaaagugcuuauagugcagguag hsa-miR-18a hsa-miR-18a uaaggugcaucuagugcagauag uaaggugcaucuagugcagauag hsa-miR-126 hsa-miR-126 ucguaccgugaguaauaaugcg ucguaccgugaguaauaaugcg hsa-miR-126^*hsa-miR-126^* cauuauuacuuuugguacgcg cauuauuacuuuugguacgcg hsa-miR-140-3p hsa-miR-140-3p uaccacaggguagaaccacgg uaccacaggguagaaccacgg hsa-miR-101 hsa-miR-101 uacaguacugugauaacugaa uacaguaacugugauaacugaa hsa-miR-143 hsa-miR-143 ugagaugaagcacuguagcuc ugagaugaagcacuguagcuc hsa-miR-140-5p hsa-miR-140-5p cagugguuuuacccuaugguag cagugguuuaccccuaugguag hsa-miR-29c hsa-miR-29c uagcaccauuugaaaucgguua uagcaccauuugaaaucgguua hsa-miR-199b-5p hsa-miR-199b-5p cccaguguuuagacuaucuguuc cccaguguuuagacuaucuguuc hsa-miR-125a-3p hsa-miR-125a-3p acaggugagguucuugggagcc acaggugagguucuuggagcc hsa-miR-9 hsa-miR-9 ucuuugguuaucuagcuguauga ucuuugguuaucuagcuguauga hsa-miR-200b hsa-miR-200b uaauacugccugguaaugauga uaauacugccuggaaugauga hsa-miR-210 hsa-miR-210 cugugcgugugacagcggcuga cugugcgugugacagcggcuga hsa-miR-7 hsa-miR-7 uggaagacuagugauuuuguugu uggaagacuagugauuuuguugu hsa-miR-128 hsa-miR-128 ucacagugaaccggucucuuu ucacagugaaccggucucuuu hsa-miR-106a hsa-miR-106a aaaagugcuuacagugcagguag aaaagugcuuacagugcagguag

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-375(SEQIDNO:90)、hsa-miR-335(SEQIDNO:116)、hsa-miR-23a(SEQIDNO:130)、hsa-miR-27a(SEQIDNO:131)、hsa-miR-22(SEQIDNO:137)、hsa-miR-592(SEQIDNO:140)、hsa-miR-135a(SEQIDNO:146)、hsa-miR-24(SEQIDNO:156)、hsa-miR-454(SEQIDNO:39)、hsa-miR-301a(SEQIDNO:40)、hsa-miR-199b-5p(SEQIDNO:65)、hsa-miR-9(SEQIDNO:68)、hsa-miR-34a(SEQIDNO:87)、hsa-miR-199a-5p(SEQIDNO:132)、hsa-miR-335^*(SEQIDNO:141)、hsa-miR-152(SEQIDNO:157)和hsa-miR-216b(SEQIDNO:158)的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-375 (SEQ ID NO: 90), hsa-miR-335 (SEQ ID NO: 116), hsa-miR-23a (SEQ ID NO: 130), hsa- miR-27a (SEQ ID NO: 131), hsa-miR-22 (SEQ ID NO: 137), hsa-miR-592 (SEQ ID NO: 140), hsa-miR-135a (SEQ ID NO: 146), hsa-miR-24 (SEQ ID NO: 156), hsa-miR-454 (SEQ ID NO:39), hsa-miR-301a (SEQ ID NO:40), hsa-miR-199b-5p (SEQ ID NO:65), hsa-miR-9 (SEQ ID NO:68), hsa -miR-34a (SEQ ID NO:87), hsa-miR-199a-5p (SEQ ID NO:132), hsa-miR-335^* (SEQ ID NO:141), hsa-miR-152 (SEQ ID NO:157) and hsa-miR- Any one or more nucleic acid molecules of 216b (SEQ ID NO: 158).

上述miRNA的核酸序列列于表6。The nucleic acid sequences of the above miRNAs are listed in Table 6.

表6Table 6

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-375 hsa-miR-375 uuuguucguucggcucgcguga uuuguucguucggcucgcguga hsa-miR-335 hsa-miR-335 ucaagagcaauaacgaaaaaugu ucaagagcaauaacgaaaaaugu hsa-miR-23a hsa-miR-23a aucacauugccagggauuucc aucacauugccagggauuucc hsa-miR-27a hsa-miR-27a uucacaguggcuaaguuccgc uucacaguggcuaaguuccgc hsa-miR-22 hsa-miR-22 aagcugccaguugaagaacugu aagcugccaguugaagaacugu hsa-miR-592 hsa-miR-592 uugugucaauaugcgaugaugu uugugucaauaugcgaugaugu hsa-miR-135a hsa-miR-135a uauggcuuuuuauuccuauguga uauggcuuuuuauuccuauguga hsa-miR-24 hsa-miR-24 uggcucaguucagcaggaacag uggcucaguucagcaggaacag hsa-miR-454 hsa-miR-454 uagugcaauauugcuuauagggu uagugcaauauugcuuauagggu hsa-miR-301a hsa-miR-301a cagugcaauaguauugucaaagc cagugcaauaguauugucaaagc hsa-miR-199b-5p hsa-miR-199b-5p cccaguguuuagacuaucuguuc cccaguguuuagacuaucuguuc hsa-miR-9 hsa-miR-9 ucuuugguuaucuagcuguauga ucuuugguuaucuagcuguauga hsa-miR-34a hsa-miR-34a uggcagugucuuagcugguugu uggcagugucuuagcugguugu hsa-miR-199a-5p hsa-miR-199a-5p cccaguguucagacuaccuguuc cccaguguucagacuaccuguuc hsa-miR-335^*hsa-miR-335^* uuuuucauuauugcuccugacc uuuuucauuauugcuccugacc hsa-miR-152 hsa-miR-152 ucagugcaugacagaacuugg ucagugcaugacagaacuugg hsa-miR-216b hsa-miR-216b aaaucucugcaggcaaauguga aaaucucugcaggcaaauguga

在第七方面，本发明涉及用于将腺癌肺癌、鳞状细胞肺癌或小细胞肺癌与其它两种区分开的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在腺癌肺癌、鳞状细胞肺癌或小细胞肺癌的指征。In a seventh aspect, the present invention relates to a diagnostic kit of molecular markers for distinguishing adenocarcinoma lung cancer, squamous cell lung cancer or small cell lung cancer from the other two, said kit comprising a plurality of nucleic acid molecules, each nucleic acid Molecularly encoded microRNA sequences, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in target cells and in one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a A nucleic acid expression signature that is indicative of the presence of adenocarcinoma lung cancer, squamous cell lung cancer, or small cell lung cancer.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-25(SEQIDNO:30)、hsa-miR-205(SEQIDNO:79)、hsa-miR-34a(SEQIDNO:87)、hsa-miR-375(SEQIDNO:90)、hsa-miR-29a(SEQIDNO:100)、hsa-miR-27a(SEQIDNO:131)、hsa-miR-29b(SEQIDNO:101)、hsa-miR-93(SEQIDNO:18)、hsa-miR-106b(SEQIDNO:28)、hsa-miR-15b(SEQIDNO:38)、hsa-miR-454(SEQIDNO:39)、hsa-miR-301a(SEQIDNO:40)、hsa-miR-145(SEQIDNO:58)、hsa-miR-29c(SEQIDNO:64)、hsa-miR-221(SEQIDNO:113)、hsa-miR-335(SEQIDNO:116)、hsa-miR-23a(SEQIDNO:130)、hsa-miR-199a-5p(SEQIDNO:132)、hsa-miR-22(SEQIDNO:137)、hsa-miR-592(SEQIDNO:140)、hsa-miR-95(SEQIDNO:152)、hsa-miR-24(SEQIDNO:7156)和hsa-miR-92a(SEQIDNO:160).的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-25 (SEQ ID NO: 30), hsa-miR-205 (SEQ ID NO: 79), hsa-miR-34a (SEQ ID NO: 87), hsa-miR-25 (SEQ ID NO: 87), hsa- miR-375 (SEQ ID NO:90), hsa-miR-29a (SEQ ID NO:100), hsa-miR-27a (SEQ ID NO:131), hsa-miR-29b (SEQ ID NO:101), hsa-miR-93 (SEQ ID NO: 18), hsa-miR-106b (SEQ ID NO: 28), hsa-miR-15b (SEQ ID NO: 38), hsa-miR-454 (SEQ ID NO: 39), hsa-miR-301a (SEQ ID NO: 40), hsa-miR -145 (SEQ ID NO:58), hsa-miR-29c (SEQ ID NO:64), hsa-miR-221 (SEQ ID NO:113), hsa-miR-335 (SEQ ID NO:116), hsa-miR-23a (SEQ ID NO:130 ), hsa-miR-199a-5p (SEQ ID NO: 132), hsa-miR-22 (SEQ ID NO: 137), hsa-miR-592 (SEQ ID NO: 140), hsa-miR-95 (SEQ ID NO: 152), hsa- Any one or more nucleic acid molecules of miR-24 (SEQ ID NO: 7156) and hsa-miR-92a (SEQ ID NO: 160).

特别优选地，与鳞状细胞肺癌和小细胞肺癌相比，在腺癌肺癌中编码hsa-miR-34a、hsa-miR-29a、hsa-miR-29b、hsa-miR-145、hsa-miR-29c的任一或多种核酸分子表达上调，而编码hsa-miR-25、hsa-miR-93、hsa-miR-301a、hsa-miR-106b、hsa-miR-15b、has-miR-92a的任一或多种核酸分子的表达下调。在腺癌肺癌中编码hsa-miR-205、hsa-miR-27a、hsa-miR-221的任一或多种核酸分子，与小细胞肺癌相比表达上调，与鳞状细胞肺癌相比表达下调。在腺癌肺癌中编码hsa-miR-375的核酸分子，与鳞状细胞肺癌相比表达上调，与小细胞肺癌相比表达下调。与小细胞肺癌相比，在腺癌肺癌中编码hsa-5miR-24、hsa-miR-23a、hsa-miR-22、hsa-miR-199a-5p的任一或多种核酸分子表达上调，而编码hsa-miR-592、hsa-miR-335、hsa-miR-95、hsa-miR-454的任一或多种核酸分子的表达下调。Particularly preferably, hsa-miR-34a, hsa-miR-29a, hsa-miR-29b, hsa-miR-145, hsa-miR- The expression of any one or more nucleic acid molecules of 29c is up-regulated, while those encoding hsa-miR-25, hsa-miR-93, hsa-miR-301a, hsa-miR-106b, hsa-miR-15b, has-miR-92a Expression of any one or more nucleic acid molecules is downregulated. In adenocarcinoma lung cancer, any one or more nucleic acid molecules encoding hsa-miR-205, hsa-miR-27a, hsa-miR-221 are up-regulated compared with small cell lung cancer and down-regulated compared with squamous cell lung cancer . The nucleic acid molecule encoding hsa-miR-375 was upregulated in adenocarcinoma lung cancer compared with squamous cell lung cancer and downregulated compared with small cell lung cancer. Compared with small cell lung cancer, the expression of any one or more nucleic acid molecules encoding hsa-5miR-24, hsa-miR-23a, hsa-miR-22, hsa-miR-199a-5p was up-regulated in adenocarcinoma lung cancer, while The expression of any one or more nucleic acid molecules encoding hsa-miR-592, hsa-miR-335, hsa-miR-95, hsa-miR-454 is down-regulated.

上述miRNA的核酸序列列于表7。The nucleic acid sequences of the above miRNAs are listed in Table 7.

表7Table 7

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-25 hsa-miR-25 cauugcacuugucucggucuga cauugcacuugucucggucuga hsa-miR-205 hsa-miR-205 uccuucauuccaccggagucug uccuucauuccaccggagugug hsa-miR-34a hsa-miR-34a uggcagugucuuagcugguugu uggcagugucuuagcugguugu hsa-miR-375 hsa-miR-375 uuuguucguucggcucgcguga uuuguucguucggcucgcguga hsa-miR-29a hsa-miR-29a uagcaccaucugaaaucgguua uagcaccaucugaaaucgguua hsa-miR-29b hsa-miR-29b uagcaccauuugaaaucaguguu uagcaccauuugaaaucaguguu hsa-miR-27a hsa-miR-27a uucacaguggcuaaguuccgc uucacaguggcuaaguuccgc hsa-miR-93 hsa-miR-93 caaagugcuguucgugcagguag39 --> caaagugcuguucgugcagguag39 --> hsa-miR-106b hsa-miR-106b uaaagugcugacagugcagau uaaagugcugacagugcagau hsa-miR-15b hsa-miR-15b uagcagcacaucaugguuuaca uagcagcaacaucaugguuuaca hsa-miR-454 hsa-miR-454 uagugcaauauugcuuauagggu uagugcaauauugcuuauagggu hsa-miR-301a hsa-miR-301a cagugcaauaguauugucaaagc cagugcaauaguauugucaaagc hsa-miR-145 hsa-miR-145 guccaguuuucccaggaaucccu guccaguuuucccaggaaucccu hsa-miR-29c hsa-miR-29c uagcaccauuugaaaucgguua uagcaccauuugaaaucgguua hsa-miR-221 hsa-miR-221 agcuacauugucugcuggguuuc agcuacauugucugcuggguuuc hsa-miR-335 hsa-miR-335 ucaagagcaauaacgaaaaaugu ucaagagcaauaacgaaaaaugu hsa-miR-23a hsa-miR-23a aucacauugccagggauuucc aucacauugccagggauuucc hsa-miR-199a-5p hsa-miR-199a-5p cccaguguucagacuaccuguuc cccaguguucagacuaccuguuc hsa-miR-22 hsa-miR-22 aagcugccaguugaagaacugu aagcugccaguugaagaacugu hsa-miR-592 hsa-miR-592 uugugucaauaugcgaugaugu uugugucaauaugcgaugaugu hsa-miR-95 hsa-miR-95 uucaacggguauuuauugagca uucaacggguauuuauugagca hsa-miR-24 hsa-miR-24 uggcucaguucagcaggaacag uggcucaguucagcaggaacag hsa-miR-92a hsa-miR-92a uauugcacuugucccggccugu uauugcacuuguccccggccugu

在第八方面，本发明涉及用于区分腺癌肺癌和鳞状细胞肺癌的分子标记的诊断试剂盒，所述试剂盒包含多种核酸分子，每种核酸分子编码微RNA序列，其中所述多种核酸分子的一或多种在靶细胞和在一或多种对照细胞中差异表达，并且其中所述一或多种差异表达的核酸分子一起代表一种核酸表达特征，该核酸表达特征是存在腺癌肺癌或鳞状细胞肺癌的指征。In an eighth aspect, the present invention relates to a molecular marker diagnostic kit for distinguishing adenocarcinoma lung cancer from squamous cell lung cancer, said kit comprising a variety of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein said multiple One or more of the nucleic acid molecules are differentially expressed in the target cell and in the one or more control cells, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is present Indications for adenocarcinoma lung or squamous cell lung cancer.

在更优选的实施方案中，所述核酸表达特征包括编码hsa-miR-497(SEQIDNO:14)、hsa-miR-93(SEQIDNO:18)、hsa-miR-25(SEQIDNO:30)、hsa-miR-29c(SEQIDNO:64)、hsa-miR-205(SEQIDNO:79)、hsa-miR-375(SEQIDNO:90)、hsa-miR-149(SEQIDNO:95)、hsa-miR-29a(SEQIDNO:100)、hsa-miR-1(SEQIDNO:27)、has-miR-15b(SEQIDNO:38)、hsa-miR-181c(SEQIDNO:99)、hsa-miR-29b(SEQIDNO:101)、hsa-miR-769-5p(SEQIDNO:104)和hsa-miR-221(SEQIDNO:113).的任一或多种核酸分子。In a more preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-497 (SEQ ID NO: 14), hsa-miR-93 (SEQ ID NO: 18), hsa-miR-25 (SEQ ID NO: 30), hsa-miR- miR-29c (SEQ ID NO:64), hsa-miR-205 (SEQ ID NO:79), hsa-miR-375 (SEQ ID NO:90), hsa-miR-149 (SEQ ID NO:95), hsa-miR-29a (SEQ ID NO: 100), hsa-miR-1 (SEQ ID NO: 27), has-miR-15b (SEQ ID NO: 38), hsa-miR-181c (SEQ ID NO: 99), hsa-miR-29b (SEQ ID NO: 101), hsa-miR - any one or more nucleic acid molecules of 769-5p (SEQ ID NO: 104) and hsa-miR-221 (SEQ ID NO: 113).

上述miRNA的核酸序列列于表8。The nucleic acid sequences of the above miRNAs are listed in Table 8.

表8Table 8

miRNA miRNA 序列(5'→3') Sequence (5'→3') hsa-miR-497 hsa-miR-497 cagcagcacacugugguuugu cagcagcacacuguguuugu hsa-miR-93 hsa-miR-93 caaagugcuguucgugcagguag caaagugcuguucgugcagguag hsa-miR-25 hsa-miR-25 cauugcacuugucucggucuga cauugcacuugucucggucuga hsa-miR-29c hsa-miR-29c uagcaccauuugaaaucgguua uagcaccauuugaaaucgguua hsa-miR-205 hsa-miR-205 uccuucauuccaccggagucug uccuucauuccaccggagugug hsa-miR-375 hsa-miR-375 uuuguucguucggcucgcguga uuuguucguucggcucgcguga hsa-miR-149 hsa-miR-149 ucuggcuccgugucuucacuccc ucuggcuccgugucuucacuccc hsa-miR-29a hsa-miR-29a uagcaccaucugaaaucgguua uagcaccaucugaaaucgguua hsa-miR-1 hsa-miR-1 uggaauguaaagaaguauguau uggaauguaaagaaguauguau hsa-miR-15b hsa-miR-15b uagcagcacaucaugguuuaca uagcagcaacaucaugguuuaca

hsa-miR-181c hsa-miR-181c aacauucaaccugucggugagu aacauucaaccugucggugagu hsa-miR-29b hsa-miR-29b uagcaccauuugaaaucaguguu uagcaccauuugaaaucaguguu hsa-miR-769-5p hsa-miR-769-5p ugagaccucuggguucugagcu ugagaccucuggguucugagcu hsa-miR-221 hsa-miR-221 agcuacauugucugcuggguuuc agcuacauugucugcuggguuuc

文揭示的所有miRNA序列均已经保存在miRBase数据库中(http://microrna.sanger.ac.uk/；也参见Griffiths-JonesS.etal.(2008)Nucl.AcidsRes.36,D154-D158)。All miRNA sequences disclosed herein have been deposited in the miRBase database (http://microrna.sanger.ac.uk/; see also Griffiths-Jones S. et al. (2008) Nucl. Acids Res. 36, D154-D158).

本文所用术语“改变编码miRNA序列的核酸分子的表达”是指对特定核酸分子的任何操纵以导致所述分子的表达水平改变，即与“野生型”(即未改变的对照)的表达相比产生不同量的相应miRNA。本文所用术语“不同量”既包括与未改变的对照相比较高的量，也包括较低的量。换句话说，如本文所定义的操纵可以是上调(即激活)或下调(即抑制)核酸分子的表达(即特别是转录)。The term "altering the expression of a nucleic acid molecule encoding an miRNA sequence" as used herein refers to any manipulation of a specific nucleic acid molecule to result in an altered expression level of said molecule, i.e. compared to the expression of a "wild type" (i.e. an unaltered control) Different amounts of corresponding miRNAs were produced. The term "different amount" as used herein includes both higher and lower amounts compared to an unaltered control. In other words, manipulation as defined herein may be upregulation (ie activation) or downregulation (ie inhibition) of the expression (ie in particular transcription) of a nucleic acid molecule.

在本发明中，核酸表达特征中所包含的编码微RNA序列的一或多种核酸分子的表达以这样的方式被改变，即其表达在所述一或多种靶细胞中被上调的核酸分子的表达被下调，而其表达在所述一或多种靶细胞中被下调的核酸分子的表达被上调。换句话说，编码miRNA序列的特定核酸分子的表达的改变以与所述分子在所述一或多种癌靶细胞中的调节作用的反方式(anti-cyclical)来发生，以在所述一或多种靶细胞中干扰被上调的分子的“过度活性”和/或恢复被下调的分子的“缺陷活性”。In the present invention, the expression of one or more nucleic acid molecules encoding microRNA sequences contained in the nucleic acid expression profile is altered in such a way that the expression of the nucleic acid molecules whose expression is upregulated in the one or more target cells Expression of is downregulated, and expression of the nucleic acid molecule whose expression is downregulated in the one or more target cells is upregulated. In other words, changes in the expression of a particular nucleic acid molecule encoding a miRNA sequence occur in an anti-cyclical manner to the regulation of said molecule in said one or more cancer target cells, so that in said one or more Interfering with the "overactivity" of an upregulated molecule and/or restoring the "deficit activity" of a downregulated molecule in one or more target cells.

在本发明方法的一个优选实施方案中，下调核酸分子的表达包括将编码与被下调的核酸分子编码的微RNA序列互补的序列的核酸分子导入一或多种靶细胞中。In a preferred embodiment of the method of the present invention, down-regulating the expression of the nucleic acid molecule comprises introducing a nucleic acid molecule encoding a sequence complementary to the microRNA sequence encoded by the down-regulated nucleic acid molecule into one or more target cells.

本文所用术语“互补序列”是指血液中导入一或多种细胞中的“互补”核酸分子(本文也称作“反义核酸分子”)能与上调的内源“有义”核酸分子形成碱基对，优选Watson-Crick碱基对。The term "complementary sequence" as used herein refers to a "complementary" nucleic acid molecule (also referred to herein as an "antisense nucleic acid molecule") introduced into one or more cells in the blood capable of forming a base with an upregulated endogenous "sense" nucleic acid molecule. Base pairs, preferably Watson-Crick base pairs.

两种核酸分子(即“有义”和“反义”分子)可以是完全互补的，即其不含有任何碱基错配和/或添加或缺失核苷酸。在其它实施方案中，这两种分子包含一或多个碱基错配或者其核苷酸总数不同(由于添加或缺失所致)。在另外的实施方案中，“互补”核酸分子包含一段与上调的“有义”核酸分子中包含的序列完全互补的至少十个连续核苷酸。The two nucleic acid molecules (ie "sense" and "antisense" molecules) can be perfectly complementary, ie they do not contain any base mismatches and/or added or deleted nucleotides. In other embodiments, the two molecules contain one or more base mismatches or differ in their total number of nucleotides (due to additions or deletions). In additional embodiments, a "complementary" nucleic acid molecule comprises a stretch of at least ten contiguous nucleotides that is completely complementary to a sequence comprised in an upregulated "sense" nucleic acid molecule.

“互补”核酸分子(即编码与下调的核酸分子编码的微RNA序列互补的核酸序列的核酸分子)可以是天然发生的DNA-或RNA分子或者在其序列中包含一或多个相同类型或一或多种不同类型的修饰核苷酸的合成的核酸分子。A "complementary" nucleic acid molecule (i.e., a nucleic acid molecule encoding a nucleic acid sequence complementary to the microRNA sequence encoded by the down-regulated nucleic acid molecule) may be a naturally occurring DNA- or RNA molecule or contain in its sequence one or more of the same type or a Or a synthetic nucleic acid molecule of many different types of modified nucleotides.

例如，可能这种核酸分子包含至少一个核糖核苷酸主链单位及至少一个脱氧核糖核苷酸主链单位。此外，所述核酸分子可含有一或多个RNA主链修饰为2'-O-甲基基团或者2'-O-甲氧基基团(也称作2'-O-甲基化)，其防止在培养基中核酸酶降解，并且重要地是也防止RNA诱导性沉默复合物核酸酶的内核分离，导致miRNA的不可逆抑制。另一可能的修饰(其功能等价于2'-O-甲基化)包含锁定核酸(LNAs)，代表含有一或多个LNA核苷酸单体的核酸类似物，所述单体在RNA模拟糖构象中具有锁定双环呋喃糖单位(参见例如Orom,U.A.etal.(2006)Gene372,137-141)。For example, it is possible that such a nucleic acid molecule comprises at least one ribonucleotide backbone unit and at least one deoxyribonucleotide backbone unit. In addition, the nucleic acid molecule may contain one or more RNA backbone modifications as 2'-O-methyl groups or 2'-O-methoxy groups (also known as 2'-O-methylation) , which prevents nuclease degradation in culture medium and importantly also prevents inner core dissociation of RNA-induced silencing complex nucleases, leading to irreversible inhibition of miRNAs. Another possible modification (which is functionally equivalent to 2'-O-methylation) involves locked nucleic acids (LNAs), which represent nucleic acid analogs containing one or more LNA nucleomonomers that are present in RNA The simulated sugar conformation has locked bicyclic furanose units (see eg Orom, U.A. et al. (2006) Gene 372, 137-141).

最近开发了另一类的miRNA表达沉默基因。这些称作“antagomirs”的化学工程化寡核苷酸是与胆固醇缀合的单链23个核苷酸的RNA分子(Krutzfeldt,J.etal.(2005)Nature438,685689)。作为这种化学修饰寡核苷酸的另一选择，产生了作为RNA从转基因中产生的可以在细胞中表达的微RNA抑制剂。称作“微RNA海绵(microRNAsponges)”的这些竞争性抑制剂是从强启动子表达的转录物，含有感兴趣的微RNA的多个串联结合位点(Ebert,M.S.etal.(2007)Nat.Methods4,721-726)。Another class of miRNA expression silenced genes was recently developed. These chemically engineered oligonucleotides, called "antagomirs", are single-stranded 23 nucleotide RNA molecules conjugated to cholesterol (Krutzfeldt, J. et al. (2005) Nature 438, 685689). As an alternative to such chemically modified oligonucleotides, microRNA inhibitors that can be expressed in cells are produced as RNA from transgenes. These competitive inhibitors, termed "microRNA sponges", are transcripts expressed from strong promoters, containing multiple tandem binding sites for the microRNA of interest (Ebert, M.S. et al. (2007) Nat. Methods 4, 721-726).

为了阐明癌性或癌前样品中鉴定的miRNA的任何潜在关联，可以进行关于所述miRNA可以结合的mRNA靶序列的鉴定的预备功能分析。基于发现miRNA既可以参与肿瘤阻抑也可以参与肿瘤发生(综述参见例如Esquela-Kerscher,A.andSlack,F.J(2006)如前;Calin,G.A.andCroce,C.M.(2007)如前;Blenkiron,C.andMiska,E.A.(2007)如前)，可以推测这种miRNA的mRNA靶位点包括肿瘤阻抑基因以及癌基因。To elucidate any potential association of miRNAs identified in cancerous or precancerous samples, preparatory functional analyzes can be performed with regard to the identification of mRNA target sequences to which the miRNAs can bind. Based on the discovery that miRNAs can be involved in both tumor suppression and tumorigenesis (for review see e.g. Esquela-Kerscher, A. and Slack, F.J (2006) supra; Calin, G.A. and Croce, C.M. (2007) supra; Blenkiron, C. and Miska , E.A. (2007) supra), it can be speculated that the mRNA target sites of this miRNA include tumor suppressor genes as well as oncogenes.

如果核酸分子包含含有关于转录和/或翻译调节信息的序列元件，且这种序列“可操纵地连接”于编码多肽的核苷酸序列，则称该核酸分子为“能表达核酸分子”或者能“使得核苷酸序列表达”。可操纵地连接是其中所述调节序列元件与被表达的序列(和/或互相表达的序列)以能使得基因表达的方式进行连接的连接。A nucleic acid molecule is said to be an "expressing nucleic acid molecule" or capable of "Causing expression of a nucleotide sequence". Operably linked is one in which the regulatory sequence element and the expressed sequence (and/or reciprocally expressed sequences) are linked in such a way that the gene is expressed.

为基因表达必需的调节区的确切性质在不同物种中可以不同，但是通常这些区域均包含启动子，在原核生物中其含有两个启动子perse，即指导转录起始的DNA元件以及当转录为RNA时发出翻译起始信号的DNA元件。这种启动子区域通常包括参与转录和翻译起始的5'非编码区，如在原核生物中的-35/-10盒和Shine-Dalgarno元件或者在真核细胞中的TATA盒、CAAT序列和5'-加帽元件。这些区域也可以包括增强子或阻抑子元件以及翻译信号和前导序列以将天然多肽靶向于宿主细胞的特定区室。另外，3'非编码序列可含有参与转录终止、聚腺苷酸化等的调节元件。然而，如果这些终止序列在特定的宿主细胞中的功能不令人满意，则可以用在该细胞中发挥功能的信号取代。The exact nature of the regulatory regions necessary for gene expression can vary in different species, but generally these regions contain a promoter, which in prokaryotes contains two promoters perse, the DNA element that directs the initiation of transcription and the RNA is a DNA element that signals translation initiation. Such promoter regions usually include 5' noncoding regions involved in initiation of transcription and translation, such as the -35/-10 box and Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequence and 5'-capping element. These regions may also include enhancer or repressor elements as well as translation signals and leader sequences to target the native polypeptide to specific compartments of the host cell. In addition, the 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, and the like. However, if these termination sequences do not function satisfactorily in a particular host cell, they can be replaced with signals that are functional in that cell.

此外，如本文定义的核酸分子的表达也可以通过例如存在修饰的核苷酸而影响(如上所述)。例如，锁定核酸(LNA)单体被认为通过增强对降解的抗性及通过稳定对于沉默活性关键的miRNA-靶双链体结构而增加体内miRNA的功能性半衰期(见例如Naguibneva,I.etal.(2006)Biomed.Pharmacother.60,633-638)。Furthermore, the expression of a nucleic acid molecule as defined herein may also be influenced eg by the presence of modified nucleotides (as described above). For example, locked nucleic acid (LNA) monomers are thought to increase the functional half-life of miRNAs in vivo by enhancing resistance to degradation and by stabilizing the miRNA-target duplex structure critical for silencing activity (see, e.g., Naguibneva, I. et al. (2006) Biomed. Pharmacother. 60, 633-638).

因此，被导入患者的本发明的核酸分子可包括调节序列，优选启动子序列，任选还包括转录终止序列。所述启动子可以允许组成型或者可诱导的基因表达。合适启动子包括大肠杆菌(E.coli)lacUV5和tet(四环素应答)启动子、T7启动子以及SV40启动子或者CMV启动子。Thus, a nucleic acid molecule of the invention introduced into a patient may comprise regulatory sequences, preferably a promoter sequence, and optionally also a transcription termination sequence. The promoter may allow constitutive or inducible gene expression. Suitable promoters include the E. coli lacUV5 and tet (tetracycline responsive) promoters, the T7 promoter and the SV40 promoter or CMV promoter.

本发明的核酸分子也可以包含在载体或者其它克隆载体如质粒、噬菌粒、噬菌体、粘粒或人工染色体中。在优选的实施方案中，所述核酸分子包含在载体中，特别是包含在表达载体中。这种表达载体除了上述调节序列及编码如本发明定义的遗传构建体的核酸序列以外可包括衍生自与用于表达的宿主相容的物种的复制和控制序列以及赋予转染的细胞可选择表型的选择标记。本领域已知且可商购许多合适的载体如pSUPER和pSUPERIOR。The nucleic acid molecules of the invention may also be contained in vectors or other cloning vehicles such as plasmids, phagemids, phages, cosmids or artificial chromosomes. In a preferred embodiment, said nucleic acid molecule is comprised in a vector, in particular an expression vector. Such expression vectors may comprise, in addition to the aforementioned regulatory sequences and nucleic acid sequences encoding the genetic constructs as defined in the present invention, replication and control sequences derived from species compatible with the host used for expression and conferring a selectable expression on the transfected cells. type selection markers. Many suitable vectors are known in the art and are commercially available, such as pSUPER and pSUPERIOR.

在第十一方面，本发明涉及用于预防和/或治疗肺癌的药物组合物，所述组合物包含一或多种核酸分子，每种核酸分子编码与本文限定的其表达在肺癌患者中被上调的核酸分子编码的微RNA序列至少部分互补的序列和/或相应于本文限定的其表达在肺癌患者中被下调的核酸分子编码的微RNA序列的序列。In an eleventh aspect, the present invention relates to a pharmaceutical composition for the prevention and/or treatment of lung cancer, said composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding and defined herein expressed in lung cancer patients A sequence at least partially complementary to the microRNA sequence encoded by the upregulated nucleic acid molecule and/or a sequence corresponding to the microRNA sequence defined herein encoded by the nucleic acid molecule whose expression is downregulated in lung cancer patients.

最后，在第十二方面，本发明涉及所述药物组合物在制备用于预防和/或治疗肺癌的药物中的应用。Finally, in its twelfth aspect, the present invention relates to the use of said pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of lung cancer.

在本发明范围内，合适的药物组合物包括适于口服、直肠、鼻、局部(包括经含服和舌下)、腹膜和胃肠外(包括肌内、皮下或静脉内)给予的那些组合物，或者通过吸入或吹入给予的那些组合物。可以局部或全身性给予。优选通过口服或静脉内途径给予。所述制剂可以包装为独立剂量单位。Suitable pharmaceutical compositions within the scope of the present invention include those combinations suitable for oral, rectal, nasal, topical (including buccal and sublingual), peritoneal and parenteral (including intramuscular, subcutaneous or intravenous) administration substances, or those compositions administered by inhalation or insufflation. Administration can be local or systemic. Administration is preferably by oral or intravenous routes. The formulations can be packaged as individual dosage units.

本发明的药物组合物包括本领域确定的任何药物剂量形式，例如胶囊、微囊、扁囊剂、丸剂、片剂、粉末、小丸剂(pellet)、多颗粒制剂(例如珠、颗粒或晶体)、气雾剂、喷雾剂、泡沫、溶液、分散剂、酊剂、糖浆、酏剂、悬浮液、油包水乳状液如软膏，及水包油乳状液如乳剂、洗剂和香脂。The pharmaceutical compositions of the present invention include any pharmaceutical dosage form established in the art, such as capsules, microcapsules, cachets, pills, tablets, powders, pellets, multiparticulate formulations (such as beads, granules or crystals) , aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, water-in-oil emulsions such as ointments, and oil-in-water emulsions such as creams, lotions and balms.

使用药理学可接受的成分以及已建立的制备方法可以将上述(“有义”和“反义”)核酸分子配制为药物组合物(Gennaro,A.L.andGennaro,A.R.(2000)Remington:TheScienceandPracticeofPharmacy,20thEd.,LippincottWilliams&Wilkins,Philadelphia,PA;Crowder,T.M.etal.(2003)AGuidetoPharmaceuticalParticulateScience.Interpharm/CRC,BocaRaton,FL;Niazi,S.K.(2004)HandbookofPharmaceuticalManufacturingFormulations,CRCPress,BocaRaton,FL)。The aforementioned ("sense" and "antisense") nucleic acid molecules can be formulated into pharmaceutical compositions using pharmacologically acceptable ingredients and established methods of preparation (Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed. , Lippincott Williams & Wilkins, Philadelphia, PA; Crowder, T.M. et al. (2003) A Guide to Pharmaceutical Particulate Science. Interpharm/CRC, Boca Raton, FL; Niazi, S.K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL).

为了制备药物组合物，可以使用药学惰性的无机或有机赋形剂(即载体)。为了制备例如丸剂、片剂、胶囊或颗粒，可以使用例如乳糖、滑石、硬脂酸及其盐、脂肪、蜡、固体或液体多元醇、天然油或硬化油。用于生产溶液、悬浮液、乳状液、气雾剂混合物或在使用之前重配为溶液或气雾剂混合物的粉末的合适赋形剂包括水、醇、甘油、多元醇及其合适的混合物以及植物油。For the preparation of pharmaceutical compositions, pharmaceutically inert, inorganic or organic excipients (ie carriers) may be used. For the preparation of, for example, pills, tablets, capsules or granules, it is possible to use, for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural or hardened oils. Suitable excipients for the manufacture of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols and suitable mixtures thereof and vegetable oil.

所述药物组合物也可以含有添加剂，如充填剂、结合剂、增湿剂、助流剂、稳定剂、防腐剂、乳化剂，及另外的溶剂或者增溶剂或者实现储存效应的物质。后者可理解为可以将核酸分子掺入缓释或持续释放或靶向输送系统中如脂质体、纳米颗粒和微囊中。The pharmaceutical compositions may also contain additives, such as fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and further solvents or solubilizers or substances that achieve a storage effect. The latter is understood to mean that nucleic acid molecules can be incorporated into slow or sustained release or targeted delivery systems such as liposomes, nanoparticles and microcapsules.

为了靶向体内的大多数组织，需要临床可行的无创策略以将如本文定义的这种药物组合物定向于细胞。在过去，一些方法通过将合理剂量的siRNA经静脉内注射入小鼠和灵长类动物体内已经获得重大的治疗益处，而无明显的限制毒性。In order to target most tissues in the body, clinically feasible non-invasive strategies are required to target such pharmaceutical compositions as defined herein to cells. In the past, several approaches have achieved significant therapeutic benefit by intravenously injecting reasonable doses of siRNA into mice and primates without significant limiting toxicity.

一种方法包括将miRNA的过客链(miRNA^*链)与胆固醇或其衍生物/缀合物共价偶联以促进通过遍在表达的细胞表面LDL受体的吸收(Soutschek,J.etal.(2004)Nature432,173-178)。或者，未缀合的PBS-配制的锁定核酸修饰的寡核苷酸(LNA-antimiR)可以用于全身性输送(Elmen,J.etal.(2008)Nature452,896-899)。另一种输送miRNA的方法包括使用聚乙二醇使miRNA包囊化成为特定的脂质体以降低清除细胞的吸收并增强循环时间。这些特定的核酸颗粒(稳定的核酸-脂质颗粒或者SNALP)有效地将miRNA输送至肝脏(且不到达其它器官(Zimmermann,T.S.etal.(2006)Nature441,111-114所述))。近年来，描述了一类称作lipidoids的新型脂质样输送分子(基于烷基丙烯酸酯或者烷基-丙烯酰胺与伯胺或仲胺的缀合加成而合成)作为RNAi治疗剂的输送剂(Akinc,A.etal.(2008)Nat.Biotechnol.26,561-569)。One approach involves covalently coupling the passenger strand of miRNA (miRNA^* strand) to cholesterol or its derivatives/conjugates to facilitate uptake through ubiquitously expressed cell surface LDL receptors (Soutschek, J. et al. ( 2004) Nature 432, 173-178). Alternatively, unconjugated PBS-formulated locked nucleic acid modified oligonucleotides (LNA-antimiR) can be used for systemic delivery (Elmen, J. et al. (2008) Nature 452, 896-899). Another method of miRNA delivery involves encapsulation of miRNA into specific liposomes using polyethylene glycol to reduce uptake by clearing cells and enhance circulation time. These specific nucleic acid particles (stabilized nucleic acid-lipid particles or SNALPs) efficiently deliver miRNAs to the liver (and not to other organs (described by Zimmermann, T Setal. (2006) Nature 441, 111-114)). In recent years, a new class of lipid-like delivery molecules called lipidoids (synthesized based on the conjugation addition of alkylacrylates or alkyl-acrylamides to primary or secondary amines) has been described as delivery agents for RNAi therapeutics (Akinc, A. et al. (2008) Nat. Biotechnol. 26, 561-569).

进一步的细胞特异性靶向策略包括将miRNA与一种融合蛋白混合，该融合蛋白由与鱼精蛋白连接的靶向抗体片段组成，所述鱼精蛋白是使精子中的DNA成核并通过电荷结合miRNA的碱性蛋白(Song,E.etal.(2005)Nat.Biotechnol.23,709-717)。近来已经开发了对上述基本输送方法进行的多种修改和改变。这些技术为本领域所已知，并综述在例如deFougerolles,A.etal.(2007)Nat.Rev.DrugDiscov.6,443-453;Kim,D.H.andRossi,J.J.(2007)Nat.Genet.8,173-184)中。A further cell-specific targeting strategy involves mixing miRNAs with a fusion protein consisting of targeting antibody fragments linked to protamine, which nucleates DNA in sperm and passes charge Basic protein that binds miRNA (Song, E. et al. (2005) Nat. Biotechnol. 23, 709-717). Many modifications and variations of the basic delivery method described above have been developed recently. These techniques are known in the art and are reviewed, for example, in deFougerolles, A. et al. (2007) Nat. Rev. Drug Discov. 6, 443-453; Kim, D.H. and Rossi, J.J. (2007) Nat. Genet. 8, 173-184) .

本发明通过附图和如下实施例进一步描述，所述附图和实施例只是为了例证本发明的特异的实施方案的目的，不应理解为以任何方式限制本发明范围之意。The present invention is further described by the accompanying drawings and the following examples, which are only for the purpose of illustrating specific embodiments of the present invention and should not be construed as limiting the scope of the present invention in any way.

实施例Example

实施例1：样品收集与制备Embodiment 1: sample collection and preparation

鉴别患者样品中显示肺癌的靶细胞的主要方法步骤示于图1。The main method steps for identifying target cells showing lung cancer in patient samples are shown in FIG. 1 .

126个组织样本取自2007至2009年间于上海中山医院接受手术切除的肺癌患者。样本在手术后立即处理，用最适剪切温度(optimumcuttingtemperature,OCT)包埋，于液氮中速冻，贮存于-80°C。邻近形态正常组织(距肿瘤灶至少10m)取自同一个肺癌患者。这些肿瘤样本的基本特征示于表9中。The 126 tissue samples were obtained from lung cancer patients who underwent surgical resection at Zhongshan Hospital in Shanghai between 2007 and 2009. Samples were processed immediately after surgery, embedded with optimal cutting temperature (OCT), snap-frozen in liquid nitrogen, and stored at -80°C. Adjacent tissues with normal morphology (at least 10m away from the tumor focus) were obtained from the same lung cancer patient. The basic characteristics of these tumor samples are shown in Table 9.

表9Table 9

本发明中肿瘤样本的基本特征Basic features of tumor samples in the present invention

组织样本 tissue sample 数量 quantity 正常肺组织 normal lung tissue 44 44 肺癌 lung cancer 腺癌 Adenocarcinoma 36 36 鳞状细胞癌 squamous cell carcinoma 30 30 小细胞肺癌 Small Cell Lung Cancer 16 16 总计 total 126 126

患者资料(年龄、性别、影像资料、治疗方法、其它医疗状况、家族史等)得自医院数据库用于匹配所收集的不同样品。病理学追踪研究(例如通过苏木精和伊红(H&E)染色进行的组织学分析)用于明确确定给定样品的疾病状态以及保证样本的一致分级。Patient data (age, gender, imaging data, method of treatment, other medical conditions, family history, etc.) were obtained from hospital databases and used to match the different samples collected. Pathological follow-up studies, such as histological analysis by hematoxylin and eosin (H&E) staining, are used to definitively determine the disease state of a given sample as well as to ensure consistent grading of samples.

对每个癌样品任选进行激光捕获显微切割以特异性分离肿瘤细胞群(大约200000个细胞)。简而言之，将透明的转移膜应用于组织切片或样本的表面。在显微镜下，观察置于载玻片上的薄组织切片，并鉴别细胞群以进行分离。当选择的细胞位于观察视野的中心时，激活近红外激光二极管集成显微镜光学器件(nearIRlaserdiodeintegralwiththemicroscopeoptics)。脉冲激光束激活转移膜上的斑点(spot)，使该膜与下面的选择的细胞融合。然后将具有结合的细胞的转移膜从所述薄组织切片中剥离(Emmert-Buck,M.R.etal.(1996).Science274,998-1001;Espina,V.etal.(2007)ExpertRev.Mol.Diagn.7,647-657)。基本如厂商指导制备冷冻切片并使用激光捕获显微镜(ArcturusVeritas^TMLaserCaptureMicrodissectionInstrument(MolecularDevices,Inc.,Sunnyvale,CA,USA)进行捕获步骤。Laser capture microdissection was optionally performed on each cancer sample to specifically isolate the tumor cell population (approximately 200,000 cells). Briefly, a clear transfer film is applied to the surface of a tissue section or sample. Under a microscope, thin tissue sections mounted on glass slides are viewed and cell populations are identified for isolation. When the selected cell is located in the center of the observed field of view, the nearIRlaserdiodeintegralwiththemicroscopeoptics is activated. A pulsed laser beam activates spots on the transferred membrane, causing the membrane to fuse with the underlying selected cells. The transferred membrane with bound cells was then stripped from the thin tissue section (Emmert-Buck, MR et al. (1996). Science 274, 998-1001; Espina, V. et al. (2007) Expert Rev. Mol. Diagn. 7, 647 -657). Cryosections were prepared essentially according to the manufacturer's instructions and the capture step was performed using a laser capture microscope (Arcturus Veritas^™ Laser Capture Microdissection Instrument (Molecular Devices, Inc., Sunnyvale, CA, USA).

使用mirVana^TMmiRNA分离试剂盒(Ambion,Inc.,Austin,TX)根据厂商指导从组织切片中提取总RNA。使用NanoDrop1000分光光度计(NanoDropTechnologies,Waltham,MA)对浓度进行定量。使用RNA6000PicoLabChipkit(AgilentTechnologies,SantaClara,CA)通过2100生物分析仪进行RNA的质量控制。Total RNA was extracted from tissue sections using the mirVana^™ miRNA Isolation Kit (Ambion, Inc., Austin, TX) according to the manufacturer's instructions. Concentrations were quantified using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Waltham, MA). Quality control of RNA was performed by the 2100 Bioanalyzer using the RNA6000 PicoLabChipkit (Agilent Technologies, Santa Clara, CA).

实施例2：样品中miRNA表达谱的分析Example 2: Analysis of miRNA expression profiles in samples

使用AgilentmiRNA微阵列平台(AgilentTechnologies,SantaClara,CA,USA)根据厂商指导任选对特定样品中(差异)表达的miRNA进行定性分析。该微阵列包含723个来自Sanger数据库v.10.1的针对人类miRNA的探针。136个LCM选取的肺组织中的任一个的总RNA(100ng)作为输入(inputs)，经Cy3掺入标记。微阵列芯片用XDRScan(PMT100,PMT5)扫描。标记和杂交均根据AgilentmiRNA微阵列系统(AgilentmiRNAmicroarraysystem)的说明书操作。Qualitative analysis of (differentially) expressed miRNAs in specific samples was optionally performed using the Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA) according to manufacturer's instructions. The microarray contains 723 probes against human miRNAs from the Sanger database v.10.1. The total RNA (100 ng) of any one of the 136 LCM-selected lung tissues was used as input and labeled by Cy3 incorporation. Microarray chips were scanned with XDRScan (PMT100, PMT5). Labeling and hybridization were performed according to the instructions of AgilentmiRNA microarray system (AgilentmiRNAmicroarraysystem).

126个组织样本的每个测量进行多次独立实验，确定的miRNA表达水平代表获得的各自的各个数据的平均值。Multiple independent experiments were performed for each measurement of 126 tissue samples, and the determined miRNA expression levels represent the mean value of the respective individual data obtained.

实施例3：微阵列数据的数据分析Example 3: Data Analysis of Microarray Data

通过应用Quantile方法及使用本领域已知的GeneSpringGX10软件(AgilentTechnologies,SantaClara,CA,USA)对单色(CY3)杂交获得的原始数据归一化。AgilentmiRNA微阵列的723个miRNA中，296个miRNA被选择作为后续分析的阳性信号。Raw data obtained from single-color (CY3) hybridizations were normalized by applying the Quantile method and using GeneSpring GX10 software known in the art (Agilent Technologies, Santa Clara, CA, USA). Among the 723 miRNAs in the Agilent miRNA microarray, 296 miRNAs were selected as positive signals for subsequent analysis.

miRNA差异表达的分析。使用Fisher检验(F-test)和Bonferroni相关后再用非配对t检验分别鉴定正常肺组织和肺癌间，或者肺癌不同类别间的miRNA的差异表达。用归一化值计算正常和肿瘤样本间表达信号的倍数变化。Analysis of miRNA differential expression. Fisher test (F-test) and Bonferroni correlation were used to identify the differential expression of miRNAs between normal lung tissue and lung cancer, or between different types of lung cancer, respectively, using unpaired t-test. The fold change in expression signal between normal and tumor samples was calculated using normalized values.

应用p值＜0.00001的单因素方差分析(ANOVA)鉴定正常和不同类别肺癌组织间的miRNA的差异表达。差异表达的miRNA通过Pearson相关进行层次分类(Hierarchicalclustering)。A one-way analysis of variance (ANOVA) with a p value <0.00001 was used to identify the differential expression of miRNAs between normal and different types of lung cancer tissues. Differentially expressed miRNAs were classified by Pearson correlation (Hierarchicalclustering).

对于miRNA个体作为诊断标记物的特异性和灵敏性，应用MedCalc软件分别对正常组织和癌症组织中的miRNA个体进行接受者操作特征(ROC)曲线分析。95%可信区间用于决定该特征。For the specificity and sensitivity of individual miRNAs as diagnostic markers, the receiver operating characteristic (ROC) curve analysis of individual miRNAs in normal tissues and cancer tissues was performed using MedCalc software. A 95% confidence interval was used to determine this characteristic.

为了评估一特定miRNA在癌性靶细胞中与在健康对照细胞中相比是否差异表达，使用如下标准：To assess whether a particular miRNA is differentially expressed in cancerous target cells compared to healthy control cells, the following criteria are used:

(i)在至少50%的肿瘤样品中表达水平的变化≥2的p值(概率值)≤0.05；及(i) a p-value (probability value) ≤ 0.05 for a change in expression level ≥ 2 in at least 50% of tumor samples; and

(ii)p值≤0.05/295(系数295归因于Bonferroni校正，因为在AgilentmiRNA微阵列上295个人miRNA显示阳性信号)。(ii) p-value ≤ 0.05/295 (coefficient 295 due to Bonferroni correction, since 295 human miRNAs showed positive signals on the Agilent miRNA microarray).

如果满足这些标准中的至少一个，则认为所述miRNA在靶细胞和对照细胞中分别差异表达。If at least one of these criteria is met, the miRNA is considered to be differentially expressed in target cells and control cells, respectively.

分类/预测性分析：对芯片数据的预测性分析(Tibshiranietal.(2002).ProcNatlAcadSciUSA.99,6567-6572;http://wwwstat.stanford.edu/~tibs/PAM)区分正常肺组织和肺癌组织，以及肺癌的各组织亚型。根据10倍交叉验证准确性来进行截断值(各组基因)的选择。在WEKA软件的工作环境(http://www.cs.waikato.ac.nz/~ml/weka/)下，InfoGain法用来选择miRNA的特征，并用10倍交叉验证这些特征来进行多步骤分类算法以获得分类的准确性。从WEKA中获得的PAM特征预测者将被组合起来并对这些miRNA进行多步骤分类算法。Classification/predictive analysis: Predictive analysis of microarray data (Tibshiranie et al. (2002). ProcNatlAcadSciUSA. 99, 6567-6572; http://wwwstat.stanford.edu/~tibs/PAM) to distinguish normal lung tissue from lung cancer tissue , and various histological subtypes of lung cancer. The choice of cut-off values (groups of genes) was performed based on 10-fold cross-validation accuracy. In the working environment of WEKA software (http://www.cs.waikato.ac.nz/~ml/weka/), the InfoGain method is used to select the features of miRNA, and use 10-fold cross-validation of these features for multi-step classification algorithm to obtain classification accuracy. The PAM signature predictors obtained from WEKA will be combined and subjected to a multi-step classification algorithm for these miRNAs.

实施例4：验证芯片数据Example 4: Verify chip data

为了验证(和/或定量)有芯片测得的miRNA表达数据，根据厂商操作要求，用TaqManMicroRNA探针(AppliedBiosystems,FosterCity，CA,USA)进行了定量RT-PCR的检测。简而言之，使用AppliedBiosystem公司的TaqmanmicroRNA逆转录试剂盒进行逆转录。每个反应体系中加入100ng总RNA，15ul逆转录试剂混合物，其中包括1×逆转录缓冲液，1×dNTP，4URNase抑制剂和50UMultiScribeReverseTranscriptase。逆转录反应在PCR仪(Thermalcycleralphaengine,Bio-rad)中完成，具体程序为：16°C,30分钟;42°C,30分钟;85°C,5分钟。定量PCR根据AppliedBiosystem公司的TaqManUniversalPCRMasterMixkitandandTaqmanmicroRNAassayskits的具体说明完成。反应体系中包括2ul的逆转录产物，1XTaqManUniversalPCRMasterMix,无AmpEraseUNG和1XTaqManMicroRNAAssaymix.Real-timePCR在RochLightCycling480仪器中完成，具体程序为96℃,5分钟预热;95℃,15秒;60℃,60秒45或50个循环。Cp值由RochLightCycling480仪器自带软件进行二次衍生法计算所得。miRNA的绝对定量根据标准品的Cp值得到。17个miRNA在两个不同的平台中所得到的数据的比较详见图11。所获得的正常组织与肿瘤组织的信号比值在AgilentmiRNA芯片平台和定量RT-PCR的不同平台中相关性(R)为0.90。数据结果显示从AgilentmiRNA芯片平台中所获得的miRNA信号是高度可信的。In order to verify (and/or quantify) the miRNA expression data measured by the chip, according to the manufacturer's operating requirements, quantitative RT-PCR detection was performed with TaqMan MicroRNA probes (Applied Biosystems, Foster City, CA, USA). Briefly, reverse transcription was performed using AppliedBiosystem's Taqman microRNA Reverse Transcription Kit. Add 100ng total RNA and 15ul reverse transcription reagent mixture to each reaction system, including 1× reverse transcription buffer, 1× dNTP, 4 URNase inhibitors and 50 U MultiScribeReverseTranscriptase. The reverse transcription reaction was completed in a PCR machine (Thermalcycleralphaengine, Bio-rad), and the specific program was: 16°C, 30 minutes; 42°C, 30 minutes; 85°C, 5 minutes. Quantitative PCR was completed according to the specific instructions of TaqManUniversalPCRMasterMixkit and TaqmanmicroRNAassayskits from AppliedBiosystem. The reaction system includes 2ul of reverse transcription product, 1XTaqManUniversalPCRMasterMix, no AmpEraseUNG and 1XTaqManMicroRNAAssaymix. Real-timePCR is completed in RochLightCycling480 instrument, the specific program is 96 ° C, 5 minutes preheating; 95 ° C, 15 seconds; 60 ° C, 60 seconds 45 or 50 cycles. The Cp value is calculated by the secondary derivatization method with the software that comes with the RochLightCycling480 instrument. Absolute quantification of miRNAs was obtained from the Cp values of the standards. The comparison of the data obtained by 17 miRNAs in two different platforms is shown in Figure 11. The correlation (R) of the obtained signal ratio between normal tissue and tumor tissue in different platforms of Agilent miRNA chip platform and quantitative RT-PCR was 0.90. The data results show that the miRNA signals obtained from the Agilent miRNA chip platform are highly reliable.

图11Figure 11

实施例5：第一方面确认肺癌的miRNA表达数据Example 5: Confirmation of miRNA expression data in lung cancer in the first aspect

本发明的第一方面中所述确认肺癌的miRNA表达可以区分正常肺组织和肺癌组织，具体详见表10-11。表10所列的是新的可以区分正常肺组织和肺癌组织的miRNA，而表11所示的是这些miRNA在肺癌中的表达特征。在肺癌中已知的29个miRNA中，有24个(82％)的表达与以往的发明和发表的数据中的表达是相一致的。在表格中，“t”代表肺癌组织，“n”代表正常肺组织。其中，优选的miRNA(表10中的SEQIDNO:1至SEQIDNO:7,以及表11中的SEQIDNO:54至SEQIDNO:59)用粗体标出。In the first aspect of the present invention, confirming miRNA expression in lung cancer can distinguish normal lung tissue from lung cancer tissue, see Table 10-11 for details. Table 10 lists new miRNAs that can distinguish normal lung tissue from lung cancer tissue, and Table 11 shows the expression characteristics of these miRNAs in lung cancer. Of the 29 known miRNAs in lung cancer, the expression of 24 (82%) was consistent with that in previous discovery and published data. In the table, "t" represents lung cancer tissue, and "n" represents normal lung tissue. Among them, preferred miRNAs (SEQ ID NO: 1 to SEQ ID NO: 7 in Table 10, and SEQ ID NO: 54 to SEQ ID NO: 59 in Table 11) are marked in bold.

表10Table 10

新的可以区分正常肺组织和肺癌组织的miRNANovel miRNAs that can distinguish normal lung tissue from lung cancer tissue

表11Table 11

已经报道的肺癌组织中miRNA的表达The expression of miRNAs in lung cancer tissues has been reported

本发明的第一方面中所述的可以区分正常肺组织和肺癌组织的miRNA表达的预测性分析结果具体详见表12－15。表12显示了10倍交叉验证的PAM分类法。表13列出了用WEKA演算法得到的重要miRNA选择特征。表14显示了用两种不同的演算方法所得的分类组合。表15显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的7个miRNA(SEQIDNO:1,SEQIDNO:4,SEQIDNO:54至SEQIDNO:1056,SEQIDNO:58和SEQIDNO:59)已经在表14中用粗体标出。The results of predictive analysis of miRNA expression that can distinguish normal lung tissue from lung cancer tissue described in the first aspect of the present invention are detailed in Tables 12-15. Table 12 shows the 10-fold cross-validated PAM taxonomy. Table 13 lists the important miRNA selection features obtained by the WEKA algorithm. Table 14 shows the resulting classification combinations using two different algorithms. Table 15 shows the classification characteristics of specific miRNA predictors in various algorithms. Preferred seven miRNAs (SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:54 to SEQ ID NO:1056, SEQ ID NO:58 and SEQ ID NO:59) have been marked in bold in Table 14.

表12Table 12

预测肺癌和正常肺组织的PAM分数Prediction of PAM scores in lung cancer and normal lung tissue

名称 name 肺癌分数 lung cancer score 正常组织分数 normal tissue score hsa-miR-30a hsa-miR-30a -0.3173 -0.3173 0.5914 0.5914 hsa-miR-145 hsa-miR-145 -0.1445 -0.1445 0.2693 0.2693 hsa-miR-126^*hsa-miR-126^* -0.084 -0.084 0.1565 0.1565 hsa-miR-140-3p hsa-miR-140-3p -0.0423 -0.0423 0.0788 0.0788

表13Table 13

WEKA中重要miRNA的选择特征Selection features of important miRNAs in WEKA

名称 name 平均值 average value 平均秩 average rank 特性值 characteristic value hsa-miR-126 hsa-miR-126 0.88+-0.019 0.88+-0.019 1+-0 1+-0 60 60 hsa-miR-183 hsa-miR-183 0.785+-0.022 0.785+-0.022 3.4+-0.8 3.4+-0.8 141 141 hsa-miR-451 hsa-miR-451 0.786+-0.034 0.786+-0.034 4.2+-2.14 4.2+-2.14 390 390 hsa-miR-126^*hsa-miR-126^* 0.78+-0.024 0.78+-0.024 4.5+-1.2 4.5+-1.2 61 61 hsa-miR-30a hsa-miR-30a 0.768+-0.04 0.768+-0.04 5.1+-2.95 5.1+-2.95 274 274 hsa-miR-144 hsa-miR-144 0.776+-0.018 0.776+-0.018 5.2+-1.33 5.2+-1.33 97 97 hsa-miR-96 hsa-miR-96 0.752+-0.021 0.752+-0.021 6.7+-1.55 6.7+-1.55 717 717 hsa-miR-218 hsa-miR-218 0.749+-0.02 0.749+-0.02 7.1+-1.04 7.1+-1.04 212 212 hsa-miR-486-5p hsa-miR-486-5p 0.694+-0.024 0.694+-0.024 9.4+-1.28 9.4+-1.28 404 404 hsa-miR-200c hsa-miR-200c 0.675+-0.029 0.675+-0.029 10.9+-1.14 10.9+-1.14 187 187 hsa-miR-338-3p hsa-miR-338-3p 0.673+-0.048 0.673+-0.048 10.9+-2.21 10.9+-2.21 306 306 hsa-miR-140-3p hsa-miR-140-3p 0.644+-0.075 0.644+-0.075 11.9+-3.75 11.9+-3.75 89 89 hsa-miR-145 hsa-miR-145 0.643+-0.021 0.643+-0.021 12.1+-1.22 12.1+-1.22 99 99

计算器:weka.attributeSelection.InfoGainAttributeEvalCalculator: weka.attributeSelection.InfoGainAttributeEval

计算模式:10倍交叉验证Calculation mode: 10-fold cross-validation

表14Table 14

两种不同的演算方法所得的分类组合Classification combinations obtained by two different calculus methods

名称 name PAM PAM WEKA WEKA hsa-miR-126 hsa-miR-126 X x hsa-miR-126^*hsa-miR-126^* X x X x hsa-miR-140-3p hsa-miR-140-3p X x X x hsa-miR-145 hsa-miR-145 X x X51 --> X51 --> hsa-miR-30a hsa-miR-30a X x X x hsa-miR-451 hsa-miR-451 X x hsa-miR-183 hsa-miR-183 X x hsa-miR-200c hsa-miR-200c X x hsa-miR-338-3p hsa-miR-338-3p X x hsa-miR-144 hsa-miR-144 X x hsa-miR-218 hsa-miR-218 X x hsa-miR-486-5p hsa-miR-486-5p X x hsa-miR-96 hsa-miR-96 X x

表15Table 15

特定miRNA预测者在多种演算方法中的分类特征Taxonomic features of specific miRNA predictors in multiple algorithmic approaches

实施例6：第二方面确认非小细胞肺癌的miRNA数据表达Example 6: The second aspect confirms the miRNA data expression of non-small cell lung cancer

本发明的第二方面中所述确认非小细胞肺癌(NSCLC)和正常肺组织的miRNA表达，具体详见表16－17。表16所列的是新的可以区分正常肺组织和小细胞肺癌组织的miRNA，而表17所示的是这些miRNA在NSCLC中的表达特征。在NSCLC中已知的26个miRNA中，有21个(81％)的表达与以往的发明和发表的数据中的表达是相一致的。在表格中，“t”代表肺癌组织，“n”代表正常肺组织。其中，优选的miRNA(表16中的SEQIDNO:1-SEQIDNO:6,SEQIDNO:8,SEQIDNO:14,和表17中SEQIDNO:54至SEQIDNO:59)用粗体标出。The miRNA expression in non-small cell lung cancer (NSCLC) and normal lung tissue is confirmed in the second aspect of the present invention, see Table 16-17 for details. Table 16 lists new miRNAs that can distinguish normal lung tissue from small cell lung cancer tissue, and Table 17 shows the expression characteristics of these miRNAs in NSCLC. Of the 26 known miRNAs in NSCLC, the expression of 21 (81%) was consistent with that in previous discovery and published data. In the table, "t" represents lung cancer tissue, and "n" represents normal lung tissue. Among them, preferred miRNAs (SEQ ID NO: 1-SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 14 in Table 16, and SEQ ID NO: 54 to SEQ ID NO: 59 in Table 17) are marked in bold.

表16Table 16

新的在非小细胞肺癌中的miRNA表达Novel miRNA expression in non-small cell lung cancer

表17Table 17

本发明的第二方面中所述的可以区分正常肺组织和非小细胞肺癌组织的miRNA表达的预测性分析结果具体详见表18－21。表18显示了10倍交叉验证的PAM分类法。表19列出了用WEKA演算法得到的重要miRNA选择特征。表20显示了用两种不同的演算方法所得的分类组合。表21显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的7个miRNA(SEQIDNO:1,SEQIDNO:4,SEQIDNO:14,SEQIDNO:54至SEQIDNO:59)已经在表21中用粗体标出。The results of predictive analysis of miRNA expression that can distinguish normal lung tissue from non-small cell lung cancer tissue described in the second aspect of the present invention are detailed in Tables 18-21. Table 18 shows the 10-fold cross-validated PAM taxonomy. Table 19 lists the important miRNA selection features obtained by the WEKA algorithm. Table 20 shows the resulting classification combinations using two different algorithms. Table 21 shows the classification characteristics of specific miRNA predictors in various algorithms. The preferred seven miRNAs (SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:54 to SEQ ID NO:59) have been marked in bold in Table 21.

表18Table 18

预测非小细胞肺癌和正常肺组织的PAM分数Prediction of PAM scores in non-small cell lung cancer and normal lung tissue

名称 name 正常组织分数 normal tissue score NSCLC分数 NSCLC scores hsa-miR-218 hsa-miR-218 0.5167 0.5167 -0.3444 -0.3444 hsa-miR-30a hsa-miR-30a 0.4787 0.4787 -0.3191 -0.3191 hsa-miR-140-3p hsa-miR-140-3p 0.312 0.312 -0.208 -0.208 hsa-miR-145 hsa-miR-145 0.1928 0.1928 -0.1285 -0.1285 hsa-miR-126^*hsa-miR-126^* 0.155 0.155 -0.1033 -0.1033 hsa-miR-497 hsa-miR-497 0.0946 0.0946 -0.063 -0.063

表19Table 19

WEKA中区分非小细胞肺癌和正常肺组织的重要miRNA的选择特征Selected signatures of important miRNAs in WEKA that differentiate non-small cell lung cancer from normal lung tissue

名称 name 平均值 average value 平均秩 average rank 特性值 characteristic value hsa-miR-126 hsa-miR-126 0.971+-0.003 0.971+-0.003 1+-0 1+-0 60 60 hsa-miR-218 hsa-miR-218 0.864+-0.021 0.864+-0.021 2.3+-0.64 2.3+-0.64 212 212 hsa-miR-126^*hsa-miR-126^* 0.843+-0.028 0.843+-0.028 3+-0.63 3+-0.63 61 61 hsa-miR-183 hsa-miR-183 0.81+-0.023 0.81+-0.023 4.8+-1.47 4.8+-1.47 141 141 hsa-miR-451 hsa-miR-451 0.8+-0.029 0.8+-0.029 5.5+-1.5 5.5+-1.5 390 390 hsa-miR-144 hsa-miR-144 0.791+-0.018 0.791+-0.018 6.3+-1.27 6.3+-1.27 97 97 hsa-miR-96 hsa-miR-96 0.775+-0.031 0.775+-0.031 7+-1.73 7+-1.73 717 717 hsa-miR-200c hsa-miR-200c 0.755+-0.035 0.755+-0.035 8.1+-1.58 8.1+-1.58 187 187 hsa-miR-30a hsa-miR-30a 0.738+-0.04 0.738+-0.04 8.9+-1.97 8.9+-1.97 274 274 hsa-miR-140-3p hsa-miR-140-3p 0.703+-0.044 0.703+-0.044 10.8+-2.14 10.8+-2.14 89 89 hsa-miR-497 hsa-miR-497 0.693+-0.048 0.693+-0.048 11.4+-2.11 11.4+-2.11 420 420 hsa-miR-486-5p hsa-miR-486-5p 0.69+-0.042 0.69+-0.042 11.5+-2.01 11.5+-2.01 404 404 hsa-miR-139-5p hsa-miR-139-5p 0.647+-0.03 0.647+-0.03 14.3+-2.53 14.3+-2.53 88 88

表20Table 20

表21Table 21

实施例7：第三方面确认腺癌肺癌的miRNA表达数据Example 7: The third aspect confirms the miRNA expression data of adenocarcinoma and lung cancer

本发明的第三方面中所述确认腺癌肺癌(AC)和正常肺组织的miRNA表达，具体详见表22－23。表22所列的是新的可以区分正常肺组织和腺癌肺癌组织的miRNA，而表23所示的是这些miRNA在腺癌肺癌中的表达特征。在腺癌肺癌中已知的17个miRNA中，有16个(94％)的表达与以往的发明和发表的数据中的表达是相一致的。在表格中，“t”代表肺癌组织，“n”代表正常肺组织。其中，优选的miRNA(表22中的SEQIDNO:1至SEQIDNO:5,SEQIDNO:41,表23中的SEQIDNO:54至SEQIDNO:57,SEQIDNO:73)用粗体标出。The miRNA expression in adenocarcinoma lung cancer (AC) and normal lung tissue is confirmed in the third aspect of the present invention, see Table 22-23 for details. Table 22 lists new miRNAs that can distinguish normal lung tissue from adenocarcinoma lung tissue, and Table 23 shows the expression characteristics of these miRNAs in adenocarcinoma lung cancer. Expression of 16 (94%) of 17 known miRNAs in adenocarcinoma-lung cancer was consistent with previous discovery and published data. In the table, "t" represents lung cancer tissue, and "n" represents normal lung tissue. Among them, preferred miRNAs (SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 41 in Table 22, SEQ ID NO: 54 to SEQ ID NO: 57, and SEQ ID NO: 73 in Table 23) are marked in bold.

表22Table 22

新的可以区分正常肺组织和腺癌肺癌组织的miRNANovel miRNAs that can distinguish normal lung tissue from adenocarcinoma lung tissue

表23Table 23

已经报道的腺癌肺癌组织中miRNA的表达Expression of miRNAs in adenocarcinoma and lung cancer tissues has been reported

本发明的第三方面中所述的可以区分正常肺组织和腺癌肺癌组织的miRNA表达的预测性分析结果具体详见表24－27。表24显示了10倍交叉验证的PAM分类法。表25列出了用WEKA演算法得到的重要miRNA选择特征。表26显示了用两种不同的演算方法所得的分类组合。表27显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的5个miRNA(SEQIDNO:2,SEQIDNO:54至SEQIDNO:57)已经在表26中用粗体标出。The predictive analysis results of miRNA expression that can distinguish normal lung tissue from adenocarcinoma and lung cancer tissue described in the third aspect of the present invention are detailed in Tables 24-27. Table 24 shows the 10-fold cross-validated PAM taxonomy. Table 25 lists the important miRNA selection features obtained by the WEKA algorithm. Table 26 shows the resulting classification combinations using two different algorithms. Table 27 shows the classification characteristics of specific miRNA predictors in various algorithms. Preferred 5 miRNAs (SEQ ID NO:2, SEQ ID NO:54 to SEQ ID NO:57) have been marked in bold in Table 26.

表24Table 24

预测腺癌肺癌和正常肺组织的PAM分数Prediction of PAM scores in adenocarcinoma lung and normal lung tissues

表25Table 25

WEKA中区分腺癌肺癌和正常肺组织的重要miRNA的选择特征Selected signatures of important miRNAs in WEKA that distinguish adenocarcinoma lung from normal lung tissue

名称 name 平均值 average value 平均秩 average rank 特性值 characteristic value hsa-miR-126 hsa-miR-126 0.993+-0.002 0.993+-0.002 1+-0 1+-0 60 60 hsa-miR-218 hsa-miR-218 0.845+-0.027 0.845+-0.027 3.3+-1.27 3.3+-1.27 212 212 hsa-miR-126^*hsa-miR-126^* 0.828+-0.037 0.828+-0.037 4.2+-1.54 4.2+-1.54 61 61 hsa-miR-96 hsa-miR-96 0.804+-0.036 0.804+-0.036 5+-1.26 5+-1.26 717 717 hsa-miR-30a hsa-miR-30a 0.813+-0.052 0.813+-0.052 5.1+-2.84 5.1+-2.84 274 274 hsa-miR-183 hsa-miR-183 0.804+-0.036 0.804+-0.036 5.8+-1.47 5.8+-1.47 141 141 hsa-miR-135b hsa-miR-135b 0.778+-0.038 0.778+-0.038 6.6+-2.58 6.6+-2.58 79 79 hsa-miR-200c hsa-miR-200c 0.738+-0.046 0.738+-0.046 8.6+-2.8 8.6+-2.8 187 187 hsa-miR-144 hsa-miR-144 0.746+-0.035 0.746+-0.035 8.7+-2.19 8.7+-2.19 97 97 hsa-miR-451 hsa-miR-451 0.732+-0.054 0.732+-0.054 9.2+-3.09 9.2+-3.09 390 390 hsa-miR-148a hsa-miR-148a 0.682+-0.063 0.682+-0.063 12.5+-3.04 12.5+-3.04 107 107 hsa-miR-625 hsa-miR-625 0.657+-0.038 0.657+-0.038 13+-1.41 13+-1.41 601 601 hsa-miR-21 hsa-miR-21 0.633+-0.049 0.633+-0.049 14.2+-2.4 14.2+-2.4 201 201

表26Table 26

名称 name PAM PAM WEKA WEKA hsa-miR-96 hsa-miR-96 X x hsa-miR-126 hsa-miR-126 X x hsa-miR-126^*hsa-miR-126^* X x hsa-miR-30a hsa-miR-30a X x X x hsa-miR-218 hsa-miR-218 X x X x hsa-miR-21 hsa-miR-21 X x hsa-miR-183 hsa-miR-183 X x hsa-miR-144 hsa-miR-144 X x hsa-miR-451 hsa-miR-451 X x hsa-miR-200c hsa-miR-200c X x hsa-miR-135b hsa-miR-135b X x hsa-miR-148a hsa-miR-148a X x hsa-miR-625 hsa-miR-625 X x

表27Table 27

实施例8：第四方面确认鳞状细胞肺癌的miRNA表达数据Example 8: The fourth aspect confirms the miRNA expression data of squamous cell lung cancer

本发明的第四方面中所述确认鳞状细胞肺癌(SQ)和正常肺组织的miRNA表达，具体详见表28－29。表28所列的是新的可以区分正常肺组织和鳞状细胞肺癌组织的miRNA，而表28所示的是这些miRNA在鳞状细胞肺癌中的表达特征。在鳞状细胞肺癌中已知的24个miRNA中，有19个(79％)的表达与以往的发明和发表的数据中的表达是相一致的。在表格中，“t”代表肺癌组织，“n”代表正常肺组织。其中，优选的miRNA(表28中的SEQIDNO:3至SEQIDNO:7SEQIDNO:14,表29中的SEQIDNO:57至SEQIDNO:59)用粗体标出。The confirmation of miRNA expression in squamous cell lung cancer (SQ) and normal lung tissue as described in the fourth aspect of the present invention, see Table 28-29 for details. Table 28 lists new miRNAs that can distinguish normal lung tissue from squamous cell lung cancer tissue, and Table 28 shows the expression characteristics of these miRNAs in squamous cell lung cancer. Of the 24 known miRNAs in squamous cell lung cancer, the expression of 19 (79%) was consistent with that in previous discovery and published data. In the table, "t" represents lung cancer tissue, and "n" represents normal lung tissue. Among them, preferred miRNAs (SEQ ID NO: 3 to SEQ ID NO: 7, SEQ ID NO: 14 in Table 28, and SEQ ID NO: 57 to SEQ ID NO: 59 in Table 29) are marked in bold.

表28：新的可以区分正常肺组织和鳞状细胞肺癌组织的miRNATable 28: Novel miRNAs that can distinguish normal lung tissue from squamous cell lung cancer tissue

表29Table 29

已经报道的鳞状细胞肺癌组织中miRNA的表达The expression of miRNAs in squamous cell lung cancer tissues has been reported

本发明的第四方面中所述的可以区分正常肺组织和鳞状细胞肺癌组织的miRNA表达的预测性分析结果具体详见表30－33。表30显示了10倍交叉验证的PAM分类法。表31列出了用WEKA演算法得到的重要miRNA选择特征。表32显示了用两种不同的演算方法所得的分类组合。表33显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的9个miRNA(SEQIDNO:3-SEQIDNO:6,SEQIDNO:14,SEQIDNO:5710至SEQIDNO:59)已经在表32中用粗体标出。The results of predictive analysis of miRNA expression that can distinguish between normal lung tissue and squamous cell lung cancer tissue described in the fourth aspect of the present invention are detailed in Tables 30-33. Table 30 shows the 10-fold cross-validated PAM taxonomy. Table 31 lists the important miRNA selection features obtained by the WEKA algorithm. Table 32 shows the resulting classification combinations using two different algorithms. Table 33 shows the classification characteristics of specific miRNA predictors in various algorithms. The preferred nine miRNAs (SEQ ID NO:3-SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:5710 to SEQ ID NO:59) have been marked in bold in Table 32.

表30Table 30

预测鳞状细胞肺癌和正常肺组织的PAM分数Prediction of PAM scores in squamous cell lung cancer and normal lung tissue

表31Table 31

WEKA中区分鳞状细胞肺癌和正常肺组织的重要miRNA的选择特征Selected signatures of important miRNAs in WEKA that differentiate squamous cell lung cancer from normal lung tissue

名称 name 平均值 average value 平均秩 average rank 特性值 characteristic value hsa-miR-144 hsa-miR-144 0.974+-0.002 0.974+-0.002 2.9+-1.92 2.9+-1.92 97 97 hsa-miR-486-5p hsa-miR-486-5p 0.974+-0.002 0.974+-0.002 3.6+-1.43 3.6+-1.43 404 404 hsa-miR-451 hsa-miR-451 0.974+-0.002 0.974+-0.002 3.7+-1.79 3.7+-1.79 390 390 hsa-miR-338-3p hsa-miR-338-3p 0.974+-0.002 0.974+-0.002 3.8+-1.94 3.8+-1.94 306 306 hsa-miR-218 hsa-miR-218 0.974+-0.002 0.974+-0.002 3.8+-2.09 3.8+-2.09 212 212 hsa-miR-126 hsa-miR-126 0.974+-0.002 0.974+-0.002 5.3+-2.28 5.3+-2.28 60 60 hsa-miR-126^*hsa-miR-126^* 0.89+-0.028 0.89+-0.028 8.3+-1.62 8.3+-1.62 61 61 hsa-miR-30b hsa-miR-30b 0.89+-0.028 0.89+-0.028 8.4+-1.8 8.4+-1.8 276 276 hsa-miR-103 hsa-miR-103 0.89+-0.029 0.89+-0.029 10.6+-2.84 10.6+-2.84 23 twenty three hsa-miR-93 hsa-miR-93 0.89+-0.029 0.89+-0.029 10.7+-1.27 10.7+-1.27 702 702 hsa-miR-143 hsa-miR-143 0.884+-0.03 0.884+-0.03 12.4+-4.54 12.4+-4.54 95 95 hsa-miR-145 hsa-miR-145 0.883+-0.031 0.883+-0.031 12.8+-3.09 12.8+-3.09 99 99 hsa-miR-30a hsa-miR-30a 0.883+-0.03 0.883+-0.03 13.3+-3.29 13.3+-3.29 274 274

表32Table 32

名称 name PAM PAM WEKA WEKA hsa-miR-144 hsa-miR-144 X x hsa-miR-451 hsa-miR-451 X x hsa-miR-200c hsa-miR-200c X x hsa-miR-486-5p hsa-miR-486-5p X x hsa-miR-338-3p hsa-miR-338-3p X x X x hsa-miR-497 hsa-miR-497 X x hsa-miR-218 hsa-miR-218 X x hsa-miR-145 hsa-miR-145 X x X x hsa-miR-140-3p hsa-miR-140-3p X x hsa-miR-30b hsa-miR-30b X x hsa-miR-93 hsa-miR-93 X x hsa-miR-126 hsa-miR-126 X64 --> X64 --> hsa-miR-126^*hsa-miR-126^* X x hsa-miR-30a hsa-miR-30a X x hsa-miR-143 hsa-miR-143 X x hsa-miR-103 hsa-miR-103 X x

表33Table 33

实施例9：第五方面确认小细胞肺癌的miRNA数据表达Example 9: The fifth aspect confirms the expression of miRNA data in small cell lung cancer

本发明的第五方面中所述确认小细胞肺癌(SCLC)和正常肺组织的miRNA表达，具体详见表34－35。表34所列的是新的可以区分正常肺组织和小细胞肺癌组织的miRNA，而表35所示的是这些miRNA在SCLC中的表达特征。在SCLC中已知的21个miRNA中，有20个(95％)的表达与以往的发明和发表的数据中的表达是相一致的。在表格中，“t”代表肺癌组织，“n”代表正常肺组织。其中，优选的miRNA(表34中的SEQIDNO:2～SEQIDNO:4,SEQIDNO:9,SEQIDNO:18,SEQIDNO:30,SEQIDNO:38,SEQIDNO:39,SEQIDNO:90,SEQIDNO:91和SEQIDNO:129和表35中SEQIDNO:28,SEQIDNO:56,SEQIDNO:58,SEQIDNO:74和SEQIDNO:98)用粗体标出。The confirmation of miRNA expression in small cell lung cancer (SCLC) and normal lung tissue as described in the fifth aspect of the present invention, see Table 34-35 for details. Table 34 lists new miRNAs that can distinguish normal lung tissue from small cell lung cancer tissue, and Table 35 shows the expression characteristics of these miRNAs in SCLC. Of the 21 known miRNAs in SCLC, the expression of 20 (95%) was consistent with that in previous discovery and published data. In the table, "t" represents lung cancer tissue, and "n" represents normal lung tissue. Among them, the preferred miRNA (SEQIDNO:2～SEQIDNO:4, SEQIDNO:9, SEQIDNO:18, SEQIDNO:30, SEQIDNO:38, SEQIDNO:39, SEQIDNO:90, SEQIDNO:91 and SEQIDNO:129 and In Table 35, SEQ ID NO:28, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:74 and SEQ ID NO:98) are marked in bold.

表34Table 34

新的在小细胞肺癌中的miRNA表达Novel miRNA expression in small cell lung cancer

表35Table 35

已经报道的小细胞肺癌组织中miRNA的表达The expression of miRNA in small cell lung cancer tissue has been reported

本发明的第五方面中所述的可以区分正常肺组织和小细胞肺癌组织的miRNA表达的预测性分析结果具体详见表36－39。表36显示了10倍交叉验证的PAM分类法。表37列出了用WEKA演算法得到的重要miRNA选择特征。表38显示了用两种不同的演算方法所得的分类组合。表39显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的11个miRNA(SEQIDNO:2,SEQIDNO:3,SEQIDNO:18,SEQIDNO:28,SEQIDNO:38,SEQIDNO:58,SEQIDNO:90,SEQIDNO:91,SEQIDNO:9810andSEQIDNO:129)已经在表38中用粗体标出。The results of predictive analysis of miRNA expression that can distinguish between normal lung tissue and small cell lung cancer tissue described in the fifth aspect of the present invention are detailed in Tables 36-39. Table 36 shows the 10-fold cross-validated PAM taxonomy. Table 37 lists the important miRNA selection features obtained with the WEKA algorithm. Table 38 shows the resulting classification combinations using two different algorithms. Table 39 shows the classification characteristics of specific miRNA predictors in various algorithms. Preferred 11 miRNAs (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 58, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 9810 and SEQ ID NO: 129) have been used in Table 38 Marked in bold.

表36Table 36

预测小细胞肺癌和正常肺组织的PAM分数Prediction of PAM scores in small cell lung cancer and normal lung tissue

名称 name 正常组织分数 normal tissue score 小细胞肺癌分数 Small Cell Lung Cancer Score hsa-miR-107 hsa-miR-107 -0.1539 -0.1539 0.4233 0.4233 hsa-miR-25 hsa-miR-25 -0.1487 -0.1487 0.409 0.409 hsa-miR-103 hsa-miR-103 -0.1351 -0.1351 0.3714 0.3714 hsa-miR-93 hsa-miR-93 -0.1143 -0.1143 0.3143 0.3143 hsa-miR-96 hsa-miR-96 -0.1091 -0.1091 0.3001 0.3001 hsa-miR-106b hsa-miR-106b -0.1036 -0.1036 0.285 0.285 hsa-miR-15b hsa-miR-15b -0.0164 -0.0164 0.045 0.045

表37Table 37

WEKA中区分小细胞肺癌和正常肺组织的重要miRNA的选择特征Selected signatures of important miRNAs in WEKA that differentiate small cell lung cancer from normal lung tissue

名称 name 平均值 average value 平均秩 average rank 特性值 characteristic value hsa-miR-145 hsa-miR-145 0.836+-0.013 0.836+-0.013 5+-3.41 5+-3.41 99 99 hsa-miR-144 hsa-miR-144 0.836+-0.013 0.836+-0.013 5.2+-3.92 5.2+-3.92 97 97 hsa-miR-103 hsa-miR-103 0.836+-0.013 0.836+-0.013 6+-3.13 6+-3.13 23 twenty three hsa-miR-375 hsa-miR-375 0.836+-0.013 0.836+-0.013 6.3+-3.69 6.3+-3.69 347 347 hsa-miR-20a hsa-miR-20a 0.836+-0.013 0.836+-0.013 7.1+-4.13 7.1+-4.13 197 197 hsa-miR-130b hsa-miR-130b 0.836+-0.013 0.836+-0.013 7.7+-4.2 7.7+-4.2 70 70 hsa-miR-30a hsa-miR-30a 0.836+-0.013 0.836+-0.013 8.6+-4.65 8.6+-4.65 274 274 hsa-miR-454 hsa-miR-454 0.836+-0.013 0.836+-0.013 9.6+-4.43 9.6+-4.43 394 394 hsa-miR-106b hsa-miR-106b 0.836+-0.013 0.836+-0.013 9.9+-4.37 9.9+-4.37 28 28 hsa-miR-25 hsa-miR-25 0.836+-0.013 0.836+-0.013 10.3+-4.2 10.3+-4.2 237 237 hsa-miR-15b hsa-miR-15b 0.836+-0.013 0.836+-0.013 10.3+-4.98 10.3+-4.98 125 125 hsa-miR-451 hsa-miR-451 0.836+-0.013 0.836+-0.013 10.4+-4.43 10.4+-4.43 390 390 hsa-miR-17 hsa-miR-17 0.836+-0.013 0.836+-0.013 10.6+-3.69 10.6+-3.69 130 130

表38Table 38

名称 name PAM PAM WEKA WEKA hsa-miR-96 hsa-miR-96 X x hsa-miR-144 hsa-miR-144 X x hsa-miR-93 hsa-miR-93 X x hsa-miR-106b hsa-miR-106b X x X x hsa-miR-25 hsa-miR-25 X x X x hsa-miR-15b hsa-miR-15b X x X x hsa-miR-145 hsa-miR-145 X x hsa-miR-375 hsa-miR-375 X x hsa-miR-103 hsa-miR-103 X x X x hsa-miR-20a hsa-miR-20a X x hsa-miR-107 hsa-miR-107 X x hsa-miR-451 hsa-miR-451 X x hsa-miR-130b hsa-miR-130b X x hsa-miR-454 hsa-miR-454 X x hsa-miR-30a hsa-miR-30a X x hsa-miR-17 hsa-miR-17 X x

表39Table 39

实施例10：第六方面确认小细胞肺癌和非小细胞肺癌的miRNA数据表达Example 10: The sixth aspect confirms the expression of miRNA data in small cell lung cancer and non-small cell lung cancer

本发明的第六方面中所述确认小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)的miRNA表达，具体详见表40－43。表40显示了10倍交叉验证的PAM分类法。表41列出了用WEKA演算法得到的重要miRNA选择特征。表42显示了用两种不同的演算方法所得的分类组合。表43显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的8个miRNA(SEQIDNO:90,SEQIDNO:116,SEQIDNO:130,SEQIDNO:131,SEQIDNO:137,SEQIDNO:140,SEQIDNO:146andSEQIDNO:156)已经在表42中用粗体标出。The confirmation of miRNA expression in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) as described in the sixth aspect of the present invention, see Table 40-43 for details. Table 40 shows the 10-fold cross-validated PAM taxonomy. Table 41 lists the important miRNA selection features obtained by the WEKA algorithm. Table 42 shows the resulting classification combinations using two different algorithms. Table 43 shows the classification characteristics of specific miRNA predictors in various algorithms. Preferred eight miRNAs (SEQ ID NO:90, SEQ ID NO:116, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:146 and SEQ ID NO:156) have been marked in bold in Table 42.

表40Table 40

预测小细胞肺癌和非小细胞肺癌的PAM分数Predicting PAM Scores in Small Cell Lung Cancer and Non-Small Cell Lung Cancer

名称 name NSCLC分数 NSCLC scores SCLC分数 SCLC score hsa-miR-375 hsa-miR-375 -0.158 -0.158 0.6519 0.6519 hsa-miR-92a hsa-miR-92a -0.0757 -0.0757 0.3125 0.3125 hsa-miR-23a hsa-miR-23a 0.0635 0.0635 -0.2618 -0.2618 hsa-miR-454 hsa-miR-454 -0.0605 -0.0605 0.2497 0.2497 hsa-miR-335 hsa-miR-335 -0.0585 -0.0585 0.2412 0.2412 hsa-miR-24 hsa-miR-24 0.0549 0.0549 -0.2263 -0.2263 hsa-miR-301a hsa-miR-301a -0.0287 -0.0287 0.1183 0.1183 hsa-miR-135a hsa-miR-135a -0.0227 -0.0227 0.0936 0.0936 hsa-miR-27a hsa-miR-27a 0.0106 0.0106 -0.0439 -0.0439 hsa-miR-9 hsa-miR-9 -0.008 -0.008 0.0328 0.0328 hsa-miR-592 hsa-miR-592 -0.0078 -0.0078 0.0321 0.0321 hsa-miR-216b hsa-miR-216b -0.0009 -0.0009 0.0038 0.0038 hsa-miR-22 hsa-miR-22 0.0002 0.0002 -0.0009 -0.0009

表41Table 41

WEKA中区分小细胞肺癌和非细胞肺癌的重要miRNA的选择特征Selected signatures of important miRNAs in WEKA for distinguishing small cell lung cancer from non-cell lung cancer

表42Table 42

名称 name PAM PAM WEKA WEKA hsa-miR-375 hsa-miR-375 X x X x hsa-miR-335 hsa-miR-335 X x X x hsa-miR-23a hsa-miR-23a X x X x hsa-miR-27a hsa-miR-27a X x X x hsa-miR-22 hsa-miR-22 X x X x hsa-miR-592 hsa-miR-592 X x X x hsa-miR-135a hsa-miR-135a X x X x hsa-miR-24 hsa-miR-24 X x X x hsa-miR-454 hsa-miR-454 X x hsa-miR-301a hsa-miR-301a X x hsa-miR-199b-5p hsa-miR-199b-5p X x hsa-miR-9 hsa-miR-9 X x hsa-miR-34a hsa-miR-34a X x hsa-miR-199a-5p hsa-miR-199a-5p X x hsa-miR-335^*hsa-miR-335^* X x hsa-miR-152 hsa-miR-152 X x hsa-miR-216b hsa-miR-216b X x

表43Table 43

实施例11：第七方面确认腺癌、鳞状细胞癌或小细胞肺癌的miRNA数据表达Example 11: The seventh aspect confirms the expression of miRNA data in adenocarcinoma, squamous cell carcinoma or small cell lung cancer

本发明的第七方面中所述确认腺癌(AC)、鳞状细胞癌(SQ)或小细胞肺癌(SCLC)的miRNA表达，具体详见表44－47。表44显示了10倍交叉验证的PAM分类法。表45列出了用WEKA演算法得到的重要miRNA选择特征。表46显示了用两种不同的演算方法所得的分类组合。表47显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的7个miRNA(SEQIDNO:30,SEQIDNO:79,SEQIDNO:87,SEQIDNO:90,SEQIDNO:100,SEQIDNO:101,SEQIDNO:131)已经在表47中用粗体标出。The confirmation of miRNA expression in adenocarcinoma (AC), squamous cell carcinoma (SQ) or small cell lung cancer (SCLC) as described in the seventh aspect of the present invention, see Table 44-47 for details. Table 44 shows the 10-fold cross-validated PAM taxonomy. Table 45 lists the important miRNA selection features obtained by the WEKA algorithm. Table 46 shows the resulting classification combinations using two different algorithms. Table 47 shows the classification characteristics of specific miRNA predictors in various algorithms. The preferred seven miRNAs (SEQ ID NO:30, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:131) have been marked in bold in Table 47.

表44Table 44

预测腺癌、鳞状细胞癌或小细胞肺癌的PAM分数Predicting PAM scores for adenocarcinoma, squamous cell carcinoma, or small cell lung cancer

名称 name 腺癌分数 Adenocarcinoma Score SCLC分数 SCLC score 鳞状细胞癌分数 squamous cell carcinoma fraction hsa-miR-205 hsa-miR-205 -0.0317 -0.0317 0 0 0.554 0.554 hsa-miR-375 hsa-miR-375 0 0 0.4798 0.4798 0 0 hsa-miR-25 hsa-miR-25 -0.3066 -0.3066 0 0 0 0 hsa-miR-92a hsa-miR-92a 0 0 0.2156 0.2156 0 0 hsa-miR-93 hsa-miR-93 -0.1945 -0.1945 0 0 0 0 hsa-miR-34a hsa-miR-34a 0.1718 0.1718 0 0 0 0 hsa-miR-29c hsa-miR-29c 0.1691 0.1691 0 0 0 0 hsa-miR-23a hsa-miR-23a 0 0 -0.1452 -0.1452 0 0 hsa-miR-27a hsa-miR-27a 0 0 -0.0614 -0.0614 0.1239 0.1239 hsa-miR-24 hsa-miR-24 0 0 -0.116 -0.116 0 0 hsa-miR-29b hsa-miR-29b 0.1119 0.1119 0 0 0 0 hsa-miR-454 hsa-miR-454 0 0 0.0872 0.0872 0 0 hsa-miR-335 hsa-miR-335 0 0 0.0709 0.0709 0 0 hsa-miR-29a hsa-miR-29a 0.059 0.059 0 0 0 0 hsa-miR-106b hsa-miR-106b -0.0509 -0.0509 0 0 0 0 hsa-miR-301a hsa-miR-301a 0 0 0.0073 0.0073 0 0 hsa-miR-145 hsa-miR-145 0.0068 0.0068 0 0 0 0

表45Table 45

WEKA中区分腺癌、鳞状细胞癌或小细胞肺癌的重要miRNA的选择特征Selected signatures of important miRNAs in WEKA that differentiate adenocarcinoma, squamous cell carcinoma, or small cell lung cancer

表46Table 46

名称 name PAM PAM WEKA WEKA hsa-miR-25 hsa-miR-25 X x X x hsa-miR-205 hsa-miR-205 X x X x hsa-miR-34a hsa-miR-34a X x X x hsa-miR-375 hsa-miR-375 X x X x hsa-miR-29a hsa-miR-29a X x X x hsa-miR-29b hsa-miR-29b X x X x hsa-miR-27a hsa-miR-27a X x X x hsa-miR-93 hsa-miR-93 X x hsa-miR-106b hsa-miR-106b X x hsa-miR-15b hsa-miR-15b X x hsa-miR-454 hsa-miR-454 X x hsa-miR-301a hsa-miR-301a X x hsa-miR-145 hsa-miR-145 X x73 -->73 --> hsa-miR-29c hsa-miR-29c X x hsa-miR-221 hsa-miR-221 X x hsa-miR-335 hsa-miR-335 X x hsa-miR-23a hsa-miR-23a X x hsa-miR-199a-5p hsa-miR-199a-5p X x hsa-miR-22 hsa-miR-22 X x hsa-miR-592 hsa-miR-592 X x hsa-miR-95 hsa-miR-95 X x hsa-miR-24 hsa-miR-24 X x hsa-miR-92a hsa-miR-92a X x

表47Table 47

实施例12：第七方面确认腺癌和鳞状细胞癌miRNA数据表达Example 12: The seventh aspect confirms the expression of miRNA data in adenocarcinoma and squamous cell carcinoma

本发明的第七方面中所述确认腺癌(AC)、鳞状细胞癌(SQ的miRNA表达，具体详见表48－51。表48显示了10倍交叉验证的PAM分类法。表49列出了用WEKA演算法得到的重要miRNA选择特征。表50显示了用两种不同的演算方法所得的分类组合。表51显示了特定的miRNA预测者在多种演算方法中的分类特征。优选的8个miRNA(SEQIDNO:14,SEQIDNO:18,SEQIDNO:30,SEQIDNO:1579,SEQIDNO:90,SEQIDNO:95和SEQIDNO:100)已经在表52中用粗体标出。Confirm the expression of miRNA in adenocarcinoma (AC) and squamous cell carcinoma (SQ) as described in the seventh aspect of the present invention, see Tables 48-51 for details. Table 48 shows the PAM classification method of 10-fold cross-validation. Table 49 column The important miRNA selection features obtained with the WEKA algorithm are shown. Table 50 shows the classification combinations obtained with two different algorithms. Table 51 shows the classification characteristics of specific miRNA predictors in a variety of algorithms. Preferred Eight miRNAs (SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 30, SEQ ID NO: 1579, SEQ ID NO: 90, SEQ ID NO: 95, and SEQ ID NO: 100) have been marked in bold in Table 52.

表49Table 49

预测腺癌、鳞状细胞癌的PAM分数Prediction of PAM scores in adenocarcinoma, squamous cell carcinoma

名称 name AC分数 AC score SQ分数 SQ score hsa-miR-205 hsa-miR-205 -0.2566 -0.2566 0.3079 0.3079 hsa-miR-25 hsa-miR-25 -0.1618 -0.1618 0.1942 0.1942 hsa-miR-93 hsa-miR-93 -0.0677 -0.0677 0.0812 0.0812 hsa-miR-29c hsa-miR-29c 0.0174 0.0174 -0.0209 -0.0209

表50Table 50

WEKA中区分腺癌、鳞状细胞癌的重要miRNA的选择特征Selected signatures of important miRNAs in WEKA distinguishing adenocarcinoma from squamous cell carcinoma

表51Table 51

名称 name PAM PAM WEKA WEKA hsa-miR-497 hsa-miR-497 X x hsa-miR-93 hsa-miR-93 X x hsa-miR-25 hsa-miR-25 X x X x hsa-miR-29c hsa-miR-29c X x hsa-miR-205 hsa-miR-205 X x X x hsa-miR-375 hsa-miR-375 X x hsa-miR-149 hsa-miR-149 X x hsa-miR-29a hsa-miR-29a X x hsa-miR-1 hsa-miR-1 X x hsa-miR-15b hsa-miR-15b X x hsa-miR-181c hsa-miR-181c X x hsa-miR-29b hsa-miR-29b X x hsa-miR-769-5p hsa-miR-769-5p X x hsa-miR-221 hsa-miR-221 X x

表52Table 52

所得的结果显示了肺癌中全面的高度特异调节的miRNA表达。每个亚组的特异性miRNA都代表着肺癌组织中独特的miRNA表达谱，它们不仅能显示是否存在癌症的状态还可以区分不同组织亚型的肺癌。The obtained results show a comprehensive and highly specifically regulated miRNA expression in lung cancer. The specific miRNAs of each subgroup represent a unique miRNA expression profile in lung cancer tissues, which can not only show the presence or absence of cancer status but also distinguish different tissue subtypes of lung cancer.

因此，这些miRNA的表达信号不仅仅可以确认肺癌的存在，还可以对患者启用一个可靠的风险评估或可能处于癌前阶段，无论这个癌前阶段是否会进展为浸润癌。换而言之，在此所定义的miRNA表达信号可以预测疾病的进展，或者对进展为肺癌有易感性。Thus, the expression signature of these miRNAs not only confirms the presence of lung cancer, but also enables a reliable risk assessment of patients who may be at a precancerous stage, whether or not this precancerous stage will progress to invasive cancer. In other words, miRNA expression signatures defined herein can predict disease progression, or susceptibility to progression to lung cancer.

这种癌症发展风险的评估在若干方面具有显著的临床重要性。本发明的miRNA表达特征的鉴别提供了独特的分子标记，使得可以在早期阶段检测肺癌(即在通过原位技术或者显微镜分析活检组织或切除材料还不能检测到恶性细胞的阶段)，从而是的肺癌仍可以显著有效地得以治疗。另外，癌症发展的预测可用于指导对表现出肺癌癌前病变的患者的治疗决定。The assessment of this risk of cancer development is of significant clinical importance in several ways. The identification of the miRNA expression signature of the present invention provides a unique molecular signature that allows the detection of lung cancer at an early stage (i.e. at a stage where malignant cells cannot be detected by in situ techniques or microscopic analysis of biopsy tissue or resection material), thereby being Lung cancer can still be treated remarkably effectively. Additionally, predictions of cancer development can be used to guide treatment decisions for patients exhibiting precancerous lesions of lung cancer.

在此举例描述的本发明可以适当地在不存在本文特别揭示的任何因素、限制的条件下进行。因此，例如术语“包含”、“包括”“含有”等应具有广泛含义且无限制。另外，本文采用的术语和表达法用于描述本发明而无限制之意，且使用这些术语和表达法没有排除所示和所述特征的任何等同物或其部分之意，而是能意识到在权利要求范围内可以对本发明进行各种修改。因此，应理解尽管通过实施方案和任选的特征对本发明进行了特别揭示，但是本领域技术人员可以对本发明进行修改和改变，这种修改和改变包含在本发明的范围内。The invention as exemplified herein can suitably be practiced in the absence of any of the factors, limitations specifically disclosed herein. Thus, for example, the terms "comprises," "including," "containing," etc., shall have a broad meaning and are not limiting. In addition, the terms and expressions used herein are used to describe the present invention without any limiting meaning, and the use of these terms and expressions is not intended to exclude any equivalents or parts of the features shown and described, but to realize that Various modifications of the invention are possible within the scope of the claims. Therefore, it is to be understood that although the invention has been particularly disclosed by way of embodiments and optional features, modifications and changes of the invention may be made by those skilled in the art, and such modifications and changes are included within the scope of the invention.

本文广泛及概括地描述了本发明。落入上位描述范围内的每个较窄的下位概念和亚上位集合也组成了本发明的一部分。这包括用条件或从上位中除去任何主题的否定限制对本发明的上位描述，而无论所除去的主题是否在本文中特别引述。The invention has been described broadly and generically herein. Every narrower generic concept and subgeneric collection that falls within the generic description also forms a part of this invention. This includes a generic description of the invention with a proviso or negative limitation removing any subject matter from the generic, regardless of whether the removed subject matter is specifically recited herein.

其它实施方案在如下权利要求范围内。另外，当本发明的特征或各个方面用马库什组方式描述时，本领域技术人员能意识到本发明也以马库什组的任何各个成员或成员亚组方式被描述。Other embodiments are within the scope of the following claims. In addition, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will appreciate that the invention is also described in terms of any individual member or subgroup of members of the Markush group.