CN102272330B - Kits for Lung Cancer Diagnosis, Prognosis, and Survival Improvement - Google Patents
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Abstract
Translated fromChineseDescription
Translated fromChinese技术领域technical field
本发明涉及基于microRNA(微RNA)表达的疾病的诊断方法(如肺癌)，确定疾病预后和改善患者生存。 The present invention relates to a method for diagnosing diseases based on microRNA (microRNA) expression (such as lung cancer), determining disease prognosis and improving patient survival. the
背景技术Background technique
肺癌是世界导致癌症死亡的首要原因。相似的患者肺癌可能会有不同的临床结果，很难预测病人的预后。 Lung cancer is the leading cause of cancer death in the world. Similar patients with lung cancer may have different clinical outcomes, and it is difficult to predict the prognosis of patients. the
虽然近年来对肺癌分子生物学的了解有所增加，但仍无法详细地了解潜在的分子机制。临床上需要有精确的预后指标以确定高风险患者，也需要针对性的设计最佳的治疗方法。 Although knowledge of the molecular biology of lung cancer has increased in recent years, a detailed understanding of the underlying molecular mechanisms remains elusive. Clinically, accurate prognostic indicators are needed to identify high-risk patients, and the best treatment methods need to be designed specifically. the
本文披露的所有出版物，专利，专利申请及专利申请公布已列入本文并作为参考文献。 All publications, patents, patent applications and patent application publications disclosed herein are hereby incorporated by reference. the
发明内容Contents of the invention
本发明是部分基于发现，特别癌组织样本中微RNA表达增加或减少的水平。因此，此处依据miRNAs与/或的地位相应的miRNA基因表达水平，提供的诊断癌症方法和成分，并确定癌症患者的预后(如肺癌)。本文还提供了用探针方法检测miRNA或相应的miRNA来基因诊断和预后判断的癌症(如肺癌)。 The present invention is based in part on the discovery that microRNA expression levels are increased or decreased in particular cancerous tissue samples. Accordingly, methods and components are provided herein for diagnosing cancer and determining the prognosis of cancer patients (eg, lung cancer) based on the expression levels of miRNAs and/or miRNA genes corresponding to their status. This article also provides a cancer (such as lung cancer) that uses the probe method to detect miRNA or corresponding miRNA for gene diagnosis and prognosis. the
一方面，本文件阐述了一种方法，以确定一个人有肺癌，其中包括：a)肺组织样本miRNA表达水平，其中，个人组织是被怀疑是癌症，b)比较miRNA与参考品的表达水平，和c)如果样品展示至少有一个miRNA的有特征变化，判读个人具有或可能患有肺癌。肺癌可以是小细胞肺癌，肺腺癌，或肺鳞状细胞癌。至少一个的miRNA 可以是hsa-miR-210，hsa-miR-30a，hsa-miR-182，hsa-miR-486-5p，hsa-miR-140-3p，或者相应的同源性。该方法也可包括至少在三个miRNA有特征变化，其中，至少有三个微RNA均选自组成的hsa-miR-210，hsa-miR-30a，hsa-miR-182，hsa-miR-486-5p，hsa-miR-140-3p，或其相应的同源。如果样品中hsa-miR-210，hsa-miR-30a与hsa-miR-182表达增加和hsa-miR-486-5p，hsa-miR-140-3p表达降低，表明个人患有或可能造成肺癌。可以通过微阵列分析(例如，根据杂交信号的强度，或基于的杂交信号的比值)对来确定miRNA的表达水平。miRNA的表达水平也可以通过Northern blot分析，原位杂交，或定量逆转录聚合酶链反应来确定。检测miRNA来源于淋巴结样品，血液，血清，或呼吸道拭子。 In one aspect, this document sets forth a method to determine that an individual has lung cancer comprising: a) miRNA expression levels in a lung tissue sample, wherein the individual tissue is suspected of being cancerous, b) comparing the miRNA expression levels with a reference , and c) determining that the individual has or is likely to have lung cancer if the sample exhibits a characteristic change in at least one miRNA. Lung cancer can be small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. The at least one miRNA can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, or a corresponding homology. The method may also include characteristic changes in at least three miRNAs, wherein at least three microRNAs are selected from the group consisting of hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486- 5p, hsa-miR-140-3p, or its corresponding homolog. If the expression of hsa-miR-210, hsa-miR-30a and hsa-miR-182 increases and the expression of hsa-miR-486-5p, hsa-miR-140-3p decreases in the sample, it indicates that the individual suffers from or may cause lung cancer. Expression levels of miRNAs can be determined by microarray analysis (eg, based on the intensity of hybridization signals, or based on the ratio of hybridization signals). miRNA expression levels can also be determined by Northern blot analysis, in situ hybridization, or quantitative reverse transcription-polymerase chain reaction. Detection of miRNA was obtained from lymph node samples, blood, serum, or respiratory swabs. the
在另一个方面，本文件阐述的一种方法，以确定一个人有肺癌。其中包括分析至少有一个miRNA的特征变化的水平，依次确定个人具有或可能有肺癌。通过miRNA 的删除或扩增，或根据miRNA的基因拷贝数的变化来确定基因的变化。 In another aspect, this document sets forth a method for determining that a person has lung cancer. These include analyzing the levels of at least one miRNA signature change, which in turn determines that an individual has or is likely to have lung cancer. Genetic changes are identified by deletion or amplification of miRNAs, or by changes in the gene copy number of miRNAs. the
在另一个方面，本文件采用了系统检测肺癌，其中包括至少一对引物或多个探针，其中每对引物，或每个探针能够检测不同的miRNA的样品中，和其中至少约百分之五十的引物和探针序列能够检测hsa-miR-210，hsa-miR-30a，hsa-miR-182，hsa-miR-486-5p，hsa-miR-140-3p，和其相应的同源miRNA。 In another aspect, this document employs a system for detecting lung cancer that includes at least one pair of primers or a plurality of probes, wherein each pair of primers, or each probe is capable of detecting a different miRNA in a sample, and wherein at least about Fifty primers and probe sequences are capable of detecting hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, and their corresponding isoforms Source miRNA. the
在另一个方面，本文件阐述系统检测肺癌的方法，其中，系统包括至少一对引物或多个探针，其中每对引物，或每个探针能够检测不同的miRNA或基因的表达状态，在样本，其中至少有百分之五十左右的引物或探针能够检测的hsa-miR-210，hsa-miR-30a，hsa-miR-182，hsa-miR-486-5p，hsa-miR-140-3p，和其相应的同源miRNA。符合其中的一个miRNA的表达变化提示肺癌。 In another aspect, this document describes a method for systematically detecting lung cancer, wherein the system includes at least one pair of primers or a plurality of probes, wherein each pair of primers or each probe can detect the expression status of a different miRNA or gene, in Samples in which at least 50% of the primers or probes can detect hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140 -3p, and its corresponding homologous miRNA. A change in the expression of one of the miRNAs is consistent with lung cancer. the
在另一个方面，本文件使用至少一对引物或多个探针构建制造系统来检测肺癌，其中每对引物，或每个探针能够检测不同的miRNA样品中，和其中至少有50％左右的引物或探针能够检测hsa-miR-210，hsa-miR-30a，hsa-miR-182，hsa-miR-486-5p，hsa-miR-140-3p，和其相应的同源miRNA。 In another aspect, this document uses at least one pair of primers or multiple probes to construct a manufacturing system to detect lung cancer, wherein each pair of primers, or each probe can detect different miRNA samples, and at least about 50% of them The primers or probes are capable of detecting hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, and their corresponding homologous miRNAs. the
该文件还使用一种方法诊断肺癌病人，通过肺癌患者miRNA的表达水平来诊断肺癌患者。该方法可包括：依据表1或表2的miRNA或同源基因，来确定肺癌分化级别。 The document also uses a method to diagnose lung cancer patients, and diagnose lung cancer patients by the expression level of miRNA in lung cancer patients. The method may include: determining the differentiation grade of lung cancer according to the miRNAs or homologous genes in Table 1 or Table 2. the
在另一个方面，本文件阐述的方法确定肺鳞状细胞癌患者的预后生存，其中包括：a)检测肺癌组织样本至少一条miRNA的表达水平，b)比较样品中的miRNA与参考品的表达水平，和c)miRNA的表达水平与个体预后生存。在至miRNA指hsa-miR-31或相应的同源基因。 In another aspect, the method set forth in this document determines the prognosis and survival of patients with squamous cell carcinoma of the lung, which includes: a) detecting the expression level of at least one miRNA in a lung cancer tissue sample, b) comparing the expression level of the miRNA in the sample with a reference substance , and c) miRNA expression levels and individual prognosis survival. In to miRNA refers to hsa-miR-31 or the corresponding homologous gene. the
在另一个方面，本文件确定肺鳞状细胞癌病人预后生存期的方法，其中包括分析肺鳞状细胞癌样本中miRNA或对应基因的表达，与对照肺癌样品相比，miRNA基因表达高或低与术后生存密切相关。该miRNA是hsa-miR-31或相应的同源性。miRNA表达水平的确定可通过Northern blot分析，原位杂交，或定量实时聚合酶链反应。检测miRNA来源于淋巴结样品，血液，血清，或呼吸道拭子。 In another aspect, this document provides a method for determining prognostic survival in patients with squamous cell carcinoma of the lung, which comprises analyzing the expression of miRNAs or corresponding genes in samples of squamous cell carcinoma of the lung, the miRNA gene expression being high or low compared to control lung cancer samples closely related to postoperative survival. The miRNA is hsa-miR-31 or the corresponding homologue. miRNA expression levels can be determined by Northern blot analysis, in situ hybridization, or quantitative real-time polymerase chain reaction. Detection of miRNA was obtained from lymph node samples, blood, serum, or respiratory swabs. the
在另一个方面，本文件使用的一个或多个引物或探针对确定肺鳞状细胞癌病人预后生存期，其中的一个或多个引物或探针能够被用来检测样品中miRNA，miRNA可以是hsa-miR-31或相应的同源hsa-miR-31。 In another aspect, one or more primers or probe pairs used in this document to determine the prognostic survival of patients with squamous cell carcinoma of the lung, wherein one or more primers or probes can be used to detect miRNA in a sample, miRNA can is hsa-miR-31 or the corresponding homologous hsa-miR-31. the
在另一个方面，本文件使用的一个或多个引物或探针用于构建一种试剂或系统来确定肺鳞状细胞癌病人预后生存期，其中的一个或多个引物或探针能够被用来检测样品中miRNA，miRNA可以是hsa-miR-31或相应的同源hsa-miR-31。 In another aspect, one or more primers or probes used in this document are used to construct a reagent or system to determine the prognosis and survival of patients with squamous cell carcinoma of the lung, wherein one or more primers or probes can be used To detect the miRNA in the sample, the miRNA can be hsa-miR-31 or the corresponding homologous hsa-miR-31. the
在另一个方面，本文件的方法改善肺癌个人生存，包括应用一种有效的药物剂量 减少至少一种miRNA水平。miRNA可以是hsa-miR-31或相应的同源基因，miRNA可以是hsa-miR-210，hsa-miR-30a，hsa-miR-182，或相应的同源性基因。试剂可以是反义RNA或小分子干扰核糖核酸。该方法可包括：应用一种有效的药物剂量减少至少有两种miRNA水平，miRNA可以是hsa-miR-210，hsa-miR-30a，hsa-miR-182，hsa-miR-486-5p，hsa-miR-140-3p，和其相应的同源基因。应用一种有效的药物剂量减少至少有三种miRNA水平，miRNA可以是hsa-miR-210，hsa-miR-30a，hsa-miR-182，hsa-miR-486-5p，hsa-miR-140-3p，和其相应的同源基因。 In another aspect, the methods of this document improve survival of individuals with lung cancer comprising reducing the level of at least one miRNA using an effective dose of a drug. The miRNA can be hsa-miR-31 or the corresponding homologous gene, and the miRNA can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, or the corresponding homologous gene. The reagent can be antisense RNA or small molecule interfering ribonucleic acid. The method may comprise: reducing the levels of at least two miRNAs by applying an effective drug dose, the miRNAs may be hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa - miR-140-3p, and its corresponding homologous genes. Application of an effective drug dose reduces the level of at least three miRNAs, miRNAs can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p , and its corresponding homologous genes. the
在另一个方面，本文件采用的药物改善有肺癌个人生存，药物可以是反义RNA或小分子干扰核糖核酸，可以有效的剂量减少miRNA水平。miRNA可以是hsa-miR-31或相应的同源基因，hsa-miR-210，hsa-miR-30a，hsa-miR-182，或相应的同源性基因。 In another aspect, the drug used in this document improves the survival of individuals with lung cancer. The drug can be antisense RNA or small interfering ribonucleic acid, which can reduce miRNA levels at effective doses. The miRNA can be hsa-miR-31 or the corresponding homologous gene, hsa-miR-210, hsa-miR-30a, hsa-miR-182, or the corresponding homologous gene. the
在另一个方面，本文件包含药物载体和试剂包载的成分，成分可以是反义RNA或小分子干扰核糖核酸，降低了miRNA的水平，可以有效的剂量减少miRNA水平。miRNA可以是hsa-miR-31或相应的同源基因，hsa-miR-210，hsa-miR-30a，hsa-miR-182，或相应的同源性基因。 In another aspect, this document includes components contained in drug carriers and reagents. The components can be antisense RNA or small molecule interfering ribonucleic acid, which reduces the level of miRNA, and can reduce the level of miRNA at an effective dose. The miRNA can be hsa-miR-31 or the corresponding homologous gene, hsa-miR-210, hsa-miR-30a, hsa-miR-182, or the corresponding homologous gene. the
除非另有规定，本发明涉及所有的技术和科学所用词汇具有相同涵义。虽然方法和材料类似或等同于本文所述可用于实践的发明，合适的方法和材料的说明如下。所有出版物，专利申请，专利和其他参考资料本文中提到的以提及方式纳入的全部内容。在发生冲突时，本规范，包括定义，将控制。此外，材料，方法和例子是说明性的，而不是只打算限制。 Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and not intended to be limiting. the
发明细节体现在如下所附的图和说明中。本发明的其他功能，对象和优势在描述、图和声明中显而易见。 The details of the invention are set forth in the accompanying drawings and description below. Other functions, objects and advantages of the present invention are apparent in the description, drawings and statements. the
附图说明Description of drawings
图1A到1F是分类器，可以用来区分正常组织和恶性肺部病变。分类器基于主成分分析(PCA)和支持向量机(SVM)分析来构建。圆点代表癌变组织，十字架代表邻近癌旁组织。所有病理亚型的肺癌，训练集中98.2％(127/132)可以正确诊断(图1A)，测试集中92％(92/100)可以正确诊断(图1B)，其中包括60对鳞状细胞癌(鳞癌)，43对腺癌和13成对小细胞肺癌(SCLC)病人的组织。对鳞状细胞癌患者，训练集中93.3％(56/60)可以正确诊断(图1C)，测试集中96.7％(58/60)可以正确诊断(图1C)，其中包含60对鳞状细胞癌组织。对肺腺癌患者，训练集中97.8％(45/46)可以正确诊断(图1E)，测试集中90％(36/40)可以正确诊断(见图1F)，其中包含43对腺癌组织。 Figures 1A to 1F are classifiers that can be used to differentiate between normal tissue and malignant lung lesions. Classifiers were built based on Principal Component Analysis (PCA) and Support Vector Machine (SVM) analysis. Dots represent cancerous tissues, and crosses represent adjacent paracancerous tissues. For all pathological subtypes of lung cancer, 98.2% (127/132) in the training set can be correctly diagnosed (Figure 1A), and 92% (92/100) in the test set can be correctly diagnosed (Figure 1B), including 60 pairs of squamous cell carcinoma ( squamous cell carcinoma), 43 pairs of adenocarcinoma and 13 pairs of small cell lung cancer (SCLC) patient tissues. For squamous cell carcinoma patients, 93.3% (56/60) in the training set could be correctly diagnosed (Fig. 1C), and 96.7% (58/60) in the test set (Fig. 1C), which contained 60 pairs of squamous cell carcinoma tissues . For lung adenocarcinoma patients, 97.8% (45/46) of the training set can be correctly diagnosed (Fig. 1E), and 90% (36/40) of the test set can be correctly diagnosed (see Fig. 1F), which contains 43 pairs of adenocarcinoma tissues. the
图2描述的是鳞状细胞癌，腺癌和小细胞肺癌差异表达的微RNA的非监督聚类，差异表达的miRNA由SAM选出，左图基于miRNA在癌组织与相应的癌旁正常组织 的比例，右图基于miRNA的在癌组织绝对信号。分析了60例鳞状细胞癌，43例腺癌，和13例小细胞肺癌样品。这三个病理亚型肺癌在本研究中不能清楚的被分为3组，值得注意的是小细胞肺癌样本单独集中到一个组。 Figure 2 depicts the unsupervised clustering of differentially expressed microRNAs in squamous cell carcinoma, adenocarcinoma, and small cell lung cancer. The differentially expressed miRNAs are selected by SAM. The left figure is based on the miRNAs in cancer tissues and corresponding adjacent normal tissues. The ratio of the right panel is based on the absolute signal of miRNA in cancer tissue. Sixty squamous cell carcinoma, 43 adenocarcinoma, and 13 small cell lung cancer samples were analyzed. These three pathological subtypes of lung cancer could not be clearly divided into 3 groups in this study, it is worth noting that small cell lung cancer samples were collected into one group alone. the
图3A和3B是鳞状细胞癌患者的卡普兰一迈尔生存曲线。生存曲线显示患者在测试集60例鳞状细胞癌病例；图3A)及测试集(20例鳞状细胞癌病例；图3B)中，高水平的hsa-miR-31表达的病人生存率较低，而低hsa-miR-31表达的病人生存率高。训练集P值为0.007，测试集P值为0.001。 Figures 3A and 3B are Kaplan-Meier survival curves for patients with squamous cell carcinoma. Survival curves showing that patients with high levels of hsa-miR-31 expression had lower survival rates in the test set of 60 SCC cases; Fig. 3A) and test set (20 SCC cases; Fig. 3B) , while patients with low hsa-miR-31 expression had a high survival rate. The training set P value is 0.007 and the test set P value is 0.001. the
具体实施方式Detailed ways
本文件是根据，部分研究的基因组范围的miRNA表达谱使用配对的冷冻保存正常或癌变肺部组织样本。具体来说，利用DNA寡核苷酸芯片，微RNA的表达，在癌症样本进行比较的miRNA表达相应的非癌样本。五个微RNA被确定为要么过度表达或表达的肿瘤样本相比，相应的非癌样本。某些微RNA也被确定为具有改变各级不同疾病状态。此外，各级一个miRNA的发现和相关的总体无病生存率肺癌患者。 This document is based, in part, on the study of genome-wide miRNA expression profiling using paired cryopreserved normal or cancerous lung tissue samples. Specifically, using DNA oligonucleotide microarrays, the expression of microRNAs in cancer samples was compared to the corresponding expression of miRNAs in non-cancer samples. Five microRNAs were identified as either overexpressed or overexpressed in tumor samples compared to corresponding non-cancer samples. Certain microRNAs were also identified as having altered levels in different disease states. Furthermore, levels of a miRNA were found and correlated with overall disease-free survival in lung cancer patients. the
因此，本文件提供的方法和成分进行评估诊断及预后的疾病(如癌症，如肺癌)的基础上，各级微RNA或的地位相应的miRNA基因。一方面，有提供方法和成分，以确定一个人有一种疾病(如肺癌)的基础上，各级某些微RNA。在另一个方面，有提供方法和成分进行分类的癌症患者(如肺癌患者)的病理形式，层次的分化，与肿瘤分期的基础上，各级某些微RNA。在另一个方面，有提供方法和成分的确定预后生存个人癌症(如肺癌)的基础上，各级特别是微RNA。方法的改进生存的肺癌患者和药品成分，用于这种方法也还提供了一样，系统和工具包，可用于此处所述的方法。 Accordingly, this document provides methods and components for assessing the diagnosis and prognosis of a disease (eg, cancer, eg, lung cancer) based on the status of the levels of microRNAs or the corresponding miRNA genes. On the one hand, there are methods and components provided to determine whether a person has a disease (such as lung cancer) based on the levels of certain microRNAs. In another aspect, there are provided methods and components for classifying cancer patients (eg, lung cancer patients) by pathological form, level of differentiation, and tumor staging based on certain microRNA levels. In another aspect, there are provided methods and components for determining the prognosis of survival of individuals with cancer (eg, lung cancer) based on levels of particular microRNAs. Methods of improving survival of lung cancer patients and pharmaceutical compositions for use in such methods are also provided, as are systems and kits that can be used in the methods described herein. the
由于此处使用的“a”，“an”和“在“可能意味着单一或复数(即可能意味着一个或多个)除另有说明外。 As used herein, "a", "an" and "in" may mean singular or plural (ie may mean one or more) unless otherwise stated. the
“个人”作为此处使用的是指脊椎动物(例如，哺乳动物，如人类)。哺乳动物包括但不限于人类，非人类的灵长类动物，牛，羊，猪，马，狗，猫，兔子，天竺鼠，仓鼠，沙鼠，小鼠，大鼠。在一些体现，一个人可以是一个人。在一些体现，一个人可以成为一个动物模型研究的一种疾病，如肺癌。这是可以理解的是，当个人不是人，可以在相应的微RNA同源或垂直家系人类微RNA确定此处。 "Individual" as used herein refers to a vertebrate (eg, a mammal such as a human). Mammals include, but are not limited to, humans, non-human primates, cows, sheep, pigs, horses, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, mice, and rats. In some embodiments, a person can be a person. In some embodiments, a human can be an animal model to study a disease, such as lung cancer. It is understandable that when the individual is not human, the corresponding microRNA homolog or vertical lineage of human microRNAs can be identified here. the
个人可以是男性或女性。一个人可能会或可能不会显示出任何病理表型肺癌。在一些体现，一个人可以有家族病史的肺癌。 Individuals can be male or female. A person may or may not show any pathological phenotype of lung cancer. In some embodiments, a person may have a family history of lung cancer. the
所谓“肺组织样本”是指组织样品从肺癌。组织样品可以，例如，一个新鲜的标本，冷冻样品，或保留样本，(如福尔马林保存样品或石蜡包埋样本)。如下所述，并根据具体的方法，组织可用于全部或可以受到一个或多个方法分解样品成小块，细 胞集合体，或单个细胞。 By "lung tissue sample" is meant a tissue sample from lung cancer. The tissue sample can be, for example, a fresh specimen, a frozen sample, or a preserved sample, (eg, a formalin-preserved sample or a paraffin-embedded sample). As described below, and depending on the specific method, the tissue can be used whole or can be subjected to one or more methods to disintegrate the sample into small pieces, cell aggregates, or individual cells. the
肺癌，其中包括但不限于，肺鳞癌，小细胞肺癌(非小细胞肺癌)和肺腺癌。 Lung cancer, including, but not limited to, squamous cell carcinoma of the lung, small cell lung cancer (non-small cell lung cancer) and adenocarcinoma of the lung. the
miRNAs：微RNA miRNAs: microRNAs
microRNA(微RNA)是单链非编码的RNA，通常是约22(例如19，20，21，22，23，24或25)核苷酸长度。通过部分或全部序列同源性，可以与微RNA的3-端非编码区(3-端非编码区)的靶mRNA分子(Bartel，Cell 2004，116：281-297)。miRNAs之间的相互作用与目标基因可以阻止翻译成蛋白质的基因。在某些情况下，当目标和miRNA的完全匹配，基因的降解也可能发生。 microRNA (microRNA) is a single-stranded non-coding RNA, usually about 22 (eg, 19, 20, 21, 22, 23, 24 or 25) nucleotides in length. By partial or complete sequence homology, it can be with the target mRNA molecule (Bartel, Cell 2004, 116:281-297) of the 3-terminal non-coding region (3-terminal non-coding region) of microRNA. Interactions between miRNAs and target genes can prevent genes from being translated into proteins. In some cases, when the target and miRNA are perfectly matched, gene degradation may also occur. the
了解微RNA通常可在网上找到在http://miRNA.sanger.ac.uk/。数据库的问题见Griffths-Jones et al.，Nucleic Acids Research，2006，Vol.34。微RNA有牵连广泛的细胞过程，如细胞分化，细胞的生长和细胞死亡(Cheng et al，Nucleic Acids Res 2005，33：1290-1297；John et al，PLoS Biol.2004，2：e363)。可能有多达1000微RNA在人类基因组中(Zamore and Haley，Science 2005，309：1519-1524)，多达三分之一的人类基因的预测是可能的miRNA标靶(Lewis et al，Cell 2005，115：787-798)。RNA编辑的发现说明微RNA能调控更多的基因(Blow et al，Genome Biology 2006，7：R27)。 Understanding microRNAs can generally be found online at http://miRNA.sanger.ac.uk/. For database issues, see Griffths-Jones et al., Nucleic Acids Research, 2006, Vol.34. MicroRNAs have been implicated in a wide range of cellular processes, such as cell differentiation, cell growth and cell death (Cheng et al, Nucleic Acids Res 2005, 33: 1290-1297; John et al, PLoS Biol. 2004, 2: e363). There may be as many as 1000 microRNAs in the human genome (Zamore and Haley, Science 2005, 309:1519-1524), and as many as one-third of human genes are predicted to be possible miRNA targets (Lewis et al, Cell 2005 , 115:787-798). The discovery of RNA editing suggests that microRNAs can regulate more genes (Blow et al, Genome Biology 2006, 7:R27). the
这份文件披露微RNA的水平，有关联(例如，直接或成反比)肺癌。例如微RNA列于表1，其中包括名称，序列，和染色体定位的微RNA，以及说明是否是上调或下调肺癌。方法诊断的疾病，如肺癌可根据，例如，在基因水平或地位的任何微RNA列于表1。此处所述的系统可用于确定一个或多个微RNA的水平列于表1，和/或用于诊断肺癌的水平基础上的一个或多个微RNA列于表1。 This document discloses that levels of microRNAs are associated (eg, directly or inversely) with lung cancer. Example microRNAs are listed in Table 1, which includes the name, sequence, and chromosomal location of the microRNA, as well as a description of whether it is upregulated or downregulated in lung cancer. Methods for diagnosing diseases such as lung cancer can be based, for example, on the gene level or status of any microRNA listed in Table 1. The system described herein can be used to determine the level of one or more microRNAs listed in Table 1, and/or to diagnose lung cancer based on the level of one or more microRNAs listed in Table 1. the
虽然仅通过单一的微RNA检测就可以达到可以接受的敏感性和特异性检测，，如果检测本文中所提及的至少两个以上的微RNA，则敏感性和特异性会有所提高。举例，在一些实施例中，表1中至少两个、三个、四个、或五个微RNA用于确定病人是否患有肺癌，能得到明确结果。 Although acceptable sensitivity and specificity can be achieved by detecting only a single microRNA, if at least two of the microRNAs mentioned herein are detected, the sensitivity and specificity will be improved. For example, in some embodiments, at least two, three, four, or five microRNAs in Table 1 are used to determine whether a patient has lung cancer, and definite results can be obtained. the
在一些实施例中，在编号：1-5所列顺序中至少有两个(例如，至少有两个，三个，四个或五个)的微RNA的表达水平可以确定。例如，在基因水平或地位的微RNA中所列顺序编号：1-3才能确定，或水平或基因的地位，至少有两个(例如，两个或三个)微RNA中所列顺序编号：4和5才能确定。在一些体现地位，或基因水平至少有一个微RNA选自微RNA的序列编号：1-3和至少一个微RNA选自微RNA的序列编号：4和5可以待定。在某些情况下，水平或基因的地位，至少有两个(例如，两个或三个)微RNA的序列编号：1-3和水平的序列编号：4和第5才能确定。在一些体现基因水平或地位的所有微RNA列于表1来确定。 In some embodiments, the expression levels of at least two (eg, at least two, three, four or five) of the microRNAs listed in the sequence numbered: 1-5 can be determined. For example, sequence numbers listed in the gene level or position of microRNAs: 1-3 can be determined, or levels or gene positions of at least two (eg, two or three) microRNAs listed in sequence numbers: 4 and 5 to be sure. In some embodiments, at least one microRNA selected from sequence numbers of microRNAs: 1-3 and at least one microRNA selected from sequence numbers of microRNAs: 4 and 5 at the gene level can be determined. In some cases, the level or gene position of at least two (eg, two or three) microRNAs with sequence numbers: 1-3 and levels with sequence numbers: 4 and 5 can be determined. All miRNAs listed in Table 1 were identified at some gene level or status. the
在一些体现水平的一个或多个相应的miRNA的同源此处所述才能确定。一个“相 应的同源”的miRNA的说明此处指的miRNA从非人类脊椎动物对应一个人的miRNA。相应的同源通常至少有50％左右的序列特征(例如，至少有大约50％，55％，60％，65％，70％，75％，80％，85％，90％，95％，98％，或99％)序列身份相应的miRNA所述。例如，一个相应的同源序列的miRNA的ID号：1，至少可以有50％左右的序列特征(例如，至少有大约50％，55％，60％，65％，70％，75％，80％，85％，90％，95％，98％，或99％同源)的序列ID号：1。 Homology of one or more corresponding miRNAs at some level of expression can only be determined as described herein. A "corresponding homologous" miRNA is specified here to refer to a miRNA from a non-human vertebrate that corresponds to a human miRNA. Corresponding homologs usually have at least about 50% sequence characteristics (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% %, or 99%) sequence identity of the corresponding miRNA described. For example, the ID number of a miRNA corresponding to a homologous sequence: 1, can have at least about 50% sequence characteristics (for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% %, 85%, 90%, 95%, 98%, or 99% homology) Sequence ID number: 1. the
当一个miRNA的序列指出至少有大约例如，95％的可能确定一个参考序列(例如，序列ID号：1)，其用意是，该miRNA的序列是相同的参考序列但miRNA的序列可能包括每100个核苷酸序列多达5点改变。这些多达5个点的变化可能会删除，替换，补充，并可能发生在任何地方的顺序，在一个或多个连续序列。 When a miRNA sequence is indicated with at least about e.g., 95% probability of identifying a reference sequence (e.g., Sequence ID: 1), the intent is that the miRNA sequence is identical to the reference sequence but the miRNA sequence may include every 100 A nucleotide sequence with up to 5 changes. These changes of up to 5 points may be deletions, substitutions, additions, and may occur anywhere in the sequence, in one or more consecutive sequences. the
序列之间的特定身份核酸序列和参考序列的特定身份号码顺序确定如下。首先，核酸序列进行比较的序列中所列的特定序列识别号码使用BLAST2序列(Bl2seq)程序的独立版本的BLASTZ含同源版本2.0.14和BLASTP版本2.0.14。这个独立版本的BLASTZ在万维网上在美国政府的国家生物技术信息中心网站(ncbi.nlm.nih.gov)。解释如何使用Bl2seq程序可以在自述文件所附BLASTZ。Bl2seq进行比较两个序列的同源性使用或BLASTP算法。同源性是用来比较的核酸序列，而BLASTP是用来比较的氨基酸序列。要比较两个核酸序列，选项设定如下：我是到一个文件中包含的第一个核酸序列的比较(例如：C：\seq1.txt)；-J的设置到一个文件中包含第二核酸序列的比较(例如：C：\seq2.txt)；-p是设置为同源；邻设置为任何想要的文件名(例如：C：\output.txt)；-Q是设置为-1；-R是设定为2；和所有其他选项都留在他们的默认设置。例如，下面的命令可用于产生一个输出文件，其中包含了比较两个序列中：C：\Bl2seq-i：\seq1.txt--j：\seq2.txt-p，blastn-o c：\output.txt-q-1-r 2。要比较两个氨基酸序列，选择Bl2seq设定如下：我是到一个文件中包含的第一个氨基酸序列的比较(例如：C：\seq1.txt)；-J的设置到一个文件载有第二氨基酸序列的比较(例如：C：\seq2.txt)；-p是设置为blastp；邻设置为任何想要的文件名(例如：C：\output.txt)；和所有其他选择留在他们的默认设置。例如，下面的命令可用于生成一个输出文件包含两种氨基酸序列中：C：\Bl2seq-i：\seq1.txt--j：\seq2.txt-p，blastn-o c：\output.txt-q-1-r2。输出文本。如果这两个序列同源，然后指定输出文件将介绍这些同源序列匹配。如果这两个比较序列不同源，然后指定输出文件将不存在一致序列。 The order of specific identity numbers between sequences of a specific nucleic acid sequence and a specific identification number of a reference sequence is determined as follows. First, nucleic acid sequences were compared to the specific sequence identification numbers listed in the sequence using the BLAST2 Sequence (Bl2seq) program independent versions of BLASTZ with homology version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ is available on the World Wide Web at the US Government's National Center for Biotechnology Information website (ncbi.nlm.nih.gov). An explanation of how to use the Bl2seq program can be found in the attached BLASTZ readme. Bl2seq compares the homology of two sequences using the or BLASTP algorithm. Homology is used to compare nucleic acid sequences while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: I is the comparison to a file containing the first nucleic acid sequence (for example: C:\seq1.txt); -J is set to a file containing the second nucleic acid Sequence comparison (for example: C:\seq2.txt); -p is set to homology; neighbor is set to any desired file name (for example: C:\output.txt); -Q is set to -1; -R is set to 2; and all other options are left at their default settings. For example, the following command can be used to generate an output file containing the comparison of two sequences: C:\Bl2seq -i:\seq1.txt --j:\seq2.txt -p, blastn -o c:\output .txt-q-1-r 2. To compare two amino acid sequences, select the Bl2seq settings as follows: I is to a file containing the first amino acid sequence for comparison (eg: C:\seq1.txt); -J is set to a file containing the second amino acid sequence comparison (eg: C:\seq2.txt); -p is set to blastp; ortho is set to any desired filename (eg: C:\output.txt); and all other options are left in their default setting. For example, the following command can be used to generate an output file containing two amino acid sequences: C:\Bl2seq -i:\seq1.txt -j:\seq2.txt -p, blastn -o c:\output.txt - q-1-r2. output text. If the two sequences are homologous, then the specified output file will describe those homologous sequence matches. If the two compared sequences differ from source, then no consensus sequences will be present in the specified output file. the
一旦匹配，匹配的位置相同的核苷酸或氨基酸残基是在这两个序列上。序列身份确定除以匹配的数量也由长度的序列中规定的顺序确定(例如，序列ID号：1)， 或由一个长度(例如，从连续20个核苷酸序列集提出在一个确定的顺序)，然后乘以所产生的价值100。例如，核酸序列有20匹配，匹配序列中所列顺序识别码：1是相同的百分之90.9的序列中所列顺序编号：1(即20÷22*100＝90.9)。据指出，百分之序列特征值四舍五入到最接近的十分之一。例如，75.11，75.12，75.13和75.14是四舍五入至75.1，而75.15，75.16，75.17，75.18和75.19是四舍五入至75.2。人们还注意到，该长度值将永远是一个整数。 Once matched, the matched positions are identical nucleotide or amino acid residues in both sequences. Sequence identity determined by dividing the number of matches is also determined by the length of the sequence in a specified order (e.g., Sequence ID number: 1), or by a length (e.g., from a set of consecutive 20 nucleotide sequences proposed in a defined order ), and multiply the resulting value by 100. For example, if there are 20 matches in the nucleic acid sequence, the sequence identification number: 1 listed in the matching sequence is identical to the sequence number: 1 listed in 90.9 percent of the sequences (ie 20÷22*100=90.9). It is noted that percent series eigenvalues are rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded to 75.2. It is also noted that the length value will always be an integer. the
评价miRNA水平的方法 Methods for assessing miRNA levels
该方法在一个或多个微RNA的基础上。由于此处使用中，“水平”指的是miRNA的分子或其前体的数额或积累率。这个词可以用来指绝对数量的样品中的miRNA(所代表的杂交信号的强度)，或与对照的miRNA的比值(所代表的与对照的miRNA的杂交信号的比值)。对照可以是从同一样品的不同的miRNA，其水平并没有在肺癌组织样本改变，也可以是从不同的样本同一的miRNA(如癌组织样品来自同一个人，或组织样本另一个人没有患肺癌)。 The method is based on one or more microRNAs. As used herein, "level" refers to the amount or accumulation rate of miRNA molecules or their precursors. The term can be used to refer to the absolute number of miRNAs in a sample (representing the strength of the hybridization signal), or the ratio to the control miRNA (representing the ratio of the hybridization signal to the control miRNA). The control could be a different miRNA from the same sample whose level did not change in the lung cancer tissue sample, or it could be the same miRNA from a different sample (such as a cancer tissue sample from the same person, or a tissue sample from another person who did not have lung cancer) . the
一个“前体”的miRNA的分子，或“miRNA前体”，指的是未经加工的miRNA基因转录，通常包括一个RNA转录物的大约70个核苷酸长。miRNA的前体通常是RNA酶(如Dicer，Argonaut，or RNAase III)处理的活化的miRNA分子，通常是19-25(例如，19，20，21，22，23，24，或25)核苷酸长度。 A "precursor" miRNA molecule, or "premiRNA", refers to the unprocessed miRNA gene transcript, typically comprising an RNA transcript approximately 70 nucleotides in length. Precursors of miRNAs are usually 19-25 (e.g., 19, 20, 21, 22, 23, 24, or 25) nucleosides of activated miRNA molecules treated with RNases (such as Dicer, Argonaut, or RNAase III) acid length. the
“miRNA在肺组织样本水平”是指的miRNA水平的组织样品。虽然在大多数情况下根据直接测量在肺组织样本的miRNA水平来确定miRNA水平，设想miRNA的水平不仅在肺组织样品而且在其他样品也可以反映出的miRNA的水平，例如在淋巴结样品(如淋巴结或淋巴液)，血清，血液或其他生物流体材料，如痰。一个miRNA的水平可在淋巴结样品(例如，切片或淋巴结针吸)血液或血清，或肺组织拭子的基础上来确定。一个miRNA的水平可从经内镜超声引导下取得的样品来确定(例如用RT-PCR分析)。内镜超声引导下细针穿刺(活检)是一种微创技术的非手术取样，从而能够进行更加详细的分子标记分析。测定样品中的miRNA水平，不仅可以测肺癌组织，也可以测肺癌组织以外的其他肺癌组织。例如，可以先确定的血清样本miRNA水平，以及后续可以进行区域淋巴结miRNA的分析。这种多步分析可能提供更多的信息，增加诊断可信度。 "miRNA levels in lung tissue samples" refers to miRNA levels in tissue samples. Although in most cases miRNA levels are determined based on direct measurement of miRNA levels in lung tissue samples, it is contemplated that miRNA levels not only in lung tissue samples but also in other samples may reflect miRNA levels, for example in lymph node samples (e.g. lymph node or lymph), serum, blood or other biological fluid materials such as sputum. The level of an miRNA can be determined on the basis of a lymph node sample (eg, section or lymph node needle aspiration), blood or serum, or a lung tissue swab. The level of an miRNA can be determined from a sample taken under endoscopic ultrasound guidance (eg, by RT-PCR analysis). Endoscopic ultrasonography-guided fine-needle aspiration (biopsy) is a minimally invasive technique for nonsurgical sampling that enables more detailed analysis of molecular markers. Determination of miRNA levels in samples can not only detect lung cancer tissues, but also other lung cancer tissues other than lung cancer tissues. For example, serum sample miRNA levels can be determined first, and regional lymph node miRNA analysis can be performed subsequently. This multi-step analysis may provide more information and increase diagnostic confidence. the
miRNA的水平可从各个阶段来确定。例如，一个miRNA的水平可在手术之前，手术中，手术后，肿瘤治疗前，在肿瘤治疗中，和/或肿瘤治疗后来确定。一个miRNA的水平可在肺组织拭子的基础上来确定。 The level of miRNA can be determined from each stage. For example, the level of an miRNA can be determined before surgery, during surgery, after surgery, before tumor treatment, during tumor treatment, and/or after tumor treatment. A miRNA level can be determined on the basis of a lung tissue swab. the
测定微RNA水平的方法包括那些已知的工艺。例如，Northern杂交，原位杂交，RT-PCR技术，和/或微阵列分析来确定miRNA的水平。见，Einat，Methods Mol.Biol. 2006，342：139-157；and Thompson et al.，Genes Dev.2006，20：2202-2207. Methods of determining microRNA levels include those known in the art. For example, Northern hybridization, in situ hybridization, RT-PCR, and/or microarray analysis to determine miRNA levels. See, Einat, Methods Mol. Biol. 2006, 342: 139-157; and Thompson et al., Genes Dev. 2006, 20: 2202-2207.
据示范的方法，细胞总RNA的纯化，在核酸提取缓冲液的细胞离心。沉淀核酸，DNA酶处理去除DNA和沉淀。根据标准技术RNA分子可以在凝胶电泳琼脂糖凝胶分离，并转移至硝酸纤维素过滤器，例如，Northern杂交技术。RNA然后可以固定在过滤器加热。检测和量化具体的RNA可使用适当的互补RNA的标记DNA或RNA探针。自显影检测探针杂交，以微RNA可将杂交曝光在胶卷上。光密度扫描胶卷可以提供一个准确的衡量RNA转录物的水平。另外，RNA转录物水平可以在计算机化成像杂交印迹量化。 According to the demonstrated method, total cellular RNA is purified by centrifugation of cells in nucleic acid extraction buffer. To precipitate nucleic acids, DNase treatment removes DNA and precipitates. RNA molecules can be separated on gel electrophoresis agarose gels and transferred to nitrocellulose filters according to standard techniques, eg, Northern blot techniques. RNA can then be immobilized on the filter and heated. Detection and quantification of specific RNAs can be performed using appropriately complementary RNA-labeled DNA or RNA probes. Autographic detection probe hybridization can be exposed on the film with microRNA. Densitometric scanning of film can provide an accurate measure of RNA transcript levels. Alternatively, RNA transcript levels can be quantified in computerized imaging hybridization blots. the
除了Northern和其他核酸杂交技术，原位杂交来衡量RNA的转录水平。这项技术涉及将整个细胞或组织点到微观盖玻片和并制定存在细胞或组织中有放射性标记的核酸探针(如cRNA探针)。 In addition to Northern and other nucleic acid hybridization techniques, in situ hybridization is used to measure RNA transcript levels. This technique involves spotting whole cells or tissues onto microscopic coverslips and formulating radioactively labeled nucleic acid probes (such as cRNA probes) present in the cells or tissues. the
微RNA的水平也可在反转录和扩增聚合酶链反应(RT-PCR)确定。微RNA水平可以用内参量化(例如，从存在于同一样本“看家”基因的mRNA表达水平)。合适的“管家”基因用作内参包括肌球蛋白，甘油三磷酸脱氢酶(G3PDH)，和人U6。定量RT-PCR和变异是众所周知的普通的工艺。RT-PCR引物，见表1。在某些情况下，实时定量PCR(qRT-PCR)技术分析的微RNA可能比传统组织切片和在某些早期癌症染色检测微RNA更敏感。该qRT-PCR检测的miRNA水平，可能提供了敏感和特异的诊断，分类，及预后肺癌的工具。一方面，本文件提供RT-PCR的方法来确定的miRNA个人的水平(例如，一个人有疾病，如癌症)的。miRNA水平的是由qRT-PCR检测。在某些情况下，微RNA水平可用芯片检测。 The levels of microRNAs can also be determined in reverse transcription and amplification polymerase chain reaction (RT-PCR). MicroRNA levels can be internally quantified (eg, from mRNA expression levels of "housekeeping" genes present in the same sample). Suitable "housekeeping" genes to use as loading controls include myosin, glycerol triphosphate dehydrogenase (G3PDH), and human U6. Quantitative RT-PCR and mutations are well known and common techniques. RT-PCR primers, see Table 1. In some cases, microRNAs analyzed by quantitative real-time PCR (qRT-PCR) may be more sensitive than traditional tissue sections and stains for detection of microRNAs in some early cancers. The qRT-PCR detection of miRNA levels may provide a sensitive and specific diagnostic, classification, and prognostic tool for lung cancer. In one aspect, this document provides a method for RT-PCR to determine the level of miRNA in an individual (eg, an individual with a disease such as cancer). miRNA levels were detected by qRT-PCR. In some cases, microRNA levels can be detected using microarrays. the
核酸探针可以用此处所述的方法生产，也可以使用化学合成的方法，是众所周知的工艺。此外，杂交探针可以标记各种探测标签，例如，包括放射性同位素，荧光标记，报告酶，生物素，和其他配体。这种可探测标签可以放大，例如，可用光度法检测热或光指标物质。标记方法和检测这种探测器是众所周知的工艺。 Nucleic acid probes can be produced by the methods described herein, or by chemical synthesis, which are well known techniques. In addition, hybridization probes can be labeled with various detection labels, including, for example, radioisotopes, fluorescent labels, reporter enzymes, biotin, and other ligands. Such detectable labels can be amplified, for example, by photometric detection of heat or light indicator substances. Labeling methods and detection of such detectors are well known processes. the
核酸探针可用于检测样品中严格的条件下微RNA杂交。根据特别检测，严格的条件可以不同，在某一样本优化检测特定的miRNA。 Nucleic acid probes can be used to detect hybridization of microRNAs in samples under stringent conditions. Depending on the particular assay, stringent conditions can be varied to optimize detection of specific miRNAs in a given sample. the
一般情况下，杂交的稳定依赖离子浓度和温度。通常情况下，杂交反应的条件严格性降低，其次是清洗不同，但条件更高，更严格。中度严格的杂交条件允许核酸分子探针等结合互补核酸分子。核酸分子杂交，一般都至少有60％的识别(例如，至少有60％，65％，70％，75％，80％，85％，90％，95％，或95％以上的识别)。 In general, the stability of hybridization depends on ion concentration and temperature. Typically, the conditions of the hybridization reaction are less stringent, followed by different washes, but the conditions are higher and more stringent. Moderately stringent hybridization conditions allow nucleic acid molecular probes and the like to bind complementary nucleic acid molecules. Nucleic acid molecules generally hybridize with at least 60% recognition (eg, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% recognition). the
低严格条件杂交指是指使用10％甲酰胺，5xDenhart溶液，6xSSPE，0.2％的SDS，于22℃进行杂交，然后在1xSSPE，0.2％的SDS，于37℃进行洗涤。 温和的严格条件是指使用50％甲酰胺，5xDenhart的溶液，5xSSPE，0.2％的SDS在42℃进行杂交然后使用0.2xSSPE，0.2％的SDS，在42℃洗涤后。高苛刻条件是指使用50％甲酰胺，5xDenhart的溶液，5xSSPE，0.2％的SDS在42℃进行杂交，然后在0.1xSSPE，和0.1％的SDS于65℃进行洗涤。其他适合的温和和高苛刻条件是众所周知的。例如，在Sambrook et al.，Molecular Cloning：A Laboratory Manual，2nd ed.，Cold Spring Harbor Press，Plainview，N.Y.(1989)；and Ausubel et al.，supra，1999。Denhardt溶液包含1％Ficoll，1％polyvinylpyrolidone，和1％牛血清白蛋白(BSA)。20xSSPE(氯化钠，磷酸钠，乙烯胺四乙酸(乙二胺四乙酸))包含3M氯化钠，0.2M磷酸钠和0.025M(乙二胺四乙酸)。 Hybridization under low stringency conditions refers to the use of 10% formamide, 5xDenhart solution, 6xSSPE, 0.2% SDS, hybridization at 22°C, and then washing in 1xSSPE, 0.2% SDS, at 37°C. Mild stringent conditions refer to the use of 50% formamide, 5xDenhart's solution, 5xSSPE, 0.2% SDS at 42°C for hybridization followed by 0.2xSSPE, 0.2% SDS, after washing at 42°C. Highly stringent conditions refer to the use of 50% formamide, 5xDenhart's solution, 5xSSPE, 0.2% SDS for hybridization at 42°C, followed by washing in 0.1xSSPE, and 0.1% SDS at 65°C. Other suitable mild and high severity conditions are known. For example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al., supra, 1999. Denhardt's solution contained 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20xSSPE (Sodium Chloride, Sodium Phosphate, Ethylenediaminetetraacetic Acid (EDTA)) contains 3M Sodium Chloride, 0.2M Sodium Phosphate and 0.025M (EDTA). the
在一些实施例中，一个或多个微RNA水平可从在一个以上的时间点检测。这种“串行”取样可以适合个人肺癌进展监测。串行采样可以执行任何想要的时间，如每半年，每年，每两年或更多或较少。测量水平和参考水平的miRNA能进行比较，每次一个新的样本测试，或有关的数据水平可能举行较少分析。 In some embodiments, one or more microRNA levels can be detected from more than one time point. This "serial" sampling may be suitable for individual lung cancer progression monitoring. Serial sampling can be performed at any desired time, such as every six months, every year, every two years or more or less. Measured levels and reference levels of miRNA can be compared each time a new sample is tested, or the relevant data levels may be held for less analysis. the
对某一特定的miRNA，参考水平一般被视为“正常”的。参考水平根据自同一个体非癌肺组织miRNA水平。参考水平根据个人没有肺癌miRNA水平。参考水平可根据非肺癌的人口miRNA水平。在某些情况下，参考水平可以从一组样本，包括正在测试的样本，可预先或确定样品进行测试。 Reference levels are generally considered "normal" for a particular miRNA. Reference levels were based on miRNA levels in non-cancerous lung tissue from the same individual. Reference levels are based on individuals without lung cancer miRNA levels. Reference levels can be based on non-lung cancer population miRNA levels. In some cases, the reference level can be tested from a set of samples, including the sample being tested, which can be predetermined or determined. the
正如本文中所使用的“参考”价值可以是绝对值，相对值，一个值，有一个上限或下限，一系列的值，平均值，一个中间值，平均值，或比值，尤其是控制或基准值。 As used herein, a "reference" value may be an absolute value, a relative value, a value with an upper or lower limit, a range of values, an average value, a median value, or a ratio, especially a control or benchmark value. the
参考价值比较可能在任何(例如，一，二，三，四，或五)列于表1微RNA或其相应的同源进行。参考水平和miRNA水平的比较过程可以适当的任何方式的类型测量进行例如，当杂交信号被用来作为衡量miRNA的水平，水平可以直观定性比较强度的杂交信号。对于定量措施，可通过检查的数值数据和申述的数据(例如，检查图形，如柱状图或线图)进行比较，。比较的过程中可以人工(如目视)，或者可以是自动的。 Reference value comparisons may be performed on any (eg, one, two, three, four, or five) of the microRNAs listed in Table 1 or their corresponding homologues. The comparison process between reference levels and miRNA levels can be performed in any manner appropriate for the type of measurement. For example, when hybridization signals are used as a measure of miRNA levels, the levels can be visually compared qualitatively to the strength of the hybridization signals. For quantitative measures, comparisons can be made by examining numerical data and representational data (for example, examining graphs such as histograms or line graphs). The comparison process can be manual (eg, visual), or it can be automated. the
在一些实施例中，比较是确定测量值和参考水平差异之间的规模(例如，比较“倍”或百分比之间的测量值和参考水平之间差异)。由于此处使用的，“倍的差异”是指miRNA测量值和参考水平差异之间的规模。 In some embodiments, the comparison is to determine the magnitude of the difference between a measured value and a reference level (eg, comparing a "fold" or percentage difference between a measured value and a reference level). As used herein, "fold difference" refers to the scale of difference between miRNA measurement and reference levels. the
一个“特性的变化”中的miRNA水平，与参考水平比较，可以大幅度减少或大量增加。由于此处使用，“大幅度增加”是指增加的miRNA水平至少约5％(例如，至少有5％，6％，7％，8％，9％，10％，15％，20％，30％，40％，50％，或50％以上)。同样，“大幅度减少”作为此处使用指的是减少了miRNA的水平至少约5％(例如，至少有5％，6％，7％，8％，9％， 10％，15％，20％，30％，40％，50％，或50％以上)。 A "characteristic change" in which miRNA levels can be substantially reduced or substantially increased compared to a reference level. As used herein, "substantially increased" refers to increased miRNA levels by at least about 5% (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% %, 40%, 50%, or more than 50%). Likewise, "substantially reducing" as used herein refers to reducing the level of miRNA by at least about 5% (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% %, 30%, 40%, 50%, or more than 50%). the
表1列出了微RNA特征的变化。一个或多个微RNA水平的特点变化可作为诊断肺癌依据。例如，至少有一种序列编号：1-3微RNA的水平可以决定，至少有一个可以测量微RNA的数额大幅增加能指示肺癌。至少有一种序列编号：4，5，至少有一个可以测量微RNA的数额大幅增加能指示肺癌。当至少有一种序列编号：1-3，至少有一种序列编号：4，5微RNA的水平可以决定时，至少有一个可以测量微RNA的数额大幅增加和少有一个可以测量微RNA的数额大幅减少能指示肺癌。 Table 1 lists the changes in microRNA characteristics. The characteristic changes of one or more microRNA levels can be used as the basis for diagnosing lung cancer. For example, levels of at least one sequence number: 1-3 microRNA can be determined, and a substantial increase in the amount of at least one measurable microRNA can be indicative of lung cancer. At least one sequence number: 4, 5, at least one measurable increase in the amount of microRNA can be indicative of lung cancer. When there is at least one sequence number: 1-3, at least one sequence number: 4, 5 the level of microRNA can be determined, at least one measurable amount of microRNA is greatly increased and there is less one can be measured The amount of microRNA is greatly increased A decrease can be indicative of lung cancer. the
在一些实施例中，各级所有微RNA水平在表1中列出来，至少有一个可以测量序列编号：1-3微RNA的数额大幅增加和少有一个可以测量序列编号：4，5微RNA的数额大幅减少能指示肺癌，至少有二个可以测量序列编号：1-3微RNA的数额大幅增加和少有二个可以测量序列编号：4，5微RNA的数额大幅减少能指示肺癌。 In some embodiments, the levels of all microRNAs at all levels are listed in Table 1, at least one measurable sequence number: 1-3 The amount of microRNA is substantially increased and at least one measurable sequence number: 4, 5 microRNA A large decrease in the amount of at least two measurable sequence numbers: 1-3 and a large increase in the amount of at least two measurable sequence numbers: 4, 5 microRNAs can indicate lung cancer. the
在一些实施例中，使用一个以上的微RNA，但微RNA水平并不一致建议或表示有诊断肺癌，是“多数”的建议或表示可考虑的结果分析。例如，当该方法利用五个微RNA，其中三个表明肺癌，其结果可能被视为暗示或表明个人可诊断肺癌。但是，一个至少一个或多个特别是微RNA特征性改变可诊断肺癌。例如，当微RNA是hsa-miR-210，大幅度增加的hsa-miR-210可能是诊断肺癌一个先决条件。 In some embodiments, more than one microRNA is used, but the microRNA levels are not consistent to suggest or indicate a diagnosis of lung cancer, and a "majority" is suggested or indicated to be considered in the analysis of the results. For example, when the method utilizes five microRNAs, three of which are indicative of lung cancer, the results may be viewed as suggestive or indicative of a diagnosis of lung cancer in the individual. However, one or more characteristic changes of at least one or more microRNAs can be diagnosed as lung cancer. For example, when the microRNA is hsa-miR-210, a substantial increase in hsa-miR-210 may be a prerequisite for the diagnosis of lung cancer. the
评价miRNA基因状态的方法 Methods for assessing miRNA gene status
本文还提供了至少有一个微RNA的基因(或其相应的同源性)为基础的诊断肺癌的方法，列于表1。通过分析至少有一个的miRNA基因样品中的删除或扩增，可对基因状态进行评估。相对于对照样品一缺失或扩增中的miRNA基因的减少可以表明个体存在肺癌。 This article also provides a method for diagnosing lung cancer based on at least one microRNA gene (or its corresponding homologue), listed in Table 1. Gene status can be assessed by analyzing at least one miRNA gene sample for deletion or amplification. A decrease in a deleted or amplified miRNA gene relative to a control sample can indicate the presence of lung cancer in an individual. the
通过从疑似个人肺癌组织确定肺组织样品结构或序列，和对照样品结构或序列的比较，可以检测删除或放大了的miRNA基因。适用于任何检测改变基因结构或序列技术可用于本方法。例如，存在的miRNA基因缺失和扩增可利用核酸探针特异性的miRNA序列Southern杂交检测。序列分析和单链构象多态性分析也可用。 Deleted or amplified miRNA genes can be detected by determining the structure or sequence of a lung tissue sample from a suspected individual lung cancer tissue and comparing the structure or sequence of a control sample. Any technique suitable for detecting altered gene structure or sequence can be used in this method. For example, the presence of miRNA gene deletions and amplifications can be detected using nucleic acid probes specific for miRNA sequence Southern hybridization. Sequence analysis and single-strand conformation polymorphism analysis are also available. the
缺失或扩增的miRNA的基因也可以用扩增片段的基因的聚合酶链反应(PCR)检测，如果个人的DNA样本不同于样本DNA可通过测序或电泳确定扩增片段序列或长度。miRNA的基因删除还可以通过密切相关的miRNA的基因染色体标记的缺失来测。 The gene of the deleted or amplified miRNA can also be detected by polymerase chain reaction (PCR) of the gene of the amplified fragment. If the DNA sample of the individual is different from the sample DNA, the sequence or length of the amplified fragment can be determined by sequencing or electrophoresis. Genetic deletion of miRNAs can also be detected by the absence of chromosomal markers for closely related miRNA genes. the
细胞中的个人miRNA基因在也可通过测量的样本中至少一个miRNA的基因拷贝数来评估，其中一个基因拷贝数以外的其他两个为对任何性别体细胞染色体或者女性性染色体miRNA的基因，或一个以上的其他miRNA基因中的性染色体是男性，可以表明个人有肺癌。 The presence of individual miRNA genes in cells can also be assessed by measuring the gene copy number of at least one miRNA in the sample, where one gene copy number and the other two are genes for any sex somatic chromosome or female sex chromosome miRNA, or More than one other miRNA gene in a sex chromosome that is male can indicate that an individual has lung cancer. the
任何适合于检测基因拷贝数的技术都可用，包括Southern杂交和PCR扩增技术。另一种来确定的肺组织样本miRNA基因拷贝数的方法依赖于一个事实，即许多微RNA或基因簇与染色体标记或其他基因密切相关。杂合的染色体标记或其他基因密切相关的基因的丢失说明染色体标记或其他基因密切相关的基因有杂合性。测定缺失的染色体标记的方法技本文也说明了。 Any technique suitable for detecting gene copy number can be used, including Southern hybridization and PCR amplification techniques. Another approach to determine the copy number of miRNA genes in lung tissue samples relies on the fact that many microRNAs or gene clusters are closely related to chromosomal markers or other genes. Heterozygous loss of a chromosomal marker or other closely related genes indicates heterozygosity for the chromosomal marker or other closely related genes. Methods for determining missing chromosomal markers are also described herein. the
可用于确定miRNA基因其他技术，例如，等位基因特异性引物延伸的芯片，聚合酶链反应/低剂量辐射普遍阵列，微球的单碱基链延伸，序列标签分子探针反演和组合序列杂交。 Other techniques that can be used to identify miRNA genes, for example, microarrays with allele-specific primer extension, polymerase chain reaction/low-dose radiation universal arrays, single-base strand extension from microspheres, sequence-tagged molecular probe inversion and combinatorial sequences hybridize. the
在一些实施例中，“对照样品”可以是一个个人没有肺癌组织样品。另外，对照样品可以是一个人群组织样本的集合(例如，已知没有肺癌的个人)。 In some embodiments, a "control sample" can be a tissue sample from an individual without lung cancer. Alternatively, a control sample can be a collection of tissue samples from a population (eg, individuals known to be free of lung cancer). the
至少有一个(例如，一，二，三，四，五)的微RNA列于表1或其相应的同源才能确定基因状态。在体现在基因状态的一个以上的微RNA的使用，但没有一致建议或表示有诊断肺癌的“多数”的建议或表示可考虑的结果分析。例如，当该方法利用基因五个微RNA，其中三个表明肺癌，其结果可能被视为暗示或表明诊断肺癌。然而，在某些情况，诊断为肺癌要求一个或多个特定的miRNA基因的改变。 At least one (eg, one, two, three, four, five) of the microRNA listed in Table 1 or its corresponding homologue was required to determine gene status. The use of more than one microRNA was reflected in the gene status, but there was no consistent recommendation or indication that there was a "majority" of diagnoses for lung cancer or an outcome analysis that could be considered. For example, when the method utilizes five microRNA genes, three of which indicate lung cancer, the results may be considered suggestive or indicative of a diagnosis of lung cancer. However, in some instances, a diagnosis of lung cancer requires alterations in one or more specific miRNA genes. the
诊断疾病方法 Methods of diagnosing diseases
本文件提供的方法，以确定一个人有一种疾病(如癌症)，其中包括：a)确定的水平至少一个miRNA的生物样品中的个人，和b)与参考水平比较，其中的一个特点变化的水平，这表明疾病。可疾病诊断包括，但不仅限于，癌症(如肺癌，乳腺癌，食道癌，胃癌，肝癌，大肠癌，胰腺癌，白血病，淋巴瘤，肾癌，膀胱癌，子宫颈癌癌，子宫内膜癌，卵巢癌和睾丸癌)，心血管疾病(如冠心病，高血压和动脉粥样硬化)，以及与年龄有关的疾病(如帕金森氏症，阿尔茨海默氏症，糖尿病)。 This document provides a method for determining that a person has a disease (such as cancer) that includes: a) determining the level of at least one miRNA in a biological sample of the individual, and b) comparing a characteristic of the individual to a reference level, wherein the level, which indicates disease. Disease diagnoses include, but are not limited to, cancers (such as lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, leukemia, lymphoma, kidney cancer, bladder cancer, cervical cancer, endometrial cancer , ovarian cancer and testicular cancer), cardiovascular diseases (such as coronary heart disease, hypertension and atherosclerosis), and age-related diseases (such as Parkinson's disease, Alzheimer's disease, diabetes). the
例如，该文件提供的方法诊断肺癌的个人，其中包括：a)确定的水平至少一个miRNA的(例如，至少有一个miRNA的表1中所列，或相应的同源基因)的肺组织样本从个人，其中，组织是被怀疑是癌症，和b)与参考水平比较，其中的一个特点变化的水平，表明miRNA的是肺癌。 For example, this document provides a method for diagnosing lung cancer in individuals, which includes: a) determining the level of at least one miRNA (eg, at least one miRNA listed in Table 1, or a corresponding homologous gene) from a lung tissue sample An individual in which the tissue is suspected to be cancerous, and b) compared to a reference level, wherein a characteristically altered level of the miRNA is indicative of lung cancer. the
在一些实施例中，一种方法诊断肺癌的个人可以包括：1)比较的参考水平，至少有一个miRNA的(例如，至少有一个miRNA的表1中所列，或相应的同源基因)的水平，至少在一个miRNA的肺组织样品从个人，其中，组织是被怀疑是癌症，和b)确定是否个人肺癌基于特征变化的水平，至少有一个miRNA的。该方法可以进一步提供了包括肺组织样本从个人和/或孤立微RNA的组织样品。 In some embodiments, a method for diagnosing lung cancer in an individual can include: 1) comparing a reference level of at least one miRNA (eg, at least one miRNA listed in Table 1, or a corresponding homologous gene) Levels of at least one miRNA in a lung tissue sample from an individual, wherein the tissue is suspected of being cancerous, and b) determining whether the individual has lung cancer based on a characteristic change in the level of at least one miRNA. The method may further provide a tissue sample comprising a lung tissue sample from an individual and/or an isolated microRNA. the
该文件还设想了一种提供信息的诊断肺癌的个人，其中包括：a)确定的水平至少一个miRNA的表1中所列，或相应的同源性，在肺组织样本个人，其中该组织被 怀疑癌变，并b)提供信息的水平的miRNA的诊断肺癌，其中一级的miRNA的作为基础，诊断肺癌，和其中的一个特点变化的水平至少有一个指示性的miRNA是肺癌。 This document also envisages providing an informative diagnosis of lung cancer in an individual, including: a) determining the level of at least one miRNA listed in Table 1, or the corresponding homology, in a lung tissue sample of the individual in which the tissue is Carcinogenesis is suspected, and b) provide information on the levels of miRNAs for the diagnosis of lung cancer, wherein a level of miRNA is used as the basis for the diagnosis of lung cancer, and wherein a characteristic change in the level of at least one miRNA is indicative of lung cancer. the
在一些实施例中，在水平至少有一个(例如，一个，两个或三个)的微RNA的序列编号：1-3才能确定，并大幅度增加的数额至少一个测量微RNA可指示肺癌。在水平至少有一个(例如，一个或两个)的微RNA的序列编号：4和5来确定，并大幅度减少的水平至少有一个可衡量的指示微RNA肺癌。在水平至少有一个(例如，一个，两个或三个)的微RNA的序列编号：1-3和至少有一个(例如，一个或两个)的微RNA的序列编号的：4和5才能确定，并大幅度增加的数额至少有一种微RNA的序列编号：1-3和大幅度减少的水平至少有一种微RNA的序列编号：4和5可以表明肺癌。 In some embodiments, levels of at least one (eg, one, two or three) of the microRNA's sequence number: 1-3 can be determined, and a substantially increased amount of at least one of the measured microRNAs can be indicative of lung cancer. Levels of at least one (eg, one or two) of the microRNA sequence numbers: 4 and 5 are determined, and substantially reduced levels of at least one measurable microRNA are indicative of lung cancer. At least one (eg, one, two or three) of the sequence numbers of microRNAs at levels: 1-3 and at least one (eg, one or two) of the sequence numbers of microRNAs: 4 and 5 can be Determined, and substantially increased amounts of at least one microRNA with sequence numbers: 1-3 and substantially reduced levels of at least one microRNA with sequence number: 4 and 5 can indicate lung cancer. the
在一些实施例中，各级各微RNA表1中列出来确定，并大幅度增加的数额至少有一种微RNA的序列编号：1-3，结合大量减少的水平上至少有一个微RNA的序列编号：4和5可以表明肺癌。在某些情况下，大幅度增加各级至少有两个(例如，两个或三个)的微RNA的序列编号：1-3，结合大量减少的水平微RNA酶的序列编号：4和5可以表明肺癌。在某些情况下，大幅度增加各级微RNA的序列编号：1-3，结合大幅度减少的水平微RNA的序列编号：4和5可以表明肺癌。 In some embodiments, the levels of each microRNA listed in Table 1 are identified, and substantially increased amounts have at least one microRNA sequence number: 1-3, combined with substantially reduced levels of at least one microRNA sequence Numbers: 4 and 5 can indicate lung cancer. In some cases, substantially increased levels of at least two (e.g., two or three) microRNAs with sequence numbers: 1-3, combined with substantially reduced levels of microRNA enzymes with sequence numbers: 4 and 5 Can indicate lung cancer. In some cases, substantially increased levels of miRNAs with seq#: 1-3, combined with substantially reduced levels of miRNAs with seq#: 4 and 5 can indicate lung cancer. the
改变各级的miRNA的一个组织样品也可能反映了变化中的miRNA基因。基因状态可以反映，例如，删除或扩增的miRNA基因，或通过改变拷贝数的miRNA的基因。 Altered levels of miRNAs in a tissue sample may also reflect changes in miRNA genes. Gene status can reflect, for example, deletion or amplification of miRNA genes, or by changing copy number of miRNA genes. the
因此，在一些提供了一个方法诊断肺癌的个人，包括分析基因状况至少有一个miRNA的基因(例如，至少有一个基因编码的miRNA列于表1)在肺组织样本被怀疑是癌变在个人，其中一个特点改变基因的相对地位的相应的miRNA基因对照样品可以表明肺癌。在改变基因的地位来决定的基础上删除或放大了的miRNA基因。在某些情况下，基因的变化来确定地位的基础上改变了拷贝数的miRNA基因。 Thus, in some individuals a method for diagnosing lung cancer is provided that includes analysis of the genetic status of at least one miRNA gene (eg, at least one gene encoding an miRNA listed in Table 1) in lung tissue samples suspected to be cancerous in individuals in which A control sample characterized by altered gene relative status to the corresponding miRNA gene can be indicative of lung cancer. Deleted or amplified miRNA genes are determined based on altered gene status. In some cases, genetic changes were determined based on the status of the altered copy number of the miRNA gene. the
该文件还提供了一个方法诊断个人肺癌，包括分析删除或扩增miRNA的基因编码的miRNA列于表1，其中的miRNA基因分析在肺组织样本，被怀疑癌变。缺失或扩增的miRNA基因与相应的miRNA基因控制样品可以表明肺癌。例如，一种方法可以包括分析，至少有一个相应的miRNA基因的miRNA到了核苷酸序列中所列顺序识别码：1，2，3的扩增，其中扩增的miRNA基因与相应的miRNA基因控制样品可以表明肺癌。在一种方法可以包括分析，至少有一个相应的miRNA基因至少有一个miRNA的核苷酸序列中所列顺序识别码：4，5删除，其中删除了相对的miRNA基因相应的miRNA基因控制样品可以表明肺癌。在某些情况下，一种方法可以进一步提供了包括肺组织样本被怀疑癌细胞从个人和/或孤立DNA的肺组织样本。 This document also provides a method for diagnosing lung cancer in individuals, including analysis of deleted or amplified miRNA genes encoding the miRNAs listed in Table 1, wherein the miRNA genes analyzed in lung tissue samples are suspected to be cancerous. Deletion or amplification of miRNA genes with corresponding miRNA gene control samples can indicate lung cancer. For example, a method can include analyzing at least one miRNA corresponding to the miRNA gene for the amplification of the miRNA to the sequence identification code listed in the nucleotide sequence: 1, 2, 3, wherein the amplified miRNA gene is associated with the corresponding miRNA gene Control samples may indicate lung cancer. One method may include analyzing that at least one corresponding miRNA gene has at least one miRNA nucleotide sequence identification code listed in the sequence: 4, 5 deletion, wherein a control sample of the corresponding miRNA gene that is deleted relative to the miRNA gene can be Indicates lung cancer. In some cases, a method may further provide a lung tissue sample comprising DNA from an individual suspected of being cancerous and/or isolated from the lung tissue sample. the
该文件还提供了一个方法诊断肺癌的个人，包括确定基因拷贝数的至少有一个相应的miRNA基因的miRNA列于表1(或相应的同源基因)的肺组织样本，是从个人和被怀疑癌变。其他的副本数目超过两个的miRNA基因位于染色体两种性别或性染色体为女性，以及其他超过的miRNA基因位于性染色体为男性，可以表明肺癌。例如，一个方法可以包括确定该基因拷贝数的至少有一个相应的miRNA基因中的至少一个微RNA中所列顺序编号：1-3样品中的个人，以及拷贝数超过两个对miRNA的基因位于染色体两种性别或性染色体为女性，或一个以上的miRNA的基因位于性染色体为男性，可以表明肺癌。在一些体现，一种方法可以包括确定基因拷贝数的至少有一个相应的miRNA基因中的至少一个微RNA中所列顺序编号：4和5中的示例，并拷贝数少于两个为miRNA的基因位于染色体两种性别或性染色体为女性，或少于1个miRNA的基因位于性染色体为男性，可以表明肺癌。在一些体现，一种方法可以进一步提供了包括肺组织样本被怀疑癌细胞从个人和/或孤立DNA的肺组织样本。 The document also provides a method for diagnosing lung cancer in an individual, including determining the gene copy number for at least one of the corresponding miRNA genes listed in Table 1 (or the corresponding homologous gene) in a lung tissue sample from the individual and suspected cancerous. Other miRNA genes with more than two copy numbers on both sexes or sex chromosomes for females, and other miRNA genes with more than two sex chromosomes for males, can indicate lung cancer. For example, a method may include determining the gene copy number of at least one miRNA gene corresponding to at least one microRNA listed in the sequence number: 1-3 individuals in the sample, and the copy number of more than two pairs of miRNA genes located in Chromosomes for both sexes or sex chromosomes for females, or more than one miRNA gene located on the sex chromosomes for males, can indicate lung cancer. In some embodiments, a method can include determining the gene copy number of at least one microRNA corresponding to at least one miRNA gene listed in the sequence number: 4 and 5 for example, and the copy number is less than two for the miRNA Genes located on chromosomes of both sexes or sex chromosomes for females, or genes with less than 1 miRNA located on the sex chromosomes for males, can indicate lung cancer. In some embodiments, a method may further provide a lung tissue sample comprising DNA from an individual suspected of being cancerous and/or isolated from the lung tissue sample. the
此处所述的微RNA也可用于一个或多个如下：分类肺癌患者，预测风险，肺癌，监测肿瘤进展肺癌患者，并监测治疗的肺癌患者的基础上，各级或更多的微RNA在肺组织样本，或者根据基因地位的一个或多个微RNA在肺组织样本。 The microRNAs described herein can also be used for one or more of the following: classifying lung cancer patients, predicting risk of lung cancer, monitoring tumor progression in lung cancer patients, and monitoring treatment of lung cancer patients based on the level or more of the microRNA in A lung tissue sample, or one or more microRNAs in a lung tissue sample according to gene status. the
任何方法可以进一步披露包括录音或安排记录(例如，通过手写，电脑，或音频手段)有关的个人状况的评估。例如，一个方法可以包括记录有关的信息是否是个人被确定为具有肺癌预后生存的个人，或个人分类到特定的肺癌组。这些步骤将更为详细地讨论如下。 Any method may be further disclosed including recording or arranging to record (eg, by handwritten, computer, or audio means) the assessment of the individual's condition. For example, a method may include recording information pertaining to whether the individual is identified as having a lung cancer prognosis of survival, or the individual is classified into a specific lung cancer group. These steps are discussed in more detail below. the
用于确定肺癌患者预后的材料与方法 Materials and methods for determining the prognosis of patients with lung cancer
这份文件提供的方法来确定预后的肺癌患者，其中包括，例如，确定的生存预后的个人有肺癌方法。预后的方法可用于确定一个适当的疗程为个别有肺癌。例如，测定存活的可能性可以帮助确定是否更为保守或更激进的方法来治疗，应考虑，还是治疗方式应合并。此外，这种预后可以帮助确定是否为改善生存(如所述)可能是必要的和/或有效的。 This document provides methods to determine prognosis for patients with lung cancer, including, for example, methods for determining survival prognosis for individuals with lung cancer. Prognostic methods can be used to determine an appropriate course of treatment for individuals with lung cancer. For example, measuring the likelihood of survival can help determine whether a more conservative or more aggressive approach to treatment should be considered, or if treatment modalities should be combined. In addition, this prognosis can help determine whether improving survival (as described) may be necessary and/or effective. the
在一些实施例中，一种方法，确定预后生存个别有肺癌可以包括：(a)数额确定至少一个的miRNA在肺癌组织样本个人，和(b)水平的比较在样品中的miRNA一个阈值水平，其中的水平相比，miRNA的阈值的相关性或相关成反比的预期生存的个体。由于此处使用“关联”是指一个低级别的miRNA的比较阈值表明低的生存机会为个别有肺癌，反之亦然。由于此处使用“反向关联”是指高水平的miRNA的比较阈值表明成活率低，反之亦然。 In some embodiments, a method of determining the prognosis of survival for an individual with lung cancer may comprise: (a) determining the amount of at least one miRNA in a lung cancer tissue sample of the individual, and (b) comparing the level of the miRNA in the sample to a threshold level, Where levels are compared to miRNA threshold correlations or correlations are inversely proportional to expected survival of individuals. As used here "association" refers to the comparison of a low-level miRNA threshold indicating a low chance of survival for individuals with lung cancer and vice versa. Since "inverse correlation" is used here it is meant that the comparison threshold of high levels of miRNAs indicates low survival and vice versa. the
某些方法，并使用本文描述可以包括确定预后生存的miRNA水平的基础上相对阈值水平。一个起点水平可通过任何一个多元化的方法，但由此产生的阈值水平提供 某种程度的miRNA的上述存在的第一批病人的存活率有不同的存活率第二组患者miRNA的水平低于阈值水平。 Certain methods and uses described herein can include determining prognostic survival based on relative threshold levels of miRNA levels. A starting level can be provided by any one of the multiplex methods, but the resulting threshold level provides some degree of miRNA above the presence of the first group of patients with different survival rates than the second group of patients with miRNA levels below threshold level. the
阈值可以用以下方法来确定，例如，测量的miRNA水平的一个或多个非癌肺癌组织样本。一个起点水平还可以通过分析确定了各级的miRNA在一个人口的个人肺癌。这可以，例如，直方图分析，在整个队列的考验个人介绍，其中一轴代表的水平的miRNA，第二轴代表的存活率个人。两个或多个独立的个人团体可评价鉴定子人口的队列是有着相同或相似的水平的一个特定的miRNA。确定一个阈值然后可以根据miRNA的水平，最好的区分这些不同的群体，或miRNA的水平，最好的区分不同的群体。例如，一个阈值可根据平均值的miRNA的平均水平，一批具有高存活率和平均水平的miRNA的一组存活率低。一个起点水平还可以代表水平的两个或两个以上的微RNA。两个或两个以上的微RNA可以代表，例如，该比例值为每个miRNA的水平。 Threshold values can be determined by, for example, measuring miRNA levels in one or more non-cancerous lung cancer tissue samples. A starting level can also be determined by analyzing the levels of miRNAs in a population of individual lung cancers. This could, for example, be a histogram analysis, where one axis represents the levels of miRNAs and the second axis represents the survival rates of individuals across the entire cohort of test individuals. Two or more independent groups of individuals can be evaluated to identify subpopulation cohorts that share the same or similar levels of a particular miRNA. Determining a threshold can then best discriminate these different populations based on the level of miRNA, or the level of miRNA that best discriminates different populations. For example, a threshold may be based on the average level of the miRNA, a group with high survival rates and the average level of miRNAs in a group with low survival rates. A starting level can also represent levels of two or more microRNAs. Two or more microRNAs can represent, for example, the ratio value for the level of each miRNA. the
阈值可以是一个单一的数字，也同样适用于每个人有肺癌，或依特定亚群的个人。例如，老年男子可能比年轻男人有不同的阈值，女人可能有高于男子不同的阈值水平。此外，阈值可以是一个级别确定的每一个人。例如，一个阈值可在一定比例的miRNA的肺癌组织中的miRNA的相对水平的非癌组织内的同一个人。 The threshold can be a single number, equally applicable to everyone with lung cancer, or to specific subgroups of individuals. For example, older men may have different thresholds than younger men, and women may have higher threshold levels than men. Additionally, thresholds can be determined for each individual at a level. For example, a threshold can be a certain percentage of miRNAs in lung cancer tissue relative to the level of miRNAs in non-cancerous tissue within the same individual. the
核查的阈值水平区分的可能性生存肺癌患者表达低于阈值水平与患者表达上述阈值可以进行单变量或使用多变量分析。这些方法可以用来确定的可能性之间的关系的一个或多个变量和一个特定的结果。在本案中，该方法可以判断的可能性之间的相关性的miRNA水平和无病或总生存率的癌症病人。任何一个多元化的方法，众所周知，那些普通的技巧进行这些分析都可以使用。例子是单变量分析的Kaplan-Meier法和Cox回归模型。 Checking the threshold level for the probability of distinguishing surviving lung cancer patients expressing below the threshold level from patients expressing above the threshold can be performed univariately or using multivariate analysis. These methods can be used to determine the likelihood of a relationship between one or more variables and a particular outcome. In the present case, the method could determine the likelihood of a correlation between miRNA levels and disease-free or overall survival in cancer patients. Any of a variety of methods, well known, and common techniques for performing these analyzes can be used. Examples are the Kaplan-Meier method and Cox regression models for univariate analysis. the
人口为基础的测定阈值水平(例如，通过直方图分析)可以进行使用队列的患者是足够的规模，以确定两个或多个独立团体的患者有不同的miRNA水平。通常情况下，这样一个队列包括，至少有25例(例如，至少有25，30，40，50，60，75，100，125，150，200，或超过200例)。同样，一个队列核查确定阈值水平也至少有25例(例如，至少有25，30，40，50，60，75，100，125，150，200，或超过200例)。 Population-based determination of threshold levels (eg, by histogram analysis) can be performed using cohorts of patients that are of sufficient size to identify two or more independent groups of patients with different miRNA levels. Typically, such a cohort includes at least 25 cases (eg, at least 25, 30, 40, 50, 60, 75, 100, 125, 150, 200, or more than 200 cases). Likewise, a cohort check determines a threshold level of at least 25 cases (eg, at least 25, 30, 40, 50, 60, 75, 100, 125, 150, 200, or more than 200 cases). the
此外，尽管一个单一的阈值可以单独两组患者，有几个可能存在阈值，可以单独多数人口。例如，两个不同的阈值可以第一批患者高水平的miRNA的从第二组病人的中间水平的miRNA，并从第三组病人的低层次的miRNA的。在许多不同的阈值水平就足以取缔曲线，如连续线，它可以描述的可能性，无病或整体存活率的病人作为一个功能的miRNA水平。这种曲线可以构成一个“持续的”miRNA的水平，在那 里的可能性，无病或整体存活率的病人是成正比的miRNA水平。两个或两个以上的miRNA水平可以代表这样的曲线。 Furthermore, although a single threshold can separate two groups of patients, there may be several thresholds that can separate the majority of the population. For example, two different thresholds could be high levels of miRNAs from the first group of patients, intermediate levels of miRNAs from the second group of patients, and low levels of miRNAs from the third group of patients. At many different threshold levels it is sufficient to outlaw the curve, such as a continuous line, which can describe the likelihood of disease-free or overall patient survival as a function of miRNA levels. Such a curve can constitute a "sustained" miRNA level, where the likelihood of disease-free or overall patient survival is directly proportional to the miRNA level. Two or more miRNA levels can represent such a curve. the
在一些实施例中，微RNA可以结合使用，以确定此处提供的生存预后的癌症病人。利用相结合的两个或两个以上的微RNA可以提供更多的预后意义或预后。 In some embodiments, microRNAs can be used in combination to determine the survival prognoses provided herein for cancer patients. The use of two or more microRNAs in combination can provide additional prognostic significance or prognosis. the
miRNA水平还可以肺癌患者无病或整体存活率的统计学的一个指标，包括病理指标(如年龄，肿瘤大小，肿瘤组织学，临床分期，家族病史等)。例如，在临床阶段的癌症可能是一个统计学指标无病或总生存率和阈值可以根据不同的临床阶段。因此，阈值的不同的miRNA可以作为一个功能的指标，统计无病或整体存活率。 The level of miRNA can also be an indicator of the statistics of the disease-free or overall survival rate of lung cancer patients, including pathological indicators (such as age, tumor size, tumor histology, clinical stage, family medical history, etc.). For example, the clinical stage of cancer may be a statistical indicator of disease-free or overall survival and the threshold may vary according to the clinical stage. Therefore, the threshold of different miRNAs can be used as an indicator of function, statistical disease-free or overall survival. the
在某些情况下，卡普兰曲线分析可以用来确定存活率和miRNA的水平之间的相关性。 In some cases, Kaplan curve analysis can be used to determine the correlation between survival and miRNA levels. the
一种方法可以包括：(a)确定的水平，至少有一名的miRNA在肺癌组织中的个人，(b)分类个人属于不是第一或第二组的个人的肺癌，其中第一批的个人水平低的至少一个的miRNA被列为有一个生存的可能性增加，而第二组的个人高水平的至少一个的miRNA。在某些情况下，至少有一名的miRNA可以是hsa-miR-31。 A method may comprise: (a) determining the level of at least one miRNA in an individual in lung cancer tissue, (b) classifying the individual's lung cancer as belonging to an individual other than the first or second group, wherein the level of the first group of individuals Low levels of at least one miRNA were classified as having an increased likelihood of survival, whereas a second group of individuals had high levels of at least one miRNA. In some cases, at least one miRNA can be hsa-miR-31. the
根据各级一个或多个微RNA在病人样本已被确定并比较了阈值的微RNA水平，病人可以分为有一定无病或整体存活率的可能性一组。病人无病或整体存活率的可能性可以在该组无病或整体存活率的病人基础上评估。 Based on the levels of one or more microRNAs in a patient sample that have been identified and compared to threshold microRNA levels, patients can be classified into groups that have a certain likelihood of being disease-free or overall survival. The likelihood of a patient being disease-free or overall surviving can be assessed on the basis of the group of patients who are disease-free or overall surviving. the
例如，一个病人样本可确定某一特定的miRNA为低水平。病人然后可分为一组低水平的miRNA。如果已经建立患者表达水平低的miRNA可增加无病或整体存活率的可能性，具体的癌症患者将被视为有无病或整体存活率的可能性。 For example, a patient sample can be determined to have low levels of a particular miRNA. Patients can then be classified into a group with low levels of miRNAs. A particular cancer patient would be considered as likely to have disease-free or overall survival if it has been established that patients expressing low levels of miRNAs increase the likelihood of disease-free or overall survival. the
此处所述的方法可以进一步包括为个人确定适当的治疗过程的步骤。这些技能可能在估计早期阶段的癌症患者生存预后指标不同于估计晚期阶段的癌症患者生存预后指标。例如，I期预后可能是癌症患者面向持续增长的可能性和/或转移的癌症，而IV期预后病人可面向有效力的治疗方法治疗癌症。因此采取确定适当的治疗要把这些变数考虑进去。 The methods described herein may further comprise the step of determining an appropriate course of treatment for the individual. These skills may be different in estimating survival prognostic indicators for early-stage cancer patients than in late-stage cancer patients. For example, a stage I prognosis may be for a cancer patient towards the likelihood of continued growth and/or metastatic cancer, while a stage IV prognosis may be for an effective treatment for the cancer. These variables must therefore be taken into account to determine appropriate treatment. the
在某些情况下，肺癌预后的测定方法可以包括：(a)确定至少一个miRNA在肺癌组织样本的水平，和(b)miRNA水平与阈值水平比较，其中的水平相比，miRNA的阈值与个人生存反向相关。至少有一名的miRNA可以是hsa-miR-31或相应的同源性。 In some instances, the method for determining prognosis of lung cancer may comprise: (a) determining the level of at least one miRNA in a lung cancer tissue sample, and (b) comparing the miRNA level to a threshold level, wherein the level of the miRNA threshold is compared to that of the individual Survival is inversely correlated. At least one miRNA may be hsa-miR-31 or the corresponding homologue. the
所述的各级微RNA变化也可能反映在微RNA基因状态(如表1和2中列出的微RNA)。在一些实施例中，这里提供了一个确定个别有肺癌预后生存方法，其中包括分析至少有一个miRNA基因的基因状况(如编码基因的hsa-miR-31或相应同源)，与对照样本相比，表明个人相对偏高或偏低的生存机会。例如，提供了一个确定预后 生存方法，其中包括分析了相应的miRNA基因hsa-miR-31的扩增，其中一个扩增的miRNA基因与照样品关联提示低存活率。在一些实施例中，提供了一个确定的生存预后方法，包括确定该miRNA基因相应hsa-miR-31的拷贝数，其中拷贝数量高在表明成活率低。 The microRNA changes at all levels may also be reflected in the microRNA gene status (such as the microRNAs listed in Tables 1 and 2). In some embodiments, provided herein is a method for determining the prognostic survival of individuals with lung cancer, which includes analyzing the genetic status of at least one miRNA gene (such as the gene encoding hsa-miR-31 or the corresponding homologue), compared to a control sample , indicating a relatively high or low chance of survival for an individual. For example, a method for determining prognostic survival is provided that includes analysis of the amplification of the corresponding miRNA gene hsa-miR-31, one of the amplified miRNA genes associated with control samples suggesting low survival. In some embodiments, a method for determining survival prognosis is provided, comprising determining the copy number of the miRNA gene corresponding to hsa-miR-31, wherein a high copy number indicates a low survival rate. the
本文还提供了利用探针，能够探测微RNA水平(或相应的miRNA基因)，并利用包括一个或多个探针确定预后生存。例如，利用一个或多个探针(或一个系统，包括一个或多个探针)，以确定生存的预后，其中探针能检测样品中的miRNA，miRNA的阈值水平的相关性或反向关联可预测个体的生存。一个或多个探针可用于确定预后生存，miRNA的阈值水平的相关性或反向关联可预测个体的生存，其中至少有一个miRNA是hsa-miR-31或相应的同源。 Also provided herein are probes capable of probing microRNA levels (or corresponding miRNA genes) and utilizing one or more probes to determine prognostic survival. For example, using one or more probes (or a system comprising one or more probes) to determine a prognosis of survival, wherein the probes detect miRNAs in a sample, correlation or inverse correlation of threshold levels of miRNAs Can predict individual survival. One or more probes can be used to determine prognostic survival, and correlation or inverse correlation of threshold levels of miRNAs predictive of survival for individuals in which at least one miRNA is hsa-miR-31 or a corresponding homolog. the
在一些实施例中，提供了一个利用一个或多个探针用于制造试剂或系统确定为生存预后的个人有肺癌，其中探针能够检测样品中的miRNA，并在其中的水平相比，miRNA的阈值水平的相关性或反向关联可预测个体的生存。提供了一个利用一个或多个探针用于制造试剂或系统，以确定生存预后的个人有肺癌，其中的水平相比，miRNA的阈值水平的相关性或反向关联可预测个体的生存，其中至少有一人hsa-miR-31或相应的同源。 In some embodiments, there is provided a reagent or system utilizing one or more probes for the manufacture of a reagent or system for determining a prognosis for survival in an individual with lung cancer, wherein the probes are capable of detecting a miRNA in a sample, and comparing the levels therein to the miRNA A threshold level of correlation or inverse association predicts individual survival. Provided is a reagent or system for the manufacture of a reagent or system utilizing one or more probes to determine a prognosis of survival in an individual with lung cancer, wherein a correlation or inverse correlation of the level of a miRNA compared to a threshold level is predictive of survival of the individual, wherein At least one human hsa-miR-31 or corresponding homologue. the
协助医疗和专业人士研究的材料与方法 Materials and methods to assist medical and professional research
该文件还提供了方法和材料，协助医疗或研究人员为确定是否一个人罹患肺癌。医疗专业人员可以，例如，医生，护士，医疗化验师和药剂师。研究人员可以，例如，调查员，研究技术人员，博士后研修生，本科生和研究生。专业可以协助：(1)确定的水平的miRNA在肺组织样本个人，和(2)沟通信息，专业水平。 The document also provides methods and materials to assist medical or research personnel in determining whether a person has lung cancer. Medical professionals can be, for example, doctors, nurses, medical laboratory technologists and pharmacists. Researchers can be, for example, investigators, research technicians, postdoctoral fellows, undergraduates and postgraduates. Professionals can assist in: (1) determining the levels of miRNAs in lung tissue samples to individuals, and (2) communicating information to a professional level. the
在miRNA水平(或地位的相应的miRNA基因)报告后，医疗专业可以采取可能会影响病人的护理一个或一个以上的行动。例如，医疗专业可以在病人的医疗纪录记录miRNA的水平。在某些情况下，医疗专业可以记录诊断肺癌，或以其他方式改变了病人的医疗纪录，以反映患者的医疗条件。在某些情况下，专业医疗可以审查和评价病人的全部医疗记录，并评估多种治疗策略，为临床干预的病人的病情。 After the miRNA level (or status of the corresponding miRNA gene) is reported, the healthcare professional may take one or more actions that may affect the patient's care. For example, a medical professional can record miRNA levels in a patient's medical records. In some cases, a medical professional may record a lung cancer diagnosis, or otherwise alter a patient's medical records to reflect the patient's medical condition. In some cases, a medical professional may review and evaluate a patient's entire medical record and evaluate multiple treatment strategies for clinical intervention in a patient's condition. the
得到病人的miRNA的水平后，医疗人员可以启动或修改治疗肺癌的资料，。在某些情况下，医疗专业可以比较以前的报告中miRNA的水平与最近通报的miRNA的水平，并建议改变治疗。在某些情况下，可以招收医学专业的病人在临床试验新的治疗来干预肺癌。在某些情况下，医疗专业可以选择等待，直到开始治疗的病人症状需要临床干预。 After obtaining the patient's miRNA level, the medical staff can initiate or modify the data for the treatment of lung cancer. In some cases, medical professionals can compare previously reported miRNA levels to recently reported miRNA levels and recommend changes in treatment. In some cases, medical professionals can enroll patients in clinical trials of new treatments to intervene in lung cancer. In some cases, healthcare professionals may choose to wait until the patient's symptoms require clinical intervention to initiate treatment. the
医学人员可以与病人或病人家属就miRNA的水平(或相应的miRNA基因)沟通。在某些情况下，医疗专业人员可以提供一个病人和/或病人家属与肺癌有关得信息，其 中包括治疗方法，预后，并转介到专家，如肿瘤学家。在某些情况下，医疗专业可以为专家提供一份患者病历，与病人或病人家属就miRNA的水平(或相应的miRNA基因)沟通。 Medical personnel can communicate with the patient or the patient's family about the level of the miRNA (or the corresponding miRNA gene). In some cases, a medical professional can provide a patient and/or patient's family with information related to lung cancer, including treatment, prognosis, and referral to specialists such as an oncologist. In some cases, the medical profession may provide the specialist with a copy of the patient's medical records to communicate with the patient or patient's family about the levels of miRNAs (or corresponding miRNA genes). the
研究的人员可以应用有关的miRNA的水平和/或的miRNA基因的地位信息，推动肺癌研究。例如，研究人员可以编译数据，miRNA的水平和/或的miRNA基因地位有关疗效的药物治疗肺癌的症状，以确定一种有效的治疗。在某些情况下，研究的人员能获得一个miRNA的水平来评价一个入组，或继续参与研究或临床试验。在某些情况下，研究的人员进行分类的严重性，基于一个或多个微RNA水平。在某些情况下，可以研究专业有关的题目的miRNA的水平和/或地位的miRNA基因医学专业。在某些情况下，可以参考专业研究的主题，以医疗专业的临床评价肺癌，治疗肺癌的症状。 Researchers can apply information about miRNA levels and/or miRNA gene status to advance lung cancer research. For example, researchers can compile data on miRNA levels and/or miRNA gene status related to the efficacy of drugs to treat lung cancer symptoms in order to identify an effective treatment. In some cases, study personnel can obtain an miRNA level to evaluate a person's enrollment, or continued participation in a research or clinical trial. In some cases, research personnel classified severity based on the level of one or more microRNAs. In some cases, the miRNA levels and/or status of the miRNA gene can be studied professionally on the topic of the medical profession. In some cases, it may be possible to refer to the subject of professional research, clinical evaluation of lung cancer by a medical professional, and treatment of symptoms of lung cancer. the
任何适当的方法可用于将信息传达给其他人。例如，信息可以得到直接或间接地向专业人员。例如，一个实验室技术员的miRNA水平可以输入到一个基于计算机的纪录。在某些情况下，传达的信息是做出实际改变的医疗或研究记录。例如，医疗人员可以与其他医疗专业人员就医疗记录进行沟通。此外，任何类型的通信可以用来沟通信息。例如，邮件，电子邮件，电话和面对面的互动都可以使用。这些信息还可以传递给一个专业的决策信息，以电子方式提供的专业。例如，信息可以传达一个专业的信息给计算机上的数据库，例如，该专业可获取信息。此外，该信息可以传达给医院，诊所，或研究机构作为代理人的医师。 Any suitable method may be used to communicate the information to others. For example, information can be obtained directly or indirectly from professionals. For example, a lab technician's miRNA levels can be entered into a computer-based record. In some cases, the information communicated was medical or research records that actually changed. For example, medical personnel may communicate with other medical professionals regarding medical records. Also, any type of communication may be used to communicate information. For example, mail, email, phone calls, and face-to-face interactions can all be used. This information can also be passed to a professional for decision-making information by providing the professional electronically. For example, information can convey information about a profession to a database on a computer, for example, that profession can obtain information. In addition, this information may be communicated to a physician acting as a proxy for a hospital, clinic, or research institution. the
为改善肺癌患者生存的材料和方法 Materials and methods for improving survival of lung cancer patients
本文提供使用药物改变(例如，增加或减少)的某些微RNA水平改进肺癌患者的生存的方法，包括列在表1和表2微RNA。在此处提供了任何可以增加或减少微RNA的水平的方法。例如，可以用来抑制基因表达的miRNA包括，但不仅限于，双链RNA(例如，短链或小分子干扰RNA或“RNA干扰”)，反义核酸，酶的RNA分子，如核酶，小分子化合物和蛋白质。这些可用于单独或与其他联合应用。可以减少直接的miRNA的水平(例如，通过抑制miRNA的表达或功能)或间接(例如，通过影响的相应的miRNA基因)。 Provided herein are methods of improving survival of lung cancer patients using pharmaceutically altered (eg, increased or decreased) levels of certain microRNAs, including the microRNAs listed in Table 1 and Table 2. Any method that can increase or decrease the level of microRNA is provided herein. For example, miRNAs that can be used to inhibit gene expression include, but are not limited to, double-stranded RNA (e.g., short or small interfering RNA or "RNA interference"), antisense nucleic acids, enzymatic RNA molecules such as ribozymes, small Molecular compounds and proteins. These can be used alone or in combination with others. The level of the miRNA can be reduced directly (eg, by inhibiting the expression or function of the miRNA) or indirectly (eg, by affecting the corresponding miRNA gene). the
改善个人肺癌生存的方法可以包括，例如，应用有效剂量降低了miRNA的水平，与miRNA的阈值水平相比，成反比生存的个体。miRNA的制造药物应用有效剂量降低了miRNA的水平，与miRNA的阈值水平相比，成反比生存的个体。 A method of improving lung cancer survival in an individual can include, for example, administering an effective dose that reduces the level of an miRNA, compared to a threshold level of the miRNA, inversely proportional to the survival of the individual. Application of effective doses of miRNA-manufacturing drugs reduces miRNA levels, compared to threshold levels of miRNA, inversely proportional to survival of individuals. the
在一些实施例中，应用有效剂量降低了miRNA的水平改善个人肺癌生存，降低了水平的miRNA选自hsa-miR-31，hsa-miR-210，hsa-miR-30a，hsa-miR-182，及其相应的同源。miRNA的制造药物应用有效剂量降低了miRNA的水平，其中，miRNA选 自hsa-miR-31，hsa-miR-210，hsa-miR-30a，hsa-miR-182，及其相应的同源。降低hsa-miR-31，或降低hsa-miR-31结合的一个或多个hsa-miR-210，hsa-miR-30a，hsa-miR-182可特别有用。 In some embodiments, application of an effective dose reduces the level of miRNA to improve individual lung cancer survival, and the reduced level of miRNA is selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and its corresponding cognates. Manufacture of miRNAs wherein the miRNA is selected from the group consisting of hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and corresponding homologues using an effective dose to reduce the level of the miRNA. Reducing hsa-miR-31, or one or more of hsa-miR-210, hsa-miR-30a, hsa-miR-182 that reduces hsa-miR-31 binding may be particularly useful. the
此处所述的方法可以进一步包括确定的生存预后的个人(例如，此处所述的方法)之前。 The methods described herein can further comprise prior to determining a survival prognosis for the individual (eg, the methods described herein). the
在一些实施例中，微RNA以上水平可以减少，以改善肺癌病人生存。能做到这一点，例如，一个试剂，降低了两个或两个以上的微RNA水平。另外，两个或两个以上的试剂可以用来减少两个或两个以上的微RNA水平。例如，提供了一个方法，提高肺癌生存，包括减少至少有两个微RNA选自hsa-miR-31，hsa-miR-210，hsa-miR-30a，hsa-miR-182，其相应的同源。提供利用一个或多个制造药物改善肺癌生存，其中减少至少有两个微RNA选自hsa-miR-31，hsa-miR-210，hsa-miR-30a，hsa-miR-182，其相应的同源。在一些实施例中，提供了提高个人肺癌生存的方法，包括减少至少有三个微RNA选自hsa-miR-31，hsa-miR-210，hsa-miR-30a，hsa-miR-182，其相应的同源。提供利用一个或多个制造药物改善肺癌生存，包括减少至少有三个微RNA选自hsa-miR-31，hsa-miR-210，hsa-miR-30a，hsa-miR-182，其相应的同源。 In some embodiments, the above levels of microRNA can be reduced to improve the survival of lung cancer patients. This can be done, for example, with an agent that reduces the levels of two or more microRNAs. Alternatively, two or more reagents can be used to reduce the levels of two or more microRNAs. For example, a method for improving lung cancer survival comprising reducing at least two microRNAs selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and their corresponding homologous . Provided is the use of one or more manufactured drugs to improve lung cancer survival, wherein at least two microRNAs are selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and their corresponding counterparts source. In some embodiments, a method for improving lung cancer survival in individuals is provided, comprising reducing at least three microRNAs selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and their corresponding of the same origin. Provides the use of one or more manufactured drugs to improve lung cancer survival, including reducing at least three microRNAs selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and their corresponding homologs . the
在一些实施例中，在某些情况下，这为促进肺癌患者的存活提供了一个方法，包括个体化使用一种或多种药物的有效剂量，降低hsa-miR-31，hsa-miR-210，hsa-miR-30a，和hsa-miR-182的水平。在某些情况下，这为降低hsa-miR-31，hsa-miR-210，hsa-miR-30a，和hsa-miR-182的水平，促进肺癌患者的存活的药物生产应该使用一种还是多种成分提供了依据。 In some embodiments, in some cases, this provides a method for promoting the survival of lung cancer patients, including individualizing the effective dose of one or more drugs, reducing hsa-miR-31, hsa-miR-210 , hsa-miR-30a, and hsa-miR-182 levels. In some cases, this is whether one or more should be used in the manufacture of drugs that reduce the levels of hsa-miR-31, hsa-miR-210, hsa-miR-30a, and hsa-miR-182 to promote survival in lung cancer patients ingredients provided the basis. the
在一些实施例中，在某些情况下，这里提供了一个药物组成，包括能够降低某个miRNA的水平的药剂和一种在治药学上可接受的载体，其中至少有一个miRNA是hsa-miR-31，hsa-miR-210，hsa-miR-30a，或hsa-miR-182。在某些情况下，至少一个miRNA是hsa-miR-31。在某些情况下，至少一个miRNA是hsa-miR-210。在某些情况下，至少一个miRNA是hsa-miR-30a。在某些情况下，至少一个miRNA是hsa-miR-182。在某些情况下，此药剂可以是双链RNA(例如，短的或小干扰RNA，或“siRNA”)，核酸的反义链，或具有酶活性性的RNA分子比如核酶。促进存活的方法和药物将在此详述。 In some embodiments, in some cases, there is provided a pharmaceutical composition comprising an agent capable of reducing the level of an miRNA and a pharmaceutically acceptable carrier, wherein at least one miRNA is hsa-miR -31, hsa-miR-210, hsa-miR-30a, or hsa-miR-182. In certain instances, at least one miRNA is hsa-miR-31. In certain instances, at least one miRNA is hsa-miR-210. In certain instances, at least one miRNA is hsa-miR-30a. In certain instances, at least one miRNA is hsa-miR-182. In certain instances, the agent can be double-stranded RNA (eg, short or small interfering RNA, or "siRNA"), the antisense strand of a nucleic acid, or an enzymatically active RNA molecule such as a ribozyme. Methods and agents to promote survival are described in detail herein. the
存活的情况可以分为两种：一种是无疾病的存活，另一种是全面存活。“无疾病存活”指的是个体诊断后没有肿瘤复发和/或发展，例如，一个肿瘤没有复发的患者。“全面存活”指的是个体诊断后的存活时间，无论其肿瘤复发与否。 Survival can be divided into two categories: one is disease-free survival and the other is overall survival. "Disease-free survival" refers to the absence of tumor recurrence and/or progression after diagnosis in an individual, eg, a patient whose tumor has not recurred. "Overall survival" refers to the time an individual survives after diagnosis, whether or not their tumor has recurred. the
在某些情况下，一个特定的miRNA的表达能被诱导的针对此miRNA的RNA干扰抑制。RNA干扰是通过加入分离的与miRNA基因的一部分有至少70％(例如至少 70％，75％，80％，85％，90％，95％，98％，99％，或100％)同源性的双链RNA(dsRNA)产品来实现的。在某些情况下，这个dsRNA分子可以是“短的或小干扰RNA”或“siRNA”。 In some cases, the expression of a specific miRNA can be inhibited by RNA interference induced against that miRNA. RNA interference is by adding a portion of the isolated miRNA gene that has at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) homology This is achieved by double-stranded RNA (dsRNA) products. In certain instances, this dsRNA molecule can be a "short or small interfering RNA" or "siRNA." the
可以有效用于当前这些方法的siRNA可以是10-30个核苷酸长度的短双链RNA(例如，约12-28，14-26，16-24，或18-22核苷酸)。siRNA可以含有一条正义RNA链和一条互补配对的反义RNA链，二者按照Watson-Crick碱基互补配对原则通过退火形成双链。正义链含有与靶标miRNA基本相同的核酸序列。siRNA的正义链和反义链可以由互补配对的两条单链RNA组成，也可以由一个分子互补配对的两个部分通过单链的“发卡”结构连在一起组成。 siRNAs useful in the current methods can be short double-stranded RNAs of 10-30 nucleotides in length (eg, about 12-28, 14-26, 16-24, or 18-22 nucleotides). siRNA can contain a sense RNA strand and a complementary paired antisense RNA strand, which form a double strand by annealing according to the Watson-Crick base pairing principle. The sense strand contains substantially the same nucleic acid sequence as the target miRNA. The sense strand and antisense strand of siRNA can be composed of two complementary paired single-stranded RNAs, or can be composed of two complementary parts of a molecule linked together through a single-stranded "hairpin" structure. the
通过一个或多个核苷酸的插入、缺失、替换和/或转换，siRNA可能与自然形成的RNA不同。这些改变包括非核苷酸物质的加入(如加在siRNA的末端或内部)，导致siRNA能抵抗核酶消化的修饰，或者将siRNA中的一个或多个核苷酸替换为脱氧核苷酸。在某些情况下，siRNA的一条或两条链可以包含3’突出末端。siRNA可以通过化学或生物学的方法得到，或者从重组的质粒或病毒载体表达得到，这将在下文进行阐述。 siRNAs may differ from naturally occurring RNAs by insertions, deletions, substitutions and/or transitions of one or more nucleotides. These changes include the addition of non-nucleotide substances (such as added to the end or inside of the siRNA), modifications that make the siRNA resistant to ribozyme digestion, or the replacement of one or more nucleotides in the siRNA with deoxynucleotides. In some cases, one or both strands of the siRNA may contain 3' overhangs. siRNA can be obtained by chemical or biological methods, or expressed from recombinant plasmids or viral vectors, which will be described below. the
一个特定的miRNA的表达也能被反义核酸抑制。在此，“反义核苷酸”指的是能够通过RNA-RAN或RNA-DNA相互作用的方式结合目标RNA，从而改变目标RNA活性的核酸分子。适用于当前方法的反义核苷酸可以是包含与miRNA毗邻序列互补的单链核酸(如RNA，DNA，RNA-DNA嵌合体，PNA和LNA)。在某些情况下，反义核酸包含与miRNA毗邻序列至少有70％(例如至少70％，75％，80％，85％，90％，95％，98％，99％，或100％)互补配对的核酸序列。在某些情况下，反义核酸的长度大约为10-30核苷酸(例如约12-28，14-26，16-24，或18-22核苷酸)。 The expression of a specific miRNA can also be inhibited by antisense nucleic acids. Here, "antisense nucleotide" refers to a nucleic acid molecule that can bind to a target RNA through RNA-RAN or RNA-DNA interaction, thereby changing the activity of the target RNA. Antisense nucleotides suitable for use in the present method may be single-stranded nucleic acids (such as RNA, DNA, RNA-DNA chimeras, PNA and LNA) that contain complementarity to adjacent sequences of the miRNA. In certain instances, the antisense nucleic acid comprises at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) complementarity to a sequence adjacent to the miRNA paired nucleic acid sequences. In certain instances, the antisense nucleic acid is about 10-30 nucleotides in length (eg, about 12-28, 14-26, 16-24, or 18-22 nucleotides). the
反义核酸也可以包含对核酸骨架或糖和碱基(或它们的等价物)的修饰，从而增强靶标特异性、核酸酶抵抗能力、运输或其他功效相关的特征。这些修饰包括胆固醇半族，双螺旋插入物如吖啶，或者一个或多个具有核酸酶抗性的组分。 Antisense nucleic acids may also contain modifications to the nucleic acid backbone or sugars and bases (or their equivalents) to enhance target specificity, nuclease resistance, trafficking or other efficacy-related characteristics. These modifications include cholesterol moieties, duplex insertions such as acridine, or one or more nuclease-resistant components. the
反义核酸可以通过化学或生物学的方法得到，或者从重组的质粒或病毒载体表达得到，这将在下文进行阐述。 Antisense nucleic acids can be obtained chemically or biologically, or expressed from recombinant plasmids or viral vectors, as will be described below. the
一个特定的miRNA的表达也能被具有酶活性的核酸抑制。在此，“具有酶活性的核酸”指的是含有地物结合区域的核酸，该区域与miRNA的毗邻序列互补配对，可以特异性地剪切miRNA。在某些情况下，具有酶活性的核酸结合区域与miRNA毗邻序列有50-100％的互补性(例如75-95％互补性或95-100％互补性)。一个酶活性的核酸也可以在碱基、糖、磷酸组分上有修饰。一个典型的可以用于当前方法的具有酶活性的核酸就是核酶。 The expression of a specific miRNA can also be inhibited by an enzymatically active nucleic acid. Here, "nucleic acid with enzymatic activity" refers to a nucleic acid that contains an object-binding region that is complementary to an adjacent sequence of miRNA and can specifically cleave miRNA. In certain instances, the enzymatically active nucleic acid binding region is 50-100% complementary (eg, 75-95% complementary or 95-100% complementary) to a contiguous sequence of the miRNA. An enzymatically active nucleic acid can also have modifications in base, sugar, or phosphate moieties. A typical enzymatic nucleic acid that can be used in the current method is a ribozyme. the
酶活性的核酸可以通过化学或生物学的方法得到，或者从重组的质粒或病毒载体表达得到，这将在下文进行阐述。 Nucleic acids with enzymatic activity can be obtained by chemical or biological methods, or expressed from recombinant plasmids or viral vectors, which will be described below. the
当前有很多方法可以将核酸分子导入到细胞中，包括癌细胞。这些方法包括显微注射，电穿孔，脂质体转染，磷酸钙介导的转染，DEAE葡聚糖介导的转染，微粒子轰击法，通过胶态分散体运输(例如大分子复合物，凝胶颗粒，水油乳化剂，胶态离子，混合胶态离子和脂质体)，以及与抗体、短杆菌肽、人造病毒外壳或其它细胞内载体如TAT偶联。 There are currently many methods for introducing nucleic acid molecules into cells, including cancer cells. These methods include microinjection, electroporation, lipofection, calcium phosphate-mediated transfection, DEAE-dextran-mediated transfection, microparticle bombardment, transport by colloidal dispersion (e.g. macromolecular complex , gel particles, water-oil emulsifiers, colloidal ions, mixed colloidal ions and liposomes), and conjugation with antibodies, gramicidin, artificial virus coats or other intracellular carriers such as TAT. the
核酸药剂也可以通过文献中已知的载体导入到或体外或活体内的哺乳动物细胞中。合适的载体有病毒载体和非病毒载体，如质粒载体。这些载体在提供诸如反义RNA或siRNA等药剂的治疗剂量上很有帮助。 Nucleic acid agents can also be introduced into mammalian cells either in vitro or in vivo by vectors known in the literature. Suitable vectors are viral vectors and non-viral vectors, such as plasmid vectors. These vectors are helpful in delivering therapeutic doses of agents such as antisense RNA or siRNA. the
基于病毒的系统的优势在于能够相对高效地导入异源的核酸到各种各样的细胞中。导入核酸的合适的病毒载体有单纯疱疹病毒载体，牛痘病毒载体，细胞巨化病毒载体，鼠莫洛尼氏白血病毒载体，腺病毒载体，腺关联病毒载体，逆转录病毒载体和慢病毒。病毒载体的趋向性可以通过使用其它病毒的包膜蛋白或表面抗原来控制。例如，腺关联病毒载体可以假借口腔小泡病毒，狂犬病毒，埃博拉病毒，Mokola等病毒的表面蛋白来假冒这些病毒。 An advantage of virus-based systems is the relatively efficient introduction of heterologous nucleic acids into a wide variety of cells. Suitable viral vectors for introducing nucleic acid are herpes simplex virus vectors, vaccinia virus vectors, cytomegalovirus vectors, murine Moloney leukemia virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors and lentiviruses. The tropism of viral vectors can be controlled by using envelope proteins or surface antigens of other viruses. For example, adeno-associated virus vectors can fake oral vesicle virus, rabies virus, Ebola virus, Mokola virus, etc. by using surface proteins of these viruses. the
核酸或载体中可以含有任意一种可诱导的启动子或增强子，从而可以通过刺激或添加分子诱导反义RNA或siRNA的表达。这些诱导系统包括诸如四环素诱导系统，重金属诱导的金属硫蛋白启动子，蜕皮激素或相关的类固醇如鼠甾酮应答的昆虫类固醇激素，类固醇如糖皮质激素和雌激素诱导的小鼠乳腺肿瘤病毒，以及温度改变诱导的热激启动子。 The nucleic acid or vector may contain any inducible promoter or enhancer, so that the expression of antisense RNA or siRNA can be induced by stimulating or adding molecules. These induction systems include insect steroid hormones such as tetracycline-inducible systems, heavy metal-inducible metallothionein promoters, ecdysone or related steroids such as murine steroids, mouse mammary tumor virus induced by steroids such as glucocorticoids and estrogens, and heat shock promoters induced by temperature changes. the
如果一个药物的剂量能够足以改变其靶标miRNA的水平(如降低)，那么这个剂量可以说是此药物的有效剂量。在某些情况下，一种药物可以将靶标miRNA的水平至少降低miRNA水平与极限水平间差异的10％(例如，至少10％，20％，30％，40％，50％，或大于50％)。此处提供的药物(如核酸药物)的典型剂量包括0.1-3000mg/kg体重，10-2000mg/kg体重，50-1000mg/kg体重，以及100-500mg/kg体重，但并不局限于这些范围。在某些情况下，药物(如核酸药物)的剂量是100-500mg/g肿瘤重量(例如大约20-300mg/g肿瘤重量，50-200mg/g肿瘤重量，或100-150mg/g肿瘤重量)。 If the dose of a drug is sufficient to change the level of its target miRNA (eg, decrease), then this dose can be said to be the effective dose of the drug. In some instances, a drug can reduce the level of a target miRNA by at least 10% of the difference between the miRNA level and the threshold level (e.g., at least 10%, 20%, 30%, 40%, 50%, or greater than 50%) ). Typical doses of the drugs provided herein (such as nucleic acid drugs) include 0.1-3000 mg/kg body weight, 10-2000 mg/kg body weight, 50-1000 mg/kg body weight, and 100-500 mg/kg body weight, but are not limited to these ranges . In some instances, the dose of drug (eg, nucleic acid drug) is 100-500 mg/g tumor weight (eg, approximately 20-300 mg/g tumor weight, 50-200 mg/g tumor weight, or 100-150 mg/g tumor weight) . the
在此领域内一个常用的技巧可以确定给个体一种或多种药物的合适剂量。典型的给药频率包括并仅限于，至少每月一次，至少每三周一次，每两周一次，每周一次，每周两次，每周三次，每周四次，每周五次，每周六次，或每天一次，或更频繁。在某些情况下，两次给药的间距可以少于一周(例如少于每六、五、四、三、二或一天)。在某些情况下，两次给药的间距是固定的。例如，可以每天，每两天，每三天，每四 天，每五天，每六天或每周给药一次。在某些情况下，可以每天给药两次，三次或更频繁。 It is a common technique in the art to determine the appropriate dosage of one or more drugs for an individual. Typical dosing frequencies include, but are limited to, at least once a month, at least once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, Saturdays, or once a day, or more often. In certain instances, the interval between administrations may be less than one week (eg, less than every six, five, four, three, two, or one day). In some cases, the interval between two doses is fixed. For example, administration can be done daily, every two days, every three days, every four days, every five days, every six days, or once a week. In some instances, two, three or more frequent daily doses may be administered. the
一种药物的给药可以是长期的，诸如从大约一个月到到三年。例如一个给药体系可以长达2、3、4、5、6、7、8、9、10、11、12、18、24、30或36个月。在某些情况下，在给药期间给药不能停。在某些情况下，每两次给药的间距不能长于一周。 Administration of a drug can be long-term, such as from about one month to up to three years. For example, a dosing system can be for up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 or 36 months. In some cases, the dosing cannot be stopped during the dosing period. In some cases, the interval between the two doses cannot be longer than one week. the
此处描述的药物可以通过此领域内的任意途径给个体给药，包括并仅限于，静脉内，腹腔内，眼内，动脉内，肺内，口服，肺泡内，肌肉，呼吸管，皮下，脑脊髓膜内，跨皮肤，跨胸膜，局部的，吸入(如喷剂)，跨粘膜(如通过鼻粘膜)，通过肠胃，关节内，尿道内，心室内，直肠内(如通过栓剂)，阴道内(如通过阴道栓剂)，颅骨内，肝内，以及肿瘤内。在某些情况下，可以全身性的给药。在某些情况下，可以局部给药。 The drugs described herein may be administered to a subject by any route known in the art, including, but not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intraalveolar, intramuscular, respiratory, subcutaneous, Intracerebrospinal, transcutaneous, transpleural, topical, inhalation (eg, spray), transmucosal (eg, via nasal mucosa), gastrointestinal, intra-articular, intraurethral, intraventricular, intrarectal (eg, via suppositories), Intravaginally (eg, via pessary), intracranial, intrahepatic, and intratumoral. In certain instances, systemic administration can be used. In some cases, topical administration can be used. the
这里还提供了由治药学尚可接受的载体和能改变(如下调)miRNA水平的试剂组成的复合药物。在某些情况下，药物复合物包含一种成分，此成分可以降低hsa-miR-210或hsa-miR-30a或它们的同源物的水平，它可以是siRNA，反义RNA或核酶。 Also provided herein is a compound drug consisting of a pharmaceutically acceptable carrier and an agent capable of altering (eg down-regulating) miRNA levels. In certain instances, the drug complex comprises a component that reduces the level of hsa-miR-210 or hsa-miR-30a or their homologues, which can be siRNA, antisense RNA or ribozyme. the
在某些情况下，药物复合物是无菌的。在某些情况下，药物复合物是无热源的。合适的治药学上可接受的载体有水、水溶液、标准盐溶液，0.4％的盐溶液，0.3％的甘氨酸和透明质酸。药物复合物还可以包含传统的药物赋形剂和/或添加剂。合适的药物赋形剂包括稳定剂、抗氧化剂、渗透性调节剂、缓冲液、pH调节剂。合适的添加剂包括，并仅限于，生理学上不排斥的缓冲液，螯合掩蔽剂(如DTPA和DTPA双酰胺)以及钙螯合复合物(如钙DTPA和CaNaDTPA双酰胺)，钙盐或钠盐(如氯化钙、抗坏血酸钙、葡萄糖酸钙和乳酸钙)。药物复合物可以是液体包装也可以是冻干品。 In some instances, the drug complex is sterile. In some instances, the drug complex is non-pyrogenic. Suitable pharmaceutically acceptable carriers are water, aqueous solution, normal saline solution, 0.4% saline solution, 0.3% glycine and hyaluronic acid. The drug complex may also contain conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolarity regulators, buffers, pH regulators. Suitable additives include, and are limited to, physiologically non-exclusive buffers, chelation masking agents (such as DTPA and DTPA bisamide) and calcium chelating complexes (such as calcium DTPA and CaNaDTPA bisamide), calcium or sodium salts (such as calcium chloride, calcium ascorbate, calcium gluconate, and calcium lactate). The drug complex can be packaged as a liquid or as a lyophilized product. the
对于固体药物复合物，可以使用制药学上可接受的传统的无毒固体载体。药学上可接受的固体载体包括制药级别的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、滑石、纤维素、葡萄糖、蔗糖、碳酸镁等等。 For solid drug complexes, pharmaceutically acceptable traditional non-toxic solid carriers can be used. Pharmaceutically acceptable solid carriers include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. the
miRNA表达水平和/或miRNA基因状况检测系统 miRNA expression level and/or miRNA gene status detection system
这里还提供了包含用于检测表1和表2中的miRNA的引物和探针(如寡核苷酸)的系统(如实时定量PCR(qPCR)引物，原位杂交，或微阵列)。包含引物和/或探针的系统(qPCR引物，原为杂交，或微阵列)可以检测相应的miRNA基因或它们的同源物的状况。这些系统在确定表1和表2中的miRNA或它们的同源物的表达水平以及肺癌评定中很有帮助。这里的一些讨论集中在miRNA的检测系统上，但是熟悉此领域的常用技巧的人很容易理解这些描述同样适用于miRNA的基因状况检测。 Also provided herein are systems (eg, real-time quantitative PCR (qPCR) primers, in situ hybridization, or microarrays) comprising primers and probes (eg, oligonucleotides) for detecting the miRNAs in Tables 1 and 2. A system comprising primers and/or probes (qPCR primers, formerly hybridization, or microarray) can detect the status of the corresponding miRNA genes or their homologues. These systems are helpful in determining the expression levels of the miRNAs in Tables 1 and 2 or their homologues and lung cancer assessment. Some discussions here focus on the detection system of miRNA, but those who are familiar with common techniques in this field can easily understand that these descriptions are also applicable to the detection of gene status of miRNA. the
这里提供的系统包括检测miRNA和/或检测其基因状况的引物和/或探针。这里 的一些讨论集中在miRNA的检测系统上，但是熟悉此领域的常用技巧的人很容易理解这些描述大部分同样适用于包含检测基因缺失、扩增和/或miRNA基因拷贝数的变化(这里统称为miRNA的基因状况)的引物和/或探针的系统。 The systems provided herein include primers and/or probes to detect miRNAs and/or to detect their genetic status. Some of the discussions here focus on detection systems for miRNAs, but those familiar with common techniques in this field will easily understand that most of these descriptions are also applicable to detection of gene deletions, amplifications, and/or changes in miRNA gene copy number (collectively referred to here as A system of primers and/or probes for miRNA gene status). the
至少检测样品中表1和表2所列的一个miRNA(或相应miRNA的基因状况)的系统(例如多引物混合物)可以包括一对或多对引物，其中每对引物可以扩增样品(如生物学样品)中的一个特定的miRNA，假设此miRNA在样品中存在的话。 A system (e.g., a multi-primer mix) for detecting at least one miRNA listed in Tables 1 and 2 (or the genetic status of the corresponding miRNA) in a sample can include one or more pairs of primers, wherein each pair of primers can amplify a sample (e.g., a biological a specific miRNA in a chemical sample), assuming that the miRNA is present in the sample. the
至少检测样品中表1和表2所列的一个miRNA(或相应miRNA的基因状况)的系统(例如定量PCR的引物、原位杂交和微阵列芯片的探针)可以包括多对引物和/或探针，其中每对引物和/或探针可以检测肺组织样品中的一个miRNA(或相应的miRNA基因状况)，同时，至少15％(例如至少15％，20％，25％，30％，40％，50％，60％，70％，80％，90％，95％，或大于95％)的引物和/或探针可以检测表1和表2所列的miRNA或它们的同源物(或确定相应miRNA的基因状况)。在某些情况下，一对引物和/或探针可以检测肺组织样品中不同的miRNA(或相应miRNA的基因状况)。 A system (such as primers for quantitative PCR, probes for in situ hybridization and microarray chips) that detects at least one miRNA listed in Table 1 and Table 2 (or the genetic status of the corresponding miRNA) in a sample may include multiple pairs of primers and/or Probes, wherein each pair of primers and/or probes can detect one miRNA (or corresponding miRNA gene status) in the lung tissue sample, and at least 15% (such as at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) of the primers and/or probes can detect the miRNAs listed in Table 1 and Table 2 or their homologues (or determine the genetic status of the corresponding miRNA). In certain instances, a pair of primers and/or probes can detect different miRNAs (or the genetic status of the corresponding miRNAs) in a lung tissue sample. the
在某些情况下，用于诊断个体肺癌的系统(如微阵列)可以包括多对引物和/或探针，其中每对引物和/或探针可以检测个体样品中的一个miRNA(或相应的miRNA基因状况)，同时，至少15％(例如至少15％，20％，25％，30％，40％，50％，60％，70％，80％，90％，95％，或大于95％)的引物和/或探针可以检测表1和表2所列的miRNA或它们的同源物(或相应miRNA的基因状况)。在某些情况下，肺癌诊断系统可以包括至少一(如一、二、三、四或五)对引物，其中每对引物可以检测表1和表2所列的miRNA或它们的同源物，或相应miRNA的基因状况。在某些情况下，肺癌诊断系统(如微阵列)可以包括至少一(如一、二、三、四或五)种探针，其中每种探针可以检测表1和表2所列的miRNA或它们的同源物，或相应miRNA的基因状况。在某些情况下，系统中的一对引物或探针可以检测肺组不同的miRNA或不同miRNA的基因状况(例如表1和表2所列的不同miRNA或相应的同源物，或相应的基因)。这里提供的系统(如微阵列)将在下文进行详细的阐述。 In some cases, a system (such as a microarray) for diagnosing lung cancer in an individual can include multiple pairs of primers and/or probes, wherein each pair of primers and/or probes can detect one miRNA (or the corresponding miRNA) in the individual sample. miRNA gene status), and at least 15% (e.g., at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) ) primers and/or probes can detect the miRNAs listed in Table 1 and Table 2 or their homologues (or the gene status of the corresponding miRNAs). In some cases, the lung cancer diagnostic system can include at least one (such as one, two, three, four or five) pairs of primers, wherein each pair of primers can detect the miRNAs listed in Table 1 and Table 2 or their homologues, or The gene status of the corresponding miRNA. In some cases, a lung cancer diagnostic system (such as a microarray) can include at least one (such as one, two, three, four or five) probes, wherein each probe can detect the miRNAs listed in Table 1 and Table 2 or their homologues, or the genetic status of the corresponding miRNA. In some cases, a pair of primers or probes in the system can detect different miRNAs or the gene status of different miRNAs in the lung group (such as the different miRNAs listed in Tables 1 and 2 or the corresponding homologues, or the corresponding Gene). The systems provided herein (eg, microarrays) are described in detail below. the
这里还提供了多种系统用于肺癌诊断。例如，使用一个系统进行肺癌诊断，此系统中包括至少一(如一、二、三、四或五)对引物和/或探针(如寡核苷酸)，其中每对引物或探针可以检测表1和表2所列的miRNA或它们的同源物，或相应miRNA的基因状况。在某些情况下，系统中的一对引物或探针可以检测表1和表2所列的不同的miRNA或其对应的同源物的水平，或不同miRNA的基因水平。至少一个表1和表2中所列的miRNA或其同源物，或相应miRNA基因的表达水平的特征性变化都可能预示着肺癌。 Also provided herein are various systems for lung cancer diagnosis. For example, lung cancer diagnosis is performed using a system comprising at least one (such as one, two, three, four or five) pairs of primers and/or probes (such as oligonucleotides), wherein each pair of primers or probes can detect The miRNAs listed in Table 1 and Table 2 or their homologues, or the gene status of the corresponding miRNAs. In some cases, a pair of primers or probes in the system can detect the levels of different miRNAs listed in Table 1 and Table 2 or their corresponding homologues, or the gene levels of different miRNAs. Characteristic changes in the expression levels of at least one of the miRNAs listed in Tables 1 and 2, or their homologues, or corresponding miRNA genes may be indicative of lung cancer. the
这里还提供了将系统(如用于qPCR的一对或多对引物，用于原位杂交的探针混 合物，或微阵列)用于肺癌诊断的方法。这些系统由多对引物和/或探针组成，其中每对引物和/或探针可以检测样品中的不同miRNA(或相应的miRNA基因状况)，同时，至少15％(例如至少15％，20％，25％，30％，40％，50％，60％，70％，80％，90％，95％，或大于95％)的引物和/或探针可以检测表1和表2所列的miRNA或它们的同源物，或相应miRNA的基因状况。至少一个miRNA或其同源物，或相应miRNA基因的表达水平的特征性变化都可能预示着肺癌。 Also provided herein are methods of using a system (such as one or more pairs of primers for qPCR, a probe mix for in situ hybridization, or a microarray) for lung cancer diagnosis. These systems are made up of multiple pairs of primers and/or probes, wherein each pair of primers and/or probes can detect different miRNAs (or corresponding miRNA gene status) in the sample, and at the same time, at least 15% (such as at least 15%, 20 %, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%) primers and/or probes can detect the listed in Table 1 and Table 2 miRNAs or their homologues, or the genetic status of the corresponding miRNAs. Characteristic changes in the expression levels of at least one miRNA or its homologues, or corresponding miRNA genes, may be indicative of lung cancer. the
这里还提供了运用检测miRNA的引物和探针制造系统的方法。在某些情况下，这里提供了一个将一对或多对引物或探针(如寡核苷酸)用于制造肺癌个体检测系统的方法。其中每对引物或探针可以检测表1和表2所列的一个miRNA或其对应的同源物的水平，或不同miRNA的基因水平。在某些情况下，这里提供了一个将一对或多对引物或探针(如寡核苷酸)用于制造肺癌检测系统(如微阵列)的方法。其中每对引物或探针可以检测样品中不同的miRNA或其对应的同源物的水平，或不同miRNA的基因水平，至少15％(例如至少15％，20％，25％，30％，40％，50％，60％，70％，80％，90％，95％，或大于95％)的探针可以检测表1和表2所列的miRNA或它们的同源物，或相应miRNA的基因状况。 Also provided herein are methods of making systems using primers and probes for detecting miRNAs. In some cases, provided herein is a method of using one or more pairs of primers or probes (eg, oligonucleotides) in the manufacture of an individual detection system for lung cancer. Wherein each pair of primers or probes can detect the level of a miRNA listed in Table 1 and Table 2 or its corresponding homologue, or the gene level of different miRNAs. In some cases, provided herein is a method of using one or more pairs of primers or probes (eg, oligonucleotides) to make a lung cancer detection system (eg, a microarray). Wherein each pair of primers or probes can detect the levels of different miRNAs or their corresponding homologs in the sample, or the gene levels of different miRNAs, at least 15% (such as at least 15%, 20%, 25%, 30%, 40% %, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) probes can detect the miRNAs listed in Table 1 and Table 2 or their homologs, or the corresponding miRNA genetic condition. the
这里提供的系统可以包含两个或多个检测相同miRNA的引物。例如，在某些情况下(当系统是微阵列时)，微阵列中的探针可以是多拷贝的(可以是二、三、四、五、六、七或更多拷贝)。在某些情况下，一个系统可以包含检测相同miRNA的不同探针。例如，两个或多个引物可能结合于同一个miRNA的不同(重叠或不重叠)的区域。 The systems provided herein can contain two or more primers that detect the same miRNA. For example, in some cases (when the system is a microarray), the probes in the microarray may be in multiple copies (can be two, three, four, five, six, seven or more copies). In some cases, a system can contain different probes that detect the same miRNA. For example, two or more primers may bind to different (overlapping or non-overlapping) regions of the same miRNA. the
能够检测miRNA水平的任意引物或探针都可以使用。在某些情况下，引物或探针可以是寡核苷酸。希望能被理解的是检测miRNA的一些序列有所变化是可以接受的。因此，寡核苷酸序列(或它们的互补序列)可能会与此处描述的miRNA序列有所不同。熟悉此领域的技术的人很容易理解序列变化并不会显著影响寡核苷酸检测miRNA的水平。例如，当用上文描述的方法比较时，同源的和改变的寡核苷酸分子可以持有相对高度的序列相同性。这里包含的寡核苷酸序列与此处描述的miRNA至少具有40％(如至少40％，50％，60％，70％，80％，90％，95％，或大于95％)的序列同源性。在某些情况下，寡核苷酸可以包含两部分，第一部分用来检测miRNA，而第二部分连接到基质上。在某些情况下，第二部分可能包含非特异的序列(如polyT)来增加互补序列与基质表面间的距离。 Any primer or probe capable of detecting miRNA levels can be used. In some cases, primers or probes can be oligonucleotides. It is hoped that it will be understood that some variation in the sequence of the detected miRNA is acceptable. Accordingly, the oligonucleotide sequences (or their complements) may vary from the miRNA sequences described herein. Those skilled in the art will readily appreciate that sequence variations do not significantly affect the level of miRNA detected by the oligonucleotide. For example, homologous and altered oligonucleotide molecules may hold a relatively high degree of sequence identity when compared using the methods described above. The oligonucleotide sequences contained herein are at least 40% (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) sequence identical to the miRNAs described herein source. In some cases, the oligonucleotide can contain two parts, the first part is used to detect the miRNA, and the second part is attached to the substrate. In some cases, the second part may contain non-specific sequences (such as polyT) to increase the distance between the complementary sequence and the substrate surface. the
这里描述的系统中的寡核苷酸可以包含DNA、RNA、PNA、LNA、联合其中几种、和/或联合后加以修饰。寡核苷酸还可以包括修饰后的骨架。在某些情况下，寡核苷酸包括至少9，10，11，12，13，14，15，16，17，18，19，20，或多于20个连续的核苷酸片段，这些片段与此处描述的miRNA序列互补或相同。一个寡核苷酸可以包含两个或 多种这种互补序列。在某些情况下，寡核苷酸的5’或3’末端可以有一个活性基团(如胺)，将寡核苷酸连接到基质上。 The oligonucleotides in the systems described here can comprise DNA, RNA, PNA, LNA, combinations of several of these, and/or be modified after combination. Oligonucleotides may also include modified backbones. In some cases, the oligonucleotide comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 contiguous stretches of nucleotides that are Complementary or identical to the miRNA sequences described herein. An oligonucleotide may contain two or more such complementary sequences. In some cases, the 5' or 3' end of the oligonucleotide can have a reactive group (such as an amine) that attaches the oligonucleotide to the substrate. the
在某些情况下，一个系统可以包含一对或多对qPCR(也称为real time PCR(RT-PCR)或动力学聚合酶链式反应)的引物。qPCR可以同时对样品中特异的DNA序列进行检测和定量，定量是绝对的拷贝数或者用加入的DNA总量或其它校正基因校正后相对的量。qPCR的一个重要特征就是实时定量在扩增过程中随着循环增加的DNA产物。定量的方法包括使用插入双链DNA的荧光染料，以及当与互补DNA杂交是发荧光的修饰的DNA寡核苷酸探针。qPCR的方法在此领域内是广为人知的。 In some cases, a system can contain one or more pairs of primers for qPCR (also known as real time PCR (RT-PCR) or kinetic polymerase chain reaction). qPCR can simultaneously detect and quantify the specific DNA sequence in the sample, and the quantification is the absolute copy number or the relative amount corrected by the total amount of DNA added or other correction genes. An important feature of qPCR is the real-time quantification of DNA products that increase with cycle during amplification. Quantitative methods include the use of fluorescent dyes that intercalate into double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized to complementary DNA. Methods of qPCR are well known in the art. the
在某些情况下，一个系统可以包含用于原位杂交检测组织样品中一个或多个miRNA的一个或多个探针。对于原位杂交，一个标记的探针用于定位组织中某部分一个特异的DNA或RNA序列。细胞和组织样品可以通过处理固定目标核酸序列，从而便于与探针杂交。探针可以是标记的DAN互补链或互补RNA(核酸探针)。探针可以在较高温度下与靶标序列杂交，然后将过剩的探针清洗干净(要先用RNA酶水解，以防有未杂交的过剩RNA探针)。通过调整溶液的参数，如温度、盐和/或去垢剂浓度，去除任何非特异结合的探针。探针可以用放射性元素、荧光、抗原标记，从而可以通过放射自显影、荧光显微镜或免疫组织化学在组织中定位和定量。原位杂交还可以通过两种或多种标记的探针同时检测两种或多种序列。 In some cases, a system may comprise one or more probes for in situ hybridization to detect one or more miRNAs in a tissue sample. For in situ hybridization, a labeled probe is used to localize a specific DNA or RNA sequence in a portion of tissue. Cell and tissue samples can be treated to immobilize target nucleic acid sequences, thereby facilitating hybridization with probes. Probes can be labeled complementary strands of DNA or complementary RNA (nucleic acid probes). The probe can be hybridized with the target sequence at a higher temperature, and then the excess probe is cleaned (the RNase should be used to hydrolyze first to prevent unhybridized excess RNA probe). Any non-specifically bound probes are removed by adjusting the parameters of the solution, such as temperature, salt and/or detergent concentration. Probes can be labeled with radioactive elements, fluorescence, or antigens, allowing localization and quantification in tissues by autoradiography, fluorescence microscopy, or immunohistochemistry. In situ hybridization can also detect two or more sequences simultaneously by two or more labeled probes. the
在某些情况下，一个系统可以是引物的微阵列。这里的“微阵列”和“阵列”可以交替使用，指的是包含具有未知特征的生化样品(靶标)推测的结合(如杂交)位点的阵列(如有序的阵列)。在某些情况下，微阵列指的是特定的寡核苷酸探针集合固定在基质上确定的位置上。 In some cases, a system can be a microarray of primers. "Microarray" and "array" are used interchangeably herein to refer to an array (eg, an ordered array) comprising putative binding (eg, hybridization) sites of biochemical samples (targets) with unknown characteristics. In some cases, a microarray refers to a collection of specific oligonucleotide probes immobilized at defined locations on a substrate. the
在某些情况下，一个微阵列可以包含多种探针，其中每种探针可以检测样品中的一个特定miRNA，其中至少15％(例如至少15％，20％，25％，30％，40％，50％，60％，70％，80％，90％，95％，或大于95％)的探针可以检测表1所列的miRNA或它们的同源物。 In some cases, a microarray can contain multiple probes, each of which can detect a specific miRNA in a sample, of which at least 15% (e.g., at least 15%, 20%, 25%, 30%, 40% %, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) probes can detect the miRNAs listed in Table 1 or their homologues. the
在某些情况下，这里提供了检测miRNA相应的miRNA基因状况的微阵列。检测基因状况的微阵列包含了此领域内已知的基因。例如，一个系统可以包含序列标记的分子倒置探针，用于检测基因状况。 In some cases, provided herein are microarrays that detect the status of miRNAs corresponding to miRNA genes. Microarrays that detect genetic status contain genes known in the art. For example, a system could include sequence-tagged molecular inversion probes for detection of genetic conditions. the
阵列可以置于纸、玻璃、塑料(如聚丙烯、尼龙、聚苯乙烯)、聚丙烯酰胺、硝化纤维、硅、光纤或其它合适的固体或半固体物质制成的基质上，形成平面(如玻璃盘子、硅芯片)或三维(如光纤、珠子、粒子、微孔、毛细管)的造型。 Arrays can be placed on substrates made of paper, glass, plastic (e.g., polypropylene, nylon, polystyrene), polyacrylamide, nitrocellulose, silicon, optical fibers, or other suitable solid or semisolid materials to form a flat surface (e.g., glass plates, silicon chips) or in three dimensions (e.g. optical fibers, beads, particles, microwells, capillaries). the
在某些情况下，探针可以是寡核苷酸。寡核苷酸形成的阵列通过任意几种技术连在基质上。这些技术包括：(i)使用照相平版印刷技术原位合成(如高密度寡核苷酸 阵列)；(ii)在介质中点/印低密度探针于玻璃、尼龙或硝化纤维上；(iii)通过掩蔽和(iv)圆点印迹在尼龙或硝化纤维杂交膜上。寡核苷酸序列也可以通过磁珠或流动相如微孔或毛细管的方式与锚杂交非共价固定在基质上。 In some cases, probes can be oligonucleotides. Arrays of oligonucleotides are attached to substrates by any of several techniques. These techniques include: (i) in situ synthesis (e.g., high-density oligonucleotide arrays) using photolithographic techniques; (ii) dot/print low-density probes in media onto glass, nylon, or nitrocellulose; (iii) ) by masking and (iv) dot blotting on nylon or nitrocellulose hybridization membranes. The oligonucleotide sequences can also be non-covalently immobilized on the matrix by means of magnetic beads or mobile phases such as microwells or capillaries hybridized to anchors. the
有几项此领域内著名的将核酸连接到固体基质如玻片上的技术。例如，其中一个方法是将扩增的核酸混入到修饰的基质或包含能够结合固体基质的表位，如氨基、氨基衍生物或其它具有正电荷的基团，的类似物中。然后扩增产物可以与固相基质(如玻片)偶联。固相基质是经过乙醛或其它活性基团包被的，它们与扩增产物间形成共价连接，这样扩增产物就共价连在了玻片上。包含扩增产物的微阵列可以用诸如Biodot(BioDot，Inc.，Irvine，CA)点样仪和乙醛包被的玻片(CEL Associates，Houston，TX)来制作。扩增产物可以点在乙醛包被的玻片上，也可以通过报道的方法加工(Schena et al.，Proc.Natl.Acad.Sci.U.S.A.(1995)93：10614-10619)。阵列也可以通过机械手印刷在玻璃、尼龙(Ramsay，Nature Biotechnol.(1998)，16：40-44)，polypropylene(Matson et al.，Anal.Biochem.(1995)，224(1)：110-6)及硅胶片上(Marshall and Hodgson，Nature Biotechnol.(1998)，16：27-31)。其它集合阵列的方式包括电动的精细微吸管(Marshall and Hodgson，supra)，和多核苷酸直接点样于正电包被的片子上。诸如使用氨基丙烷基硅表面化学之类的方法在此领域内也广为人知，如在万维网cmt.corning.com和cmgm.stanford.edu/pbrown/上有所描述。 There are several techniques known in the art for attaching nucleic acids to solid substrates such as glass slides. For example, one approach is to incorporate the amplified nucleic acid into a modified substrate or analog containing epitopes, such as amino groups, amino derivatives, or other positively charged groups, capable of binding to a solid substrate. The amplified product can then be coupled to a solid substrate such as a glass slide. The solid phase matrix is coated with acetaldehyde or other active groups, which form a covalent link with the amplification product, so that the amplification product is covalently linked to the glass slide. Microarrays containing amplification products can be fabricated using spotters such as the Biodot (BioDot, Inc., Irvine, CA) and acetaldehyde-coated slides (CEL Associates, Houston, TX). The amplified product can be spotted on an acetaldehyde-coated glass slide, or can be processed by a reported method (Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1995) 93: 10614-10619). Arrays can also be printed on glass, nylon (Ramsay, Nature Biotechnol. (1998), 16: 40-44), polypropylene (Matson et al., Anal. Biochem. (1995), 224 (1): 110-6 ) and on silica gel sheets (Marshall and Hodgson, Nature Biotechnol. (1998), 16:27-31). Other methods of assembly arrays include motorized fine micropipettes (Marshall and Hodgson, supra), and direct spotting of polynucleotides onto positively charged slides. Methods such as the use of aminopropyl silicon surface chemistry are also well known in the art, as described on the World Wide Web at cmt.corning.com and cmgm.stanford.edu/pbrown/. the
制造微阵列的方法之一是通过制造高密度的核酸阵列。所用的技术是快速多核苷酸沉积(Blanchard et al.，Biosenesors&Bioelectronics，11：687-690)。制造微阵列的另一个方法是使用掩蔽的方法(Maskos and Southern，Nucleic.Acids.Res.(1992)，20：1679-1684)。原则上，上述的任意一种阵列(如在尼龙杂交膜上点阵)都可以使用。然而，非常小的阵列具有独特的优势，因为其杂交体系很小，这将被此领域内的专业人员认可。 One of the methods of making microarrays is by making high-density arrays of nucleic acids. The technique used is rapid polynucleotide deposition (Blanchard et al., Biosenesors & Bioelectronics, 11:687-690). Another way to make microarrays is to use the masking method (Maskos and Southern, Nucleic. Acids. Res. (1992), 20: 1679-1684). In principle, any of the arrays described above (eg arrays on nylon hybrid membranes) can be used. However, very small arrays have unique advantages because the hybridization system is small, which will be recognized by those skilled in the field. the
试剂盒 Reagent test kit
此文件还提供了用于此处描述的方法的试剂盒。在某些情况下，试剂盒包括检测miRNA水平的系统(如qPCR的一对或多对引物，原位杂交的一个或多个探针，或微阵列)。在某些情况下，试剂盒还可以额外包括用于检测的试剂。试剂盒还包括详细描述此处提到的操作方法的说明书，和/或提供具有此类说明的网址。 This document also provides kits for use in the methods described herein. In some cases, the kit includes a system for detecting miRNA levels (eg, one or more pairs of primers for qPCR, one or more probes for in situ hybridization, or a microarray). In some cases, kits may additionally include reagents for detection. The kits also include instructions detailing the methods of operation mentioned herein, and/or provide a website with such instructions. the
在某些情况下，试剂盒包含此处描述的用于肺癌诊断的系统(如qPCR的一对或多对引物，原位杂交的一个或多个探针，或微阵列)。此外还可以包括一个或多个对照样品来决定参考水平，和/或如何得到参考水平的信息。在某些情况下，试剂盒还可以包括使用此试剂盒进行肺癌诊断的说明书。 In certain instances, the kit comprises a system described herein for lung cancer diagnosis (eg, one or more pairs of primers for qPCR, one or more probes for in situ hybridization, or a microarray). In addition, one or more control samples may be included to determine the reference level, and/or for information on how to obtain the reference level. In some cases, the kit may also include instructions for using the kit to diagnose lung cancer. the
在某些情况下，试剂盒可以包含此处描述的肺癌患者的分类系统(如qPCR的一 对或多对引物，原位杂交的一个或多个探针，或微阵列)。此外还可以包括一个或多个对照样品来决定个体分类，和/或关于对照样品的信息，以及在某些情况下，含有个体分类的试剂盒使用说明。 In some cases, the kit can comprise a classification system for lung cancer patients described herein (such as one or more pairs of primers for qPCR, one or more probes for in situ hybridization, or a microarray). In addition, one or more control samples to determine the classification of the individual can be included, and/or information about the control sample and, in some cases, instructions for use of the kit containing the classification of the individual. the
在某些情况下，这里提供了为肺癌患者存活预后的试剂盒。这种试剂盒包括检测一个或多个miRNA(如表1和表2所列的一个或多个miRNA)的引物和/或探针。在某些情况下，试剂盒可以包含决定极限水平的对照样品，和/或如何获得极限水平的信息。在某些情况下，还包括用此试剂盒为患者存活预后的使用说明。在某些情况下，试剂盒可以包括改变(如降低)miRNA水平的一个或多个试剂，或包含这种试剂的药物复合物，从而促进存活。 In certain instances, kits for prognosing survival in lung cancer patients are provided herein. Such kits include primers and/or probes for detecting one or more miRNAs, such as one or more miRNAs listed in Tables 1 and 2. In some cases, kits may contain control samples to determine cut-off levels, and/or information on how to obtain cut-off levels. In some cases, instructions for using the kit to predict patient survival are also included. In some cases, the kit can include one or more agents that alter (eg, reduce) miRNA levels, or drug complexes comprising such agents, thereby promoting survival. the
此处提到的试剂盒还可以包括一些试剂，如底物，标记物，引物，标记miRNA的试剂，分离miRNA的试剂，杂交和检测的阴性或阳性对照，管子和/或其它附件，收集组织样品的试剂，缓冲液，杂交盒，盖片等等，还有可能包含软件包(如使用此处提到的统计学方法分析miRNA水平和/或miRNA水平特征性变化)，和用于获得汇集数据库的任意一个密码和/或用户名。 The kits mentioned here may also include reagents such as substrates, markers, primers, reagents for labeling miRNA, reagents for isolating miRNA, negative or positive controls for hybridization and detection, tubes and/or other accessories, collection of tissue Sample reagents, buffers, hybridization kits, coverslips, etc., may also include software packages (such as using the statistical methods mentioned here to analyze miRNA levels and/or characteristic changes in miRNA levels), and for obtaining pooled Any password and/or username for the database. the
在某些情况下，试剂盒可以包含改变(如降低)表1中所列的一个miRNA水平的药物复合物，及其用它促进肺癌患者存活的使用说明。在某些情况下，试剂盒还可以包括用来输送药物复合物的一个或多个载体或其它试剂。在某些情况下，试剂盒还包括药物复合物的使用说明。 In some cases, the kit can include a drug compound that alters (eg, lowers) the level of one of the miRNAs listed in Table 1, and instructions for using it to promote survival in lung cancer patients. In some cases, the kit may also include one or more carriers or other agents for the delivery of the drug complex. In some cases, the kit also includes instructions for the use of the drug complex. the
此发明将在以下案例中进行详述，这并不限制此发明的申请范围。 This invention will be described in detail in the following case, which does not limit the scope of application of this invention. the
案例1——miRNA水平分析样品准备 Case 1—sample preparation for miRNA level analysis
病人和样品：随机选取116对原发性肺癌组织与匹配的非癌肺组织。在这116例样本中，有60例是鳞状细胞癌，43例是腺癌，13例是小细胞肺癌。这些样本都来源于尚未进行治疗就进行外科手术的病人。样品在切下后用液氮速冻，并一直保存于-80℃(大于等于5年)，直到提取RNA。另外具有跟踪信息的20例肺原癌组织与匹配的非癌肺组织样本(储存至少5年)用于独立地确认存活分析。切下的肺样品外围部分用常规的方法进行石蜡包埋，切片，然后用苏木精和曙红染色。两个病理学者对肿瘤细胞的浓度进行了评估，并对肿瘤的组织学进行了独立的确认。跟踪信息从跟踪登记处获得。所有样品都具有临床病理学信息(抽烟、年龄、性别、病理亚型、TNM分类、肿瘤阶段、淋巴结阶段、分化状态和存活者术后存活时间)。 Patients and samples: 116 pairs of primary lung cancer tissues and matched non-cancerous lung tissues were randomly selected. Of the 116 samples, 60 were squamous cell carcinomas, 43 were adenocarcinomas, and 13 were small cell lung cancers. The samples were all from patients who underwent surgery without treatment. The samples were snap-frozen with liquid nitrogen after cutting, and kept at -80°C (greater than or equal to 5 years) until RNA was extracted. An additional 20 cases of lung cancer with follow-up information and matched noncancerous lung tissue samples (stored for at least 5 years) were used for independent confirmation of survival analysis. Peripheral parts of excised lung samples were paraffin-embedded by conventional methods, sectioned, and stained with hematoxylin and eosin. Two pathologists assessed the concentration of tumor cells and independently confirmed tumor histology. Tracking information is obtained from the tracking registry. All samples had clinicopathological information (smoking, age, sex, pathological subtype, TNM classification, tumor stage, lymph node stage, differentiation status, and postoperative survival time of survivors). the
miRNA微阵列制作：miRNA微阵列设计包括509条成熟的miRNA序列。其中包括来自英国剑桥Wellcome Trust Sanger研究所(网址http://microrna.sanger.ac.uk)登记的435条人成熟miRNA(包括报道的122条预测miRNA序列(Xie et al.，2005))，196条大鼠成熟miRNA，261条小鼠成熟miRNA。此外，我们设计了8条与RNA序列无 同源性的寡核苷酸，并用Ambion的miRNA引物构建试剂盒(Cat.No.1550；Ambion，Inc.，Austin，TX)活体外合成了它们相应的miRNA。这些合成的miRNA作为内参以不同量在分析前加入到人miRNA样品中。 miRNA microarray production: The miRNA microarray design includes 509 mature miRNA sequences. These include 435 human mature miRNAs (including 122 predicted miRNA sequences reported (Xie et al., 2005)) registered from the Wellcome Trust Sanger Institute in Cambridge, UK (website http://microrna.sanger.ac.uk), 196 rat mature miRNAs, 261 mouse mature miRNAs. In addition, we designed 8 oligonucleotides with no homology to the RNA sequence and synthesized their corresponding miRNA primers in vitro using Ambion's miRNA primer construction kit (Cat.No.1550; Ambion, Inc., Austin, TX). miRNA. These synthetic miRNAs were added to human miRNA samples in varying amounts as internal controls before analysis. the
所有的miRNA探针序列设计都与它们相应的成熟的miRNA的全长完全互补配对。为了使探针易于固定到乙醛表面修饰的玻片上(CapitalBio Corp.，Beijing，China)，我们将探针序列连接到40nt(3’末端miRNA加5’末端19聚polyT)的C6 5’氨基修饰基。寡核苷酸探针在MWG Biotech(Ebersberg，Germany)合成，用EasyArrayTM点样液(CapitalBio Corp.)溶解，浓度40μM。使用SmartArrayTM microarrayer(CapitalBio Corp.)每个探针点三个重复。 All miRNA probe sequences are designed to be fully complementary to the full length of their corresponding mature miRNAs. In order to make the probe easy to immobilize on the acetaldehyde surface-modified glass slide (CapitalBio Corp., Beijing, China), we ligated the probe sequence to the C6 5' amino group of 40 nt (3' end miRNA plus 5' end 19 polyT) Modifier. Oligonucleotide probes were synthesized at MWG Biotech (Ebersberg, Germany) and dissolved in EasyArray™ spotting solution (CapitalBio Corp.) at a concentration of 40 μM. Triplicate replicates were spotted for each probe using a SmartArray™ microarrayer (CapitalBio Corp.).
目标RNA标记：用Trizol试剂(Invitrogen；Carlsbad，CA)提取总RNA，然后用Ambion的miRNA分离试剂盒分离小分子量RNA。根据Thomson的方法(Thomson etal.，2004)用T4RNA连接酶标记法标记目标RNA。简单地说2单位T4RNA连接酶(New England Biolabs，Beijing，China)将4μg小分子量RNA用500ng的5’-磷酸-胞嘧啶-脲嘧啶-cy3-3’(Dharmacon；Lafayette，CO)标记。标记反应在4℃进行2小时。标记的RNA用0.3M的醋酸钠和2.5体积乙醇沉淀，乙醇清洗、空气干燥后，用含有3×SSC，0.2％SDS和15％的甲醛的杂交液15μl重悬标记的RNA。 Target RNA labeling: Total RNA was extracted with Trizol reagent (Invitrogen; Carlsbad, CA), and small molecular weight RNA was isolated with Ambion's miRNA isolation kit. Target RNA was labeled with T4 RNA ligase labeling according to Thomson's method (Thomson et al., 2004). Briefly, 4 μg of small molecular weight RNA was labeled with 500 ng of 5'-phospho-cytosine-uracil-cy3-3' (Dharmacon; Lafayette, CO) with 2 units of T4 RNA ligase (New England Biolabs, Beijing, China). The labeling reaction was carried out at 4°C for 2 hours. The labeled RNA was precipitated with 0.3M sodium acetate and 2.5 volumes of ethanol, washed with ethanol, air-dried, and resuspended in 15 μl of hybridization solution containing 3×SSC, 0.2% SDS and 15% formaldehyde. the
玻片杂交：杂交在置于三阶段倾斜的混合仪BIOMIXER-(CapitalBio Corp.)中的杂交盒内，并于LIFTERSLIP-(Erie；Portsmouth，NH)下进行，以便持续提供杂交液，从而使杂交在整个玻片表面更均匀，避免了边缘效应。在42℃水浴中进行过夜杂交。然后将阵列连续清洗两次：第一次在0.2％SDS，2×SSC的清洗液中42℃清洗5分钟，第二次在0.2％SSC的清洗液中室温清洗5分钟。然后用共聚焦扫描仪LUXSCAN-对阵列进行扫描，得到的图片用LUXSCAN3.0-软件分析(都来源于CapitalBio Corp.)。 Slide hybridization: The hybridization was carried out in the hybridization box placed in the three-stage inclined mixer BIOMIXER-(CapitalBio Corp.), and carried out under the LIFTERSLIP-(Erie; Portsmouth, NH), so as to continuously provide the hybridization liquid, so that the hybridization More uniform across the entire slide surface, avoiding edge effects. Hybridization was performed overnight in a 42°C water bath. Then the array was washed twice in succession: the first time was washed in 0.2% SDS, 2×SSC washing solution at 42°C for 5 minutes, and the second time was washed in 0.2% SSC washing solution at room temperature for 5 minutes. Then the array was scanned with a confocal scanner LUXSCAN-, and the images obtained were analyzed with LUXSCAN3.0-software (all derived from CapitalBio Corp.). the
数据分析：对于所有的样品，每个miRNA的重复点都减去平均背景值。表达信号＜1500的点将被过滤掉。信号都使用中值法校准。差异表达的miRNA通过SAM(Significance Analysis of Microarrays，在万维网上可以找到stat.stanford.edu/～tibs/SAM/index.html.)辨别。足够区分癌和癌旁组织的最小数量的标志性miRNA是通过PCA(Principal Component Analysis)和SVM(Support Vector Machine)方法分析得到的。预测最显著的miRNA靶标是通过四个公开的软件分析得到的：miRBase(网址：microrna.sanger.ac.uk/sequences/)，MIRANDA(available on the World Wide Web at microrna.org/)，TARGETSCAN(网址：targetscan.org/)，和PICTAR(网址：pictar.bio.nyu.edu/)。为了减少假阳性，只有至少被三个软件预测到的靶标才计算。患者的存活曲线使用Kaplan-Meier的方法评估。协变量的共同作用使用Cox Proportional Hazard Regression Model检测。 Data Analysis: For all samples, replicates for each miRNA were subtracted from the mean background value. Points with an expression signal <1500 will be filtered out. Signals were all calibrated using the median method. Differentially expressed miRNAs were identified by SAM (Significance Analysis of Microarrays, available on the World Wide Web at stat.stanford.edu/~tibs/SAM/index.html.). The minimum number of marker miRNAs sufficient to distinguish cancer and paracancerous tissues was obtained by PCA (Principal Component Analysis) and SVM (Support Vector Machine) methods. The predicted most prominent miRNA targets were analyzed by four publicly available software: miRBase (website: microrna.sanger.ac.uk/sequences/), MIRANDA (available on the World Wide Web at microrna.org/), TARGETSCAN ( URL: targetscan.org/), and PICTAR (URL: pictar.bio.nyu.edu/). To reduce false positives, only targets predicted by at least three softwares were counted. Patient survival curves were estimated using the Kaplan-Meier method. Co-effects of covariates were tested using the Cox Proportional Hazard Regression Model. the
定量聚合酶链式反应分析：为了验证miRNA表达谱，我们使用定量聚合酶链式反应的方法(qRT-PCR)用特异的miRNA引物对总的细胞RNA进行分析。总RNA提取，逆转录，PCR的操作如前所述。 Quantitative polymerase chain reaction analysis: To verify miRNA expression profiles, we used quantitative polymerase chain reaction (qRT-PCR) to analyze total cellular RNA with specific miRNA primers. The operations of total RNA extraction, reverse transcription, and PCR were as described above. the
案例2——miRNA表达可以区分肺癌中的恶性组织与附近正常组织 Case 2—miRNA expression can distinguish malignant tissue from nearby normal tissue in lung cancer
为了研究miRNA表达是否可以区分肺癌、鳞状细胞癌和腺癌中的恶性组织与附近正常组织，我们使用了116对原发性肺癌(60例鳞状细胞癌，43例腺癌，13例小细胞肺癌)，60对鳞状细胞癌和43对腺癌样品进行研究，这些样品都有相应的邻近正常肺组织，至少距离肿瘤5厘米。组织先用液氮速冻，然后于-80℃保存至少5年。116对原发性肺癌，60对鳞状细胞癌和43对腺癌样品被随机分成训练组(66对原发性肺癌，30对鳞状细胞癌和23对腺癌)和检验组(50对原发性肺癌，30对鳞状细胞癌和20对腺癌)。表达信号＜1500的点被过滤掉，通过SAM分析训练组的数据，以q值等于0，变化大于等于2倍的条件筛选到29个miRNA。为了建立一个分类器，我们使用了PCA-SVM策略，并获得了取得最高分值的一组miRNA(hsa-miR-486-5p，hsa-miR-210，hsa-miR-30a，hsa-miR-140-3p，和hsa-miR-182)这些分析在最初的训练组中的准确性如下：对肺癌98.2％，对鳞状细胞癌93.3％，对腺癌97.8％(Figures 1A，1C，和1E)。在这五个miRNA中，三个miRNA(hsa-miR-210，hsa-miR-30a，和hsa-miR-182)在肿瘤中上调，两个miRNA(hsa-miR-486-5p和hsa-miR-140-3p)下调(Table 1)。建立分类器后，检验组被用来评估策略模型。在检验组中的准确性如下：对肺癌92％，对鳞状细胞癌96.7％，对腺癌90％(Figures 1B，1D，和1F)。总的来说，结果显示分类器可以利用少至5个标志性的miRNA有效地将肺癌、鳞状细胞癌和腺癌的恶性组织与正常组织分开。 To investigate whether miRNA expression could distinguish malignant tissue from nearby normal tissue in lung, squamous cell carcinoma, and adenocarcinoma, we used 116 pairs of primary lung cancers (60 squamous cell carcinoma, 43 adenocarcinoma, 13 cell lung cancer), 60 pairs of squamous cell carcinoma and 43 pairs of adenocarcinoma samples with corresponding adjacent normal lung tissue at least 5 cm away from the tumor. Tissues were snap-frozen in liquid nitrogen and then stored at -80°C for at least 5 years. 116 pairs of primary lung cancer, 60 pairs of squamous cell carcinoma and 43 pairs of adenocarcinoma samples were randomly divided into training group (66 pairs of primary lung cancer, 30 pairs of squamous cell carcinoma and 23 pairs of adenocarcinoma) and test group (50 pairs of primary lung cancer, 30 pairs of squamous cell carcinoma and 20 pairs of adenocarcinoma). The points with expression signal <1500 were filtered out, and the data of the training group were analyzed by SAM, and 29 miRNAs were screened under the condition that the q value was equal to 0 and the change was greater than or equal to 2 times. To build a classifier, we used the PCA-SVM strategy and obtained the set of miRNAs (hsa-miR-486-5p, hsa-miR-210, hsa-miR-30a, hsa-miR- 140-3p, and hsa-miR-182) The accuracy of these analyzes in the original training set was as follows: 98.2% for lung cancer, 93.3% for squamous cell carcinoma, and 97.8% for adenocarcinoma (Figures 1A, 1C, and 1E ). Among these five miRNAs, three miRNAs (hsa-miR-210, hsa-miR-30a, and hsa-miR-182) were upregulated in tumors, two miRNAs (hsa-miR-486-5p and hsa-miR -140-3p) down-regulated (Table 1). After building the classifier, the test set is used to evaluate the policy model. Accuracies in the test set were as follows: 92% for lung cancer, 96.7% for squamous cell carcinoma, and 90% for adenocarcinoma (Figures 1B, 1D, and 1F). Collectively, the results show that the classifier can effectively separate malignant tissue from normal tissue in lung cancer, squamous cell carcinoma, and adenocarcinoma using as few as 5 signature miRNAs. the
案例3——miRNA表达不区分肺癌的不同病理亚型 Case 3—miRNA expression does not distinguish different pathological subtypes of lung cancer
研究目的是看miRNA表达是否可以区分肺癌的三种病理亚型：鳞状细胞癌、腺癌和小细胞肺癌，中的恶性组织与附近正常组织。基于癌组织(C)与癌旁正常组织(N)miRNA的比率和癌组织中miRNA的信号，通过SAM分析选出每个肿瘤中差异表达的miRNA。基于C∶N比率，三种亚型中有68个差异表达的miRNA，基于癌组织中miRNA的信号，有70个差异表达的miRNA。根据差异表达的miRNA，对60例鳞状细胞癌，43例腺癌和13例小细胞肺癌样品进行无监督聚类分析。虽然小细胞肺癌样品倾向于聚到同一组，但是此研究中肺癌的三种亚型无法根据它们的miRNA表达谱清楚地被分为三组(Figure 2)。重要的是，此分析表明鳞状细胞癌的miRNA表达谱与腺癌并没有明显的差异。相反，除了少数样品外，小细胞肺癌的miRNA表达谱与非小细胞肺癌可能有差异。 The aim of the study was to see if miRNA expression could differentiate three pathological subtypes of lung cancer: squamous cell carcinoma, adenocarcinoma, and small cell lung cancer, in malignant tissue from nearby normal tissue. Differentially expressed miRNAs in each tumor were selected by SAM analysis based on the ratio of miRNAs in cancerous tissue (C) to adjacent normal tissue (N) and the signal of miRNAs in cancerous tissue. There were 68 differentially expressed miRNAs among the three subtypes based on C:N ratios and 70 differentially expressed miRNAs based on miRNA signatures in cancer tissues. According to the differentially expressed miRNAs, 60 squamous cell carcinoma, 43 adenocarcinoma, and 13 small cell lung cancer samples were subjected to unsupervised cluster analysis. Although SCLC samples tended to cluster into the same group, the three subtypes of lung cancer in this study could not be clearly divided into three groups based on their miRNA expression profiles (Figure 2). Importantly, this analysis revealed that the miRNA expression profile of squamous cell carcinoma was not significantly different from that of adenocarcinoma. In contrast, the miRNA expression profile of SCLC may differ from that of NSCLC except for a few samples. the
案例4——miRNA表达与肺癌的病理及临床特征相关联 Case 4—— miRNA expression is associated with pathological and clinical features of lung cancer
为了检测微阵列的数据能否反应临床病理学不同的肺癌亚型的miRNA特征，我们做了进一步研究。这些研究包括在多队群组间比较miRNA的表达，包括抽烟与否，不同年龄组，性别，病理学分类，分化分类，TNM分类，肿瘤阶段，淋巴结阶段，和肿瘤阶段分类，如Table 2所示。数据分析使用的是SAM系统，基于C∶N的miRNA比值或癌组织的miRNA信号的。有几个miRNA(Table 3)同时被这两种方式挑选出来了。值得注意的是，三个miRNA(hsa-miR-205，hsa-miR-203，and hsa-miR-18b)相对于腺癌来说在鳞状细胞癌中一直处于低水平的表达。两个miRNA(hsa-miR-205and hsa-miR-181a)在鳞状细胞癌中在性别间有差异表达。两个miRNA(hsa-miR-205and hsa-miR-31)在鳞状细胞癌中在高分化水平、中等分化水平、低分化水平间有差异表达。一个miRNA(hsa-miR-205)在非小细胞肺癌中在抽烟指数＜400每年与≥400每年的患者间有差异。四项临床病理学指标(组织学亚型，性别，分化和抽烟指数)没有显著的统计学关联。有意思的是，hsa-miR-205显著地与四个不同的肺癌临床病理学指标相关联。在这组数据中没有特异的miRNA与TNM或个体肿瘤阶段相关联。 We conducted a further study to examine whether microarray data could reflect miRNA signatures of clinicopathologically distinct lung cancer subtypes. These studies included the comparison of miRNA expression among multiple cohorts, including smoking or not, different age groups, sex, pathological classification, differentiation classification, TNM classification, tumor stage, lymph node stage, and tumor stage classification, as shown in Table 2. Show. Data analysis was performed using the SAM system, based on C:N miRNA ratios or miRNA signals in cancer tissues. Several miRNAs (Table 3) were simultaneously selected by both methods. Notably, three miRNAs (hsa-miR-205, hsa-miR-203, and hsa-miR-18b) were consistently expressed at low levels in squamous cell carcinomas relative to adenocarcinomas. Two miRNAs (hsa-miR-205 and hsa-miR-181a) were differentially expressed between sexes in squamous cell carcinoma. Two miRNAs (hsa-miR-205 and hsa-miR-31) were differentially expressed in squamous cell carcinoma between well-differentiated level, moderately differentiated level and poorly differentiated level. One miRNA (hsa-miR-205) differed between patients with a smoking index <400 and ≥400 per year in non-small cell lung cancer. Four clinicopathological indicators (histological subtype, sex, differentiation, and smoking index) had no statistically significant associations. Interestingly, hsa-miR-205 was significantly associated with four different clinicopathological indicators of lung cancer. No specific miRNAs were associated with TNM or individual tumor stages in this data set. the
案例5——hsa-miR-31表达与鳞状细胞癌预后的关系 Case 5——The relationship between the expression of hsa-miR-31 and the prognosis of squamous cell carcinoma
我们还研究了miRNA表达谱与病人存活之间的关系。在鳞状细胞癌与相应的邻近正常组织间有37个miRNA差异表达，在腺癌与相应的邻近正常组织间有22个miRNA差异表达，在非小细胞肺癌与相应的邻近正常组织间有29个miRNA差异表达。所有这些差异表达的miRNA都用于Kaplan-Meier存活分析。最初的60例鳞状细胞癌与43例腺癌及它们合并成的103例非小细胞肺癌的miRNA C∶N的中值作为分割点。Kaplan-Meier分析显示hsa-miR-31高C∶N值与鳞状细胞癌的存活负相关(p＝0.007，log-rank检验，训练群组60例病人样品，Figure3A)。进一步的单变量和多变量Cox分析证实了hsa-miR-31的高表达比低表达与鳞状细胞癌的低存活率更相关(Table 2)。hsa-miR-31与临床病理学因素(抽烟指数，年龄，性别，分化，TNM分类)的单变量分析显示hsa-miR-31表达水平(p＝0.011)和TNM(p＝0.013)在训练群组中具有预后显著性(Table 4)。随后，使用临床病理学和分子因子的多变量的Cox比例风险回归模型分析显示hsa-miR-31高表达是与其他临床病理学因素不相关的显著的负相预后因子(p＝0.021；风险比率3.05；95％置信区间[CI]，1.187-7.838)，而TNM与病人的不良后果没有显著关联(p＝0.179)(Table 5)。我们进一步研究了miRNA与腺癌和非小细胞肺癌病人的存活率间的关系，结果没有观察到与存活相关的miRNA。 We also investigated the relationship between miRNA expression profiles and patient survival. 37 miRNAs were differentially expressed between squamous cell carcinoma and corresponding adjacent normal tissues, 22 miRNAs were differentially expressed between adenocarcinoma and corresponding adjacent normal tissues, and 29 miRNAs were differentially expressed between non-small cell lung cancer and corresponding adjacent normal tissues Differential expression of miRNAs. All these differentially expressed miRNAs were used in Kaplan-Meier survival analysis. The median value of miRNA C:N of the first 60 squamous cell carcinomas and 43 adenocarcinomas and their merged 103 non-small cell lung cancers was used as the cut-off point. Kaplan-Meier analysis showed that a high C:N value of hsa-miR-31 was negatively correlated with the survival of squamous cell carcinoma (p=0.007, log-rank test, training group of 60 patient samples, Figure 3A). Further univariate and multivariate Cox analyzes confirmed that high expression of hsa-miR-31 was more associated with poor survival in SCC than low expression (Table 2). Univariate analysis of hsa-miR-31 and clinicopathological factors (smoking index, age, sex, differentiation, TNM classification) showed that hsa-miR-31 expression level (p=0.011) and TNM (p=0.013) were significantly different in the training group group with prognostic significance (Table 4). Subsequently, a multivariate Cox proportional hazards regression model analysis using clinicopathological and molecular factors revealed that high hsa-miR-31 expression was a significant negative prognostic factor independent of other clinicopathological factors (p=0.021; hazard ratio 3.05; 95% confidence interval [CI], 1.187-7.838), while TNM was not significantly associated with poor patient outcomes (p=0.179) (Table 5). We further investigated the relationship between miRNAs and the survival rate of patients with adenocarcinoma and non-small cell lung cancer, and no survival-related miRNAs were observed. the
为了研究hsa-miR-31与鳞状细胞癌患者预后间的关系，用独立的20例鳞状细胞癌组织进行了分析。qRT-PCR分析了miRNA的表达水平。Kaplan-Meier存活分析(Figure 3)证实了hsa-miR-31高表达的病人存活率显著下降(p＝0.001；log-rank检验，训练群组20对病人组织)。单变量(p＝0.007)和多变量(p＝0.029)Cox比例风险回归模型分析也显示了hsa-miR-31高表达是鳞状细胞癌负相预后独立的指示指标(Table5)。 In order to study the relationship between hsa-miR-31 and the prognosis of squamous cell carcinoma patients, 20 independent squamous cell carcinoma tissues were analyzed. The expression levels of miRNAs were analyzed by qRT-PCR. Kaplan-Meier survival analysis (Figure 3) confirmed that the survival rate of patients with high expression of hsa-miR-31 was significantly decreased (p=0.001; log-rank test, training cohort 20 pairs of patient tissues). Univariate (p=0.007) and multivariate (p=0.029) Cox proportional hazards regression model analysis also showed that high expression of hsa-miR-31 is an independent indicator of negative prognosis in squamous cell carcinoma (Table 5). the
此文件包含众多具体案例，但是这些并不局限此发明申请的范围，而是作为此发明具体情况下的特征描述。此文件中在不同情况下描述的一些特征也可以组合应用于某一个情况。反之，在某一情况下描述的不同特征也可以单独或部分组合应用于多种情况。此外，虽然以上一些特征可能是以组合使用的形式描述的，即使最初是这样宣布，但是组合使用的一个或多个特征在某些情况下也可以从组合中割离，描述的组合也可能包含亚组合或变化的亚组合。 This document contains numerous specific examples, but these do not limit the scope of this invention application, but rather serve as a description of the characteristics of this invention in specific cases. Some of the features described in this document in different contexts may also be applied in combination in a single context. Conversely, different features described in a certain situation can also be applied to multiple situations alone or in partial combination. In addition, although some of the above features may be described in combination, even if originally declared to be so, one or more features used in combination may in some cases be severed from the combination, and the described combination may also contain Subcombinations or variant subcombinations. the
此处具体描述的情况之一少数几个。这些情况及其他情况可以基于本文件的描述和阐述进行变化和增加。 One of the few situations specifically described here. These and other circumstances may be varied and added to based on the description and illustrations in this document. the
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