技术领域technical field
本发明属于基因工程及肿瘤学领域，涉及一种与食管鳞癌辅助诊断相关的血清miRNA标志物及其应用。The invention belongs to the fields of genetic engineering and oncology, and relates to a serum miRNA marker related to the auxiliary diagnosis of esophageal squamous cell carcinoma and its application.
背景技术Background technique
食管癌是常见的消化道恶性肿瘤。中国食管癌的死亡率居肿瘤第四位，2015年约有4.8万食管癌新发病例及3.8万食管癌相关致死患者。我国食管鳞癌病例约占食管癌总数的90％，尽管目前诊断方法、围手术期策略及放化疗手段不断进步，食管鳞癌患者术后复发转移风险仍然较大，并且五年生存率仅为15％-25％。由于食管鳞癌患者早期临床表现无明显特异性，大部分患者确诊时已处于肿瘤中晚期，因此，早期诊断早期干预能够有效地改善患者的预后及生存质量。目前食管鳞癌的主要诊断措施包括：影像学检查、内镜检查以及病理学诊断，然而影像学及内镜检查有一定的放射及创伤风险。临床上常用的肿瘤标志物，如癌胚抗原(CEA)和鳞状上皮细胞癌抗原(SCC)等，也缺乏一定的灵敏性和特异性。因此，发现新的能够早期诊断食管鳞癌的标记物从而促进食管鳞癌早期干预和治疗具有重要的临床意义。Esophageal cancer is a common malignant tumor of the digestive tract. The mortality rate of esophageal cancer in China ranks fourth among cancers. In 2015, there were approximately 48,000 new cases of esophageal cancer and 38,000 deaths related to esophageal cancer. Cases of esophageal squamous cell carcinoma account for about 90% of the total number of esophageal cancers in my country. Although the current diagnostic methods, perioperative strategies, and radiotherapy and chemotherapy methods have been continuously improved, the risk of recurrence and metastasis of patients with esophageal squamous cell carcinoma after surgery is still relatively high, and the five-year survival rate is only 10%. 15%-25%. Since the early clinical manifestations of patients with esophageal squamous cell carcinoma have no obvious specificity, most patients are already in the middle and late stage of the tumor when they are diagnosed. Therefore, early diagnosis and early intervention can effectively improve the prognosis and quality of life of patients. Currently, the main diagnostic measures for esophageal squamous cell carcinoma include: imaging examination, endoscopy, and pathological diagnosis. However, imaging and endoscopy have certain risks of radiation and trauma. Commonly used clinical tumor markers, such as carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC), also lack certain sensitivity and specificity. Therefore, it is of great clinical significance to discover new markers for early diagnosis of esophageal squamous cell carcinoma so as to promote early intervention and treatment of esophageal squamous cell carcinoma.
微小RNA(miRNAs)是一类长度在22个左右的核苷酸的小非编码RNA分子，其通过转录后调控广泛参与了各种生命活动过程，包括肿瘤的发生、侵袭及转移等。研究发现，miRNA的表达在肿瘤中有不同程度的上调和下调，为其能够作为一种新兴的肿瘤标志物奠定了基础。2008年，Mitchell在外周血中检测到游离的miRNA，发现其能够稳定存在于外周血中，并且可以作为诊断肿瘤的无创标志物。现已有研究证实了循环miRNA在多种肿瘤(包括食管鳞癌)中的潜在诊断价值。但目前关于食管鳞癌的较大样本miRNA谱系研究仍然较少。因此，本研究利用Exiqon miRNA qPCR panel芯片以及基于qRT-PCR的相对定量法，通过对大样本的食管鳞癌血清的研究，旨在寻找对食管鳞癌具有潜在诊断价值的血清miRNA。并对这些miRNA在食管鳞癌组织中以及外周血清外泌体中的表达进行验证，以进一步明确其与食管鳞癌的关系。若根据这类miRNA设计针对于食管鳞癌的诊断试剂盒，将会推动我国食管鳞癌的诊治水平，也为将来对食管鳞癌的进一步研究提供思路。MicroRNAs (miRNAs) are a class of small non-coding RNA molecules with a length of about 22 nucleotides, which are widely involved in various life processes through post-transcriptional regulation, including tumorigenesis, invasion and metastasis. Studies have found that the expression of miRNA is up-regulated and down-regulated in different degrees in tumors, which lays the foundation for it to be used as a new tumor marker. In 2008, Mitchell detected free miRNA in peripheral blood and found that it can stably exist in peripheral blood and can be used as a non-invasive marker for tumor diagnosis. Studies have demonstrated the potential diagnostic value of circulating miRNAs in a variety of tumors, including esophageal squamous cell carcinoma. However, there are still few studies on miRNA lineages in larger samples of esophageal squamous cell carcinoma. Therefore, this study uses the Exiqon miRNA qPCR panel chip and qRT-PCR-based relative quantification method to search for serum miRNAs with potential diagnostic value for esophageal squamous cell carcinoma through the study of large samples of esophageal squamous cell carcinoma serum. The expression of these miRNAs in esophageal squamous cell carcinoma tissues and peripheral serum exosomes was verified to further clarify their relationship with esophageal squamous cell carcinoma. If a diagnostic kit for esophageal squamous cell carcinoma is designed based on this type of miRNA, it will promote the diagnosis and treatment of esophageal squamous cell carcinoma in my country and provide ideas for further research on esophageal squamous cell carcinoma in the future.
发明内容Contents of the invention
本发明的目的在于提供一种与食管鳞癌辅助诊断相关的血清miRNA标志物。The purpose of the present invention is to provide a serum miRNA marker related to the auxiliary diagnosis of esophageal squamous cell carcinoma.
本发明的另一目的在于提供上述血清miRNA标志物及其引物在制备食管鳞癌辅助诊断试剂盒以及在制备治疗食管鳞癌的药物中的应用。Another object of the present invention is to provide the application of the above-mentioned serum miRNA markers and primers in the preparation of an auxiliary diagnostic kit for esophageal squamous cell carcinoma and in the preparation of drugs for treating esophageal squamous cell carcinoma.
本发明的又一目的在于提供用于食管鳞癌辅助诊断和治疗的试剂盒及药物。Another object of the present invention is to provide a kit and medicine for auxiliary diagnosis and treatment of esophageal squamous cell carcinoma.
本发明的目的可以通过以下技术方案实现：The purpose of the present invention can be achieved through the following technical solutions:
一种与食管鳞癌辅助诊断相关的血清miRNA标志物，该标志物为miR-20b-5p(CAAAGUGCUCAUAGUGCAGGUAG)、miR-28-3p(CACUAGAUUGUGAGCUCCUGGA)、miR-192-5p(CUGACCUAUGAAUUGACAGCC)、miR-223-3p(UGUCAGUUUGUCAAAUACCCCA)和miR-296-5p(AGGGCCCCCCCUCAAUCCUGU)中的一种或多种，如miR-20b-5p、miR-28-3p和miR-192-5p的组合，或miR-296-5p。该血清miRNA标志物优选为miR-10b-5p、miR-132-3p、miR-185-5p、miR-195-5p、miR-20a-3p和miR-296-5p中两种或两种以上的组合，进一步优选为miR-10b-5p、miR-132-3p、miR-185-5p、miR-195-5p、miR-20a-3p和miR-296-5p五种miRNA所组成的组合。A serum miRNA marker associated with the auxiliary diagnosis of esophageal squamous cell carcinoma. One or more of 3p (UGUCAGUUUGUCAAAUACCCCA) and miR-296-5p (AGGGCCCCCCCUCAAUCCUGU), such as a combination of miR-20b-5p, miR-28-3p and miR-192-5p, or miR-296-5p. The serum miRNA marker is preferably two or more of miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p The combination is more preferably a combination of five miRNAs including miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p.
上述的血清miRNA标志物在辅助诊断食管鳞癌中的应用。The application of the above serum miRNA markers in the auxiliary diagnosis of esophageal squamous cell carcinoma.
上述的血清miRNA标志物在制备食管鳞癌辅助诊断试剂盒或治疗食管鳞癌药物中的应用。Application of the above-mentioned serum miRNA markers in the preparation of an auxiliary diagnostic kit for esophageal squamous cell carcinoma or a drug for treating esophageal squamous cell carcinoma.
一种与食管鳞癌辅助诊断相关的血清miRNA标志物的引物，该引物包含miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p中的一种或多种miRNA的引物；优选为包含血清miRNA中miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p中两种或两种以上miRNA的引物；进一步优选为包含血清miRNA中miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p五种miRNA的引物。A primer for serum miRNA markers related to auxiliary diagnosis of esophageal squamous cell carcinoma, the primer includes miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p Primers for one or more miRNAs; preferably two or both Primers for more than one miRNA; more preferably primers for five miRNAs including miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p in serum miRNA.
上述的引物在辅助诊断食管鳞癌或制备食管鳞癌辅助诊断试剂盒中的应用。Application of the above primers in auxiliary diagnosis of esophageal squamous cell carcinoma or preparation of an auxiliary diagnostic kit for esophageal squamous cell carcinoma.
一种食管鳞癌辅助诊断试剂盒，该试剂盒用于检测血清miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p中的一种或多种miRNA；优选为用于检测血清miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p中两种或两种以上的miRNA；进一步优选为用于检测血清中的miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p五种miRNA。An auxiliary diagnostic kit for esophageal squamous cell carcinoma, which is used to detect one of serum miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p or multiple miRNAs; preferably for detecting two or more miRNAs in serum miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p; Further preferably, five miRNAs are used for detecting miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p in serum.
一种食管鳞癌辅助诊断试剂盒，该试剂盒中含有血清miRNA中miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p中的一种或多种miRNA的引物；优选为含有血清miRNA中miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p中两种或两种以上miRNA的引物；进一步优选为含有血清miRNA中miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p五种miRNA的引物。An auxiliary diagnostic kit for esophageal squamous cell carcinoma, which contains one of miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p in serum miRNA Primers for one or more miRNAs; preferably containing two or more of miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p in serum miRNA Primers for miRNA; more preferably primers containing five miRNAs in serum miRNAs: miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p.
该试剂盒还可以包括PCR技术常用试剂，如逆转录酶，缓冲液，dNTPs，MgCl2，DEPC水和Taq酶等；还可以含有标准品和/或对照品。The kit can also include reagents commonly used in PCR technology, such as reverse transcriptase, buffer, dNTPs, MgCl2 , DEPC water and Taq enzyme, etc.; it can also contain standard and/or control substances.
本发明所涉及的与食管鳞癌诊断相关的血清miRNA标志物miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p中的每种miRNA的序列均已公开，但是将各miRNA标志物进行组合作为食管鳞癌辅助诊断标志物需要本领域技术人员付出创造性劳动。各miRNA标志物的扩增引物均可通过市场购买获得，本发明实施例中使用的血清miRNA标志物的引物为购自广州锐博公司所合成生产的特异性miRNA茎环RT-PCR引物。Each miRNA in the serum miRNA markers miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p related to the diagnosis of esophageal squamous cell carcinoma involved in the present invention The sequences of all miRNA markers have been published, but the combination of miRNA markers as auxiliary diagnostic markers for esophageal squamous cell carcinoma requires creative work by those skilled in the art. The amplification primers for each miRNA marker can be purchased from the market, and the primers for serum miRNA markers used in the examples of the present invention are specific miRNA stem-loop RT-PCR primers synthesized and produced by Guangzhou Ruibo Company.
具体地说，本发明解决问题的技术方案包括：(1)建立统一标准的标本库和数据库：以标准操作程序(SOP)采集符合标准的血液样本，系统收集完整的人口学资料和临床资料。(2)血清miRNA差异表达谱分析：分析食管鳞癌和正常对照人群中差异表达的血清miRNA，并对差异表达miRNAs进行进一步大样本多阶段验证。(3)通过多阶段的验证，明确这些miRNA诊断食管鳞癌的能力。(4)血清miRNA诊断试剂盒的研制：根据食管鳞癌患者与正常人群血清中的差异表达miRNA开发miRNAs诊断试剂盒，实现对食管鳞癌患者的无创辅助诊断。(4)分析这些miRNA在食管鳞癌组织以及外泌体中的表达情况，揭示这些miRNA与食管鳞癌的关系，为将来研制可能与这些miRNA相关的治疗食管鳞癌的药物提供依据。Specifically, the technical solutions for solving the problems of the present invention include: (1) Establishing a unified standard specimen bank and database: collecting standard blood samples with standard operating procedures (SOP), and systematically collecting complete demographic and clinical data. (2) Serum miRNA differential expression profile analysis: analyze the differentially expressed serum miRNAs in esophageal squamous cell carcinoma and normal control populations, and conduct further large-sample multi-stage verification of differentially expressed miRNAs. (3) Through multi-stage verification, the ability of these miRNAs to diagnose esophageal squamous cell carcinoma was clarified. (4) Development of serum miRNA diagnostic kits: develop miRNAs diagnostic kits based on the differentially expressed miRNAs in the serum of patients with esophageal squamous cell carcinoma and normal population, and realize non-invasive auxiliary diagnosis for patients with esophageal squamous cell carcinoma. (4) Analyze the expression of these miRNAs in esophageal squamous cell carcinoma tissues and exosomes, reveal the relationship between these miRNAs and esophageal squamous cell carcinoma, and provide a basis for the future development of drugs that may be related to these miRNAs for the treatment of esophageal squamous cell carcinoma.
本发明人以标准操作程序(SOP)采集符合标准的血液样本，系统收集完整的人口学资料、临床资料，并采用了Exiqon miRNA qPCR panel芯片以及qRT-PCR方法等。The inventors used the standard operating procedure (SOP) to collect standard blood samples, systematically collected complete demographic and clinical data, and adopted Exiqon miRNA qPCR panel chips and qRT-PCR methods.
具体来说研究的实验方法主要包括以下几个部分：Specifically, the experimental method of the research mainly includes the following parts:
1.研究样本选择：初治、未行手术以及放化疗干预且经病理确认为食管鳞癌的患者。正常对照为在医院进行体检的正常人群。1. Selection of research samples: newly diagnosed patients with esophageal squamous cell carcinoma who have not undergone surgery, radiotherapy and chemotherapy intervention and confirmed by pathology. The normal control group was the normal population who underwent physical examination in the hospital.
2.Exiqon miRNA qPCR panel芯片初筛：利用TRIZOL-LS试剂对血清混合样本进行RNA提取，并进行qRT-PCR操作获得初筛结果。2. Preliminary screening with Exiqon miRNA qPCR panel chip: Use TRIZOL-LS reagent to extract RNA from serum mixed samples, and perform qRT-PCR operation to obtain the preliminary screening results.
3.训练集、验证集以及额外验证集：利用AM1556试剂盒(ABI公司)对每个血清样本进行RNA提取，通过逆转录反应得到cDNA样品，加入PCR引物和SYBR Green荧光染料进行PCR反应。通过比对标准品的Ct值，得出样本中的miRNA含量。3. Training set, validation set and additional validation set: RNA was extracted from each serum sample using AM1556 kit (ABI Company), cDNA samples were obtained by reverse transcription reaction, PCR primers and SYBR Green fluorescent dye were added for PCR reaction. By comparing the Ct value of the standard, the miRNA content in the sample is obtained.
4.利用TRIZOL-LS试剂提取食管鳞癌和癌旁组织中的RNA，ExoQuick试剂盒(SBI公司)和AM1556试剂盒(ABI公司)提取外泌体中的RNA，通过qRT-PCR的方法，检测miRNA在组织以及外泌体中的表达差异。4. Use TRIZOL-LS reagent to extract RNA in esophageal squamous cell carcinoma and paracancerous tissues, ExoQuick kit (SBI company) and AM1556 kit (ABI company) to extract RNA in exosomes, and use qRT-PCR to detect Differential expression of miRNAs in tissues and exosomes.
5.统计分析：运用χ2检验、配对t检验以及非参数秩和检验比较miRNA表达水平在不同研究组中的差异。通过计算ROC曲线分析证实血清miRNA的诊断价值。5. Statistical analysis: χ2 test, paired t test and non-parametric rank sum test were used to compare the differences of miRNA expression levels in different research groups. The diagnostic value of serum miRNA was confirmed by ROC curve analysis.
本发明研究组目前通过对食管鳞癌病人的外周血清中的miRNA进行系统的表达分析，现已发现了一组5个具有临床诊断潜能的食管鳞癌血清microRNA标记物(miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p)。The research group of the present invention has now discovered a group of 5 serum microRNA markers (miR-20b-5p, miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p and miR-296-5p).
本发明的有益效果：Beneficial effects of the present invention:
1.相较于传统的肿瘤标志物，血清miRNA作为新型的生物标志物，具有稳定性好、微创易获取、灵敏性和特异性高的特点。这类分子标志物的开发利用将为包括肿瘤在内的各种疾病的诊断以及进一步治疗提供新的方向。1. Compared with traditional tumor markers, serum miRNA, as a new type of biomarker, has the characteristics of good stability, minimal invasiveness, easy access, high sensitivity and specificity. The development and utilization of such molecular markers will provide a new direction for the diagnosis and further treatment of various diseases including tumors.
2.研究者通过Exiqon miRNA qPCR panel芯片以及基于qRT-PCR的相对定量法，对食管鳞癌和正常对照人群血清中的差异表达miRNA进行严密、多阶段的验证和评价。证实了这组miRNA作为诊断食管鳞癌的无创标记物的可靠性以及可重复性。2. The researchers used the Exiqon miRNA qPCR panel chip and the relative quantitative method based on qRT-PCR to conduct rigorous and multi-stage verification and evaluation of the differentially expressed miRNAs in the serum of esophageal squamous cell carcinoma and normal controls. The reliability and reproducibility of this group of miRNAs as non-invasive markers for the diagnosis of esophageal squamous cell carcinoma were confirmed.
3.研究者发现miR-20b-5p、miR-28-3p和miR-192-5p在食管鳞癌组织中的表达与血清中表达一致，同时miR-296-5p在血清外泌体中的表达也是高于正常对照的，显示了这组miRNA与食管鳞癌之间的紧密关系。这些结果将给未来研究这些miRNA对于食管鳞癌的机制以及针对这些miRNA对于食管鳞癌的治疗提供新的思路。3. The researchers found that the expression of miR-20b-5p, miR-28-3p and miR-192-5p in esophageal squamous cell carcinoma tissue was consistent with that in serum, and the expression of miR-296-5p in serum exosomes It is also higher than the normal control, showing the close relationship between this group of miRNAs and esophageal squamous cell carcinoma. These results will provide new ideas for the future research on the mechanism of these miRNAs in esophageal squamous cell carcinoma and the treatment of these miRNAs in esophageal squamous cell carcinoma.
附图说明Description of drawings
图1：实验流程图Figure 1: Experimental flow chart
图2：在食管鳞癌血清中高表达的5个miRNAFigure 2: Five miRNAs highly expressed in esophageal squamous cell carcinoma serum
图3：对所获得的miRNA进行ROC曲线分析Figure 3: ROC curve analysis of the obtained miRNAs
A：训练集与验证集的合集；B：训练集；C：验证集；D：外部验证集。A: the combination of training set and validation set; B: training set; C: validation set; D: external validation set.
图4：5个miRNA在食管鳞癌组织中的表达情况Figure 4: Expression of 5 miRNAs in esophageal squamous cell carcinoma tissues
图5：5个miRNA在食管鳞癌患者血清外泌体中的表达情况Figure 5: Expression of 5 miRNAs in serum exosomes of patients with esophageal squamous cell carcinoma
具体实施方式Detailed ways
发明人于2013年至2015年从南京医科大学第一附属医院及常州市第一人民医院收集了大量的食管鳞癌患者和正常体检人群的静脉血清样品，通过对样品资料的整理，从中选择了200例食管鳞癌和188例正常对照的样本作为Exiqon miRNA qPCR panel芯片初筛和后续一系列qRT-PCR验证的实验样品。同时还留取了36例食管鳞癌组织和36例癌旁组织。所选择的患者血清样本均来自于初治、未行手术以及放化疗干预且经病理确认为食管鳞癌的病人。并系统采集了这些样本的人口学资料、临床资料。The inventor collected a large number of venous serum samples from the First Affiliated Hospital of Nanjing Medical University and the First People's Hospital of Changzhou from 2013 to 2015, and selected The samples of 200 cases of esophageal squamous cell carcinoma and 188 cases of normal controls were used as the experimental samples for the initial screening of Exiqon miRNA qPCR panel chip and subsequent series of qRT-PCR verification. At the same time, 36 cases of esophageal squamous cell carcinoma tissues and 36 cases of paracancerous tissues were retained. The selected patients' serum samples were all from patients who had not undergone surgery, radiotherapy and chemotherapy intervention and were pathologically confirmed as esophageal squamous cell carcinoma. The demographic data and clinical data of these samples were collected systematically.
参照流程图(图1)，从食管鳞癌以及正常对照血清样本中随机选择了30例食管鳞癌样本以及10例正常对照，并且分别混合成了3例食管鳞癌血清混合样本和1个正常混合样本(一个混合样本由10例200ul血清样本汇合形成2ml的样本)。对这4例混合样本进行Exiqon miRNA qPCR panel芯片初筛和分析，具体步骤参照Exiqon miRNA qPCR panel芯片的说明书：Referring to the flow chart (Figure 1), 30 esophageal squamous cell carcinoma samples and 10 normal controls were randomly selected from the serum samples of esophageal squamous cell carcinoma and 10 normal controls, and were mixed into 3 mixed serum samples of esophageal squamous cell carcinoma and 1 normal control. Mixed samples (one mixed sample is composed of 200ul serum samples from 10 cases to form a 2ml sample). The Exiqon miRNA qPCR panel chip was used for preliminary screening and analysis of these 4 mixed samples. For specific steps, refer to the instruction manual of the Exiqon miRNA qPCR panel chip:
1.血清抽提1. Serum Extraction
取出血清样品，样品化冻后3000x g离心5min去除一些碎片及一些不溶成分。转移250ul上清至新的1.5ml管中，加入750ul TRIZOL-LS后，剧烈震荡5s。The serum samples were taken out, and after the samples were thawed, they were centrifuged at 3000×g for 5 minutes to remove some debris and some insoluble components. Transfer 250ul supernatant to a new 1.5ml tube, add 750ul TRIZOL-LS, shake vigorously for 5s.
2.两相分离2. Two-phase separation
匀浆后样品于15到30℃孵育5分钟。每1ml的TRIZOL-LS试剂匀浆的样品中加入0.2ml的氯仿，盖紧管盖。手动剧烈振荡管体15秒后，15到30℃孵育2到3分钟。4℃下13,000g离心15分钟。After homogenization the samples were incubated at 15 to 30°C for 5 minutes. Add 0.2ml of chloroform to every 1ml of TRIZOL-LS reagent homogenized sample, and tightly cap the tube. Shake the tube vigorously by hand for 15 seconds, and incubate at 15 to 30°C for 2 to 3 minutes. Centrifuge at 13,000g for 15 minutes at 4°C.
3.RNA沉淀3. RNA precipitation
将水相转移到新离心管中。水相与异丙醇混合以沉淀其中的RNA，加入异丙醇的量为：1ml TRIZOL-LS试剂匀浆的样品中加0.5ml的异丙醇和5ul的糖元。4℃静置半小时，让RNA尽可能的析出。于4℃13,000g离心15分钟。Transfer the aqueous phase to a new centrifuge tube. The aqueous phase is mixed with isopropanol to precipitate RNA therein. The amount of isopropanol added is: 0.5ml of isopropanol and 5ul of glycogen are added to 1ml of TRIZOL-LS reagent homogenized sample. Stand at 4°C for half an hour to let the RNA precipitate as much as possible. Centrifuge at 13,000 g for 15 minutes at 4°C.
4.RNA清洗4. RNA cleaning
移去上清液，每1ml TRIZOL-LS试剂匀浆的样品中加入至少1ml的75％(v/v)乙醇，清洗RNA沉淀。静置10分钟，然后4℃下10000g离心5分钟。Remove the supernatant, and add at least 1 ml of 75% (v/v) ethanol to each 1 ml of TRIZOL-LS reagent homogenized sample to wash the RNA pellet. Let stand for 10 minutes, then centrifuge at 10,000 g for 5 minutes at 4°C.
5.重新溶解RNA沉淀5. Redissolve the RNA pellet
去除乙醇溶液，空气中干燥RNA沉淀5-10分钟，加入无RNA酶的水用枪反复吹打几次，然后55到60℃孵育10分钟。Remove the ethanol solution, dry the RNA pellet in the air for 5-10 minutes, add RNase-free water and pipette several times with a gun, then incubate at 55 to 60°C for 10 minutes.
6.测量浓度：6. Measure the concentration:
通常能得到～5μg RNA/50ml血清。Usually ~5 μg RNA/50ml serum can be obtained.
7.cDNA合成7. cDNA synthesis
(1)稀释模板RNA：使用DEPC水将20－25ng模板RNA稀释至14ul(终浓度为1.492－1.786ng/μl)。(1) Dilute template RNA: dilute 20-25ng template RNA to 14ul with DEPC water (final concentration is 1.492-1.786ng/μl).
(2)准备反应液：将5×Reaction Buffer以及DEPC水置于冰上溶解，并震荡混匀。Enzyme mix置于-20℃冰盒，使用前轻弹混匀后置于冰上。所有试剂均离心后使用。(2) Prepare the reaction solution: dissolve 5×Reaction Buffer and DEPC water on ice, and shake to mix. Enzyme mix should be placed in a -20°C ice box, flick and mix well before use and place on ice. All reagents were used after centrifugation.
(3)配置反应液：配置下表中的反应液(3) Configure the reaction solution: configure the reaction solution in the following table
(4)混合并离心试剂：震荡或者抽吸混匀反应液后离心，以保证所有溶液彻底混合均匀。(4) Mix and centrifuge reagents: Shake or pump to mix the reaction solution and then centrifuge to ensure that all solutions are thoroughly mixed.
(5)逆转录反应及热失活：将反应液于42℃温育60分钟后，于95℃温育5分钟以失活逆转录酶。(5) Reverse transcription reaction and heat inactivation: After incubating the reaction solution at 42° C. for 60 minutes, incubate at 95° C. for 5 minutes to inactivate the reverse transcriptase.
8.Real-Time PCR8. Real-Time PCR
试剂：Reagent:
Nuclease free water(Exiqon)Nuclease free water (Exiqon)
SYBRTM Green master mix(Exiqon)SYBRTM Green master mix (Exiqon)
cDNA模板cDNA template
ROX(Invitrogen)ROX (Invitrogen)
miRNA PCR ARRAY(Exiqon)miRNA PCR ARRAY (Exiqon)
仪器：instrument:
ABI PRISM7900system(Applied Biosystems)ABI PRISM7900system (Applied Biosystems)
(1)准备Real-time PCR试剂：将制备的cDNA模板，DEPC水和SYBRTM Green mastermix置于冰上溶解15-20分钟。(1) Prepare Real-time PCR reagents: put the prepared cDNA template, DEPC water and SYBRTM Green mastermix on ice for 15-20 minutes to dissolve.
(2)稀释cDNA模板：将RT反应获得的cDNA模板用nuclease free water稀释110倍(例如，向20μl反应液中加入2180ul nuclease free water)。(2) Dilute the cDNA template: Dilute the cDNA template obtained by the RT reaction 110 times with nuclease free water (for example, add 2180ul nuclease free water to the 20μl reaction solution).
(3)混合所有反应试剂：(3) Mix all reagents:
A.将PCR板简单离心后，移去封膜。A. After centrifuging the PCR plate briefly, remove the sealing film.
B.将110倍稀释的cDNA模板与2×SYBR Green master mix按照1：1体积比混合。B. Mix the 110-fold diluted cDNA template with 2×SYBR Green master mix at a volume ratio of 1:1.
C.倒置混匀反应液并离心C. Mix the reaction solution by inversion and centrifuge
D.将混合反应液加入板中的每个孔D. Add the mixed reaction solution to each well in the plate
E.重新封好PCR板E. Reseal the PCR plate
(4)将PCR板简单低温离心(4) Simply centrifuge the PCR plate at low temperature
(5)Real-time PCR扩增：根据下表中的反应条件进行Real-time PCR扩增和溶解曲线分析。(5) Real-time PCR amplification: Real-time PCR amplification and melting curve analysis were performed according to the reaction conditions in the table below.
Real-time PCR循环条件如下表：Real-time PCR cycling conditions are as follows:
数据分析：采用ΔΔCt方法Data analysis: using the ΔΔCt method
使用PCR仪器附带的软件进行初步数据分析，获得原始的Cq值(Cp或Ct，依仪器不同名称可能不同)。Use the software attached to the PCR instrument for preliminary data analysis to obtain the original Cq value (Cp or Ct, the name may be different depending on the instrument).
我们建议使用GenEx qPCR分析软件(www.exiqon.com/mirna-pcr-analysis)对数据进行标准和深入的数据分析。We recommend standard and in-depth data analysis of the data using GenEx qPCR Analysis Software (www.exiqon.com/mirna-pcr-analysis).
a.计算每个处理组中的每个通路相关基因的ΔCt。a. Calculation of ΔCt for each pathway-related gene in each treatment group.
ΔCt(group 1)＝average Ct–average of HK genes’Ct for group 1arrayΔCt(group 1)=average Ct–average of HK genes’Ct for group 1array
ΔCt(group 2)＝average Ct–average of HK genes’Ct for group 2arrayΔCt(group 2)=average Ct–average of HK genes’Ct for group 2array
b.计算2个PCR Array(或两组)中每个基因的ΔΔCt。b. Calculate the ΔΔCt for each gene in the 2 PCR Arrays (or two sets).
ΔΔCt＝ΔCt(组2)-ΔCt(组1)ΔΔCt = ΔCt (group 2) - ΔCt (group 1)
备注：通常组1是对照，组2是实验组。Note: Usually group 1 is the control group and group 2 is the experimental group.
c.通过2-ΔΔCt计算组2与组1对应基因的表达差异。c. Calculate the expression difference of corresponding genes between group 2 and group 1 by 2-ΔΔCt.
在芯片初筛后，得到了如下表中的36个差异表达miRNA(3个食管鳞癌血清混合样本中相对于正常样本都超过了1.5倍差异)。After the primary screening by the chip, 36 differentially expressed miRNAs were obtained as shown in the following table (the difference in the three mixed serum samples of esophageal squamous cell carcinoma was more than 1.5 times compared with the normal samples).
对于初筛得到的36个差异表达miRNA，通过训练集、验证集和额外验证集，采用基于qRT-PCR的相对定量法进行验证，具体步骤为：For the 36 differentially expressed miRNAs obtained from the preliminary screening, the relative quantitative method based on qRT-PCR was used for verification through the training set, verification set and additional verification set. The specific steps are as follows:
1.血清RNA提取：选用ABI公司血清RNA提取试剂盒(AM1556)，参照试剂盒说明，每个样本吸取200ul进行提取RNA，并最后用100ul DEPC水进行溶解。1. Serum RNA extraction: Select the serum RNA extraction kit (AM1556) from ABI Company, refer to the kit instructions, absorb 200ul of each sample to extract RNA, and finally dissolve it with 100ul DEPC water.
2.cDNA的制备：2. Preparation of cDNA:
1)采用50μL反应体系进行逆转录实验1) Use 50 μL reaction system for reverse transcription experiment
以上反应体系混匀，瞬时离心后，以下列程序进行反应：The above reaction system was mixed evenly, and after brief centrifugation, the reaction was carried out according to the following procedure:
2)上述反应之后再反应体系中再加入如下反应物2) After the above reaction, add the following reactants to the reaction system
3.qPCR3.qPCR
1)采用5μL反应体系，按以下比例进行试验1) Using 5μL reaction system, conduct the test according to the following ratio
反应体系混匀，瞬时离心后，放置于实时定量PCR仪中，以下列程序进行反应：The reaction system was mixed evenly, centrifuged briefly, placed in a real-time quantitative PCR instrument, and reacted according to the following procedures:
反应结束后添加溶解曲线。Add the dissolution curve after the reaction is complete.
数据分析：通过ΔΔCt方法可以计算获得每个样本中的miRNA的相对浓度。利用SPSS16.0软件进行统计分析，得到了一组在训练集和验证集中均一致于食管鳞癌血清中高表达的5个miRNA：miR-20b-5p、miR-28-3p、miR-192-5p、miR-223-3p和miR-296-5p(在训练集和验证集中P值都小于0.05，图2)。通过这5个miRNA，可以计算每个样本的ROC曲线。如图3，这5个miRNA组成的分子标志物能够很好的区分食管鳞癌患者和正常人群。Data analysis: The relative concentration of miRNA in each sample can be calculated by the ΔΔCt method. Using SPSS16.0 software for statistical analysis, a group of five miRNAs that were consistent in the serum of esophageal squamous cell carcinoma in the training set and validation set were obtained: miR-20b-5p, miR-28-3p, miR-192-5p , miR-223-3p and miR-296-5p (P values in both the training set and the verification set are less than 0.05, Figure 2). With these 5 miRNAs, the ROC curve for each sample can be calculated. As shown in Figure 3, the molecular markers composed of these five miRNAs can well distinguish patients with esophageal squamous cell carcinoma from normal people.
研究组之后进一步检测了这5个miRNA在食管鳞癌组织以及血清中外泌体的表达，食管鳞癌组织提取RNA利用TRIZOL，外泌体提取试剂盒为ExoQuick试剂盒(SBI公司)。200ul血清所提取出的外泌体用200ul DEPC水重悬后，利用AM1556试剂盒(ABI公司)对外泌体RNA进行提取，步骤同血清RNA提取过程。The research team further detected the expression of these five miRNAs in esophageal squamous cell carcinoma tissue and exosomes in serum. RNA was extracted from esophageal squamous cell carcinoma tissue using TRIZOL, and the exosome extraction kit was ExoQuick kit (SBI company). After exosomes extracted from 200ul serum were resuspended in 200ul DEPC water, exosomal RNA was extracted using AM1556 kit (ABI Company), and the procedure was the same as that of serum RNA extraction.
运用非参数检验分析发现，miR-20b-5p、miR-28-3p和miR-192-5p在食管鳞癌组织中的表达要高于癌旁组织(图4)。miR-296-5p在食管鳞癌血清外泌体中的表达亦明显高于正常人群(图5)。Using non-parametric test analysis, it was found that the expression of miR-20b-5p, miR-28-3p and miR-192-5p in esophageal squamous cell carcinoma tissues was higher than that in adjacent tissues (Figure 4). The expression of miR-296-5p in serum exosomes of esophageal squamous cell carcinoma was also significantly higher than that of normal people (Figure 5).
试剂盒包括一批血清miRNA qRT-PCR引物，还可以有相应PCR技术所需的常用试剂，如：逆转录酶，缓冲液，dNTPs，MgCl2，DEPC水，荧光探针，RNA酶抑制剂，Taq酶等，可根据具体采用的实验方法选用，这些常用试剂都是本领域技术人员熟知的，另外还可以有标准品和对照(如定量标化的正常人样本等)。此试剂盒的价值在于只需要血清而不需要其它组织样品，通过最精简的荧光法检测血清样本中miRNA的表达含量，来辅助诊断该样本来源患者的罹患食管鳞癌的可能性。血清miRNA检测方便，且定量精确，大大提高疾病诊断的敏感性和特异性，因此将此试剂盒投入实践，可以帮助指导诊断以及进一步的个体化治疗。The kit includes a batch of serum miRNA qRT-PCR primers, as well as common reagents required for corresponding PCR techniques, such as: reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probes, RNase inhibitors, Taq Enzymes, etc. can be selected according to the specific experimental method used. These commonly used reagents are well known to those skilled in the art. In addition, there may be standards and controls (such as quantitatively standardized normal human samples, etc.). The value of this kit is that it only needs serum and no other tissue samples, and detects the expression level of miRNA in serum samples by the most streamlined fluorescence method to assist in the diagnosis of the possibility of esophageal squamous cell carcinoma in the patient from which the sample originated. Serum miRNA detection is convenient and quantitatively accurate, which greatly improves the sensitivity and specificity of disease diagnosis. Therefore, putting this kit into practice can help guide diagnosis and further individualized treatment.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610723653.1ACN106119392B (en) | 2016-08-25 | 2016-08-25 | A serum miRNA marker related to the auxiliary diagnosis of esophageal squamous cell carcinoma and its application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610723653.1ACN106119392B (en) | 2016-08-25 | 2016-08-25 | A serum miRNA marker related to the auxiliary diagnosis of esophageal squamous cell carcinoma and its application |
| Publication Number | Publication Date |
|---|---|
| CN106119392A CN106119392A (en) | 2016-11-16 |
| CN106119392Btrue CN106119392B (en) | 2019-12-31 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610723653.1AActiveCN106119392B (en) | 2016-08-25 | 2016-08-25 | A serum miRNA marker related to the auxiliary diagnosis of esophageal squamous cell carcinoma and its application |
| Country | Link |
|---|---|
| CN (1) | CN106119392B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733234A (en)* | 2019-03-25 | 2020-10-02 | 深圳大学 | A molecular marker of esophageal cancer and its application |
| CN109825575A (en)* | 2019-04-08 | 2019-05-31 | 首都医科大学附属北京胸科医院 | miRNA markers for auxiliary diagnosis of tuberculosis and their applications |
| CN110283912A (en)* | 2019-07-18 | 2019-09-27 | 暨南大学 | Application of the has-miR-3656 as esophageal squamous cell carcinoma molecular marker |
| CN110716044B (en)* | 2019-10-23 | 2023-04-18 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma |
| CN111286538B (en)* | 2020-02-13 | 2023-04-07 | 朱伟 | Circulating miRNA and carcinoembryonic miRNA markers related to pan-tumor auxiliary diagnosis and application thereof |
| CN112410428A (en)* | 2020-11-19 | 2021-02-26 | 中国医学科学院肿瘤医院 | Application of microRNA molecule in diagnosis and treatment of T1 stage esophageal squamous carcinoma lymph node metastasis |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101861401A (en)* | 2007-10-11 | 2010-10-13 | 俄亥俄州立大学研究基金会 | Methods and compositions for diagnosis and treatment of esophageal adenocarcinoma |
| WO2012012689A2 (en)* | 2010-07-22 | 2012-01-26 | The Johns Hopkins University | Circulating micrornas are biomarkers of various diseases |
| WO2015194627A1 (en)* | 2014-06-18 | 2015-12-23 | 東レ株式会社 | Esophageal cancer detection kit or device, and detection method |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101861401A (en)* | 2007-10-11 | 2010-10-13 | 俄亥俄州立大学研究基金会 | Methods and compositions for diagnosis and treatment of esophageal adenocarcinoma |
| WO2012012689A2 (en)* | 2010-07-22 | 2012-01-26 | The Johns Hopkins University | Circulating micrornas are biomarkers of various diseases |
| WO2015194627A1 (en)* | 2014-06-18 | 2015-12-23 | 東レ株式会社 | Esophageal cancer detection kit or device, and detection method |
| Publication number | Publication date |
|---|---|
| CN106119392A (en) | 2016-11-16 |
| Publication | Publication Date | Title |
|---|---|---|
| CN106119392B (en) | A serum miRNA marker related to the auxiliary diagnosis of esophageal squamous cell carcinoma and its application | |
| CN105950753B (en) | One kind blood plasma miRNA marker relevant to adenocarcinoma of lung auxiliary diagnosis and its application | |
| CN108624695B (en) | A circulating miRNA marker related to auxiliary diagnosis of papillary thyroid carcinoma and its application | |
| CN103667516B (en) | Kit or biological chip for detecting miRNAs for early colonic adenocarcinoma and rectal adenocarcinoma | |
| CN109609633B (en) | Serum miRNA marker related to breast cancer auxiliary diagnosis and application thereof | |
| CN108103198B (en) | A plasma miRNA marker related to auxiliary diagnosis of pancreatic cancer and its application | |
| CN103993093B (en) | Early bladder cancer serum miRNAs specific expression profile and diagnostic model | |
| CN106119393B (en) | Plasma miRNA marker related to esophageal squamous carcinoma auxiliary diagnosis and application thereof | |
| CN109055557B (en) | A serum miRNA marker related to auxiliary diagnosis of pancreatic cancer and its application | |
| CN108103199A (en) | A kind of Xun Huan miRNA marker relevant with oophoroma auxiliary diagnosis and its application | |
| CN103642914B (en) | Plasma/serum circulation microRNA marker related to mlignnt melnom and application of marker | |
| CN106086226B (en) | Plasma miRNA marker related to IgA nephropathy auxiliary diagnosis and application thereof | |
| CN111286538B (en) | Circulating miRNA and carcinoembryonic miRNA markers related to pan-tumor auxiliary diagnosis and application thereof | |
| CN108531586A (en) | A kind of relevant cycle miRNA marker and its application on X chromosome of and Computer-aided Diagnosis of Breast Cancer | |
| CN109609634B (en) | Circulating miRNA marker related to endometrial cancer auxiliary diagnosis and application thereof | |
| CN111424085A (en) | Application of tRNA (transfer ribonucleic acid) derived fragment in preparation of breast cancer diagnostic reagent | |
| CN108929910B (en) | A serum miRNA marker related to auxiliary diagnosis of lung adenocarcinoma and its application | |
| CN107746886B (en) | A plasma miRNA marker related to auxiliary diagnosis of colorectal cancer and its application | |
| CN109536612B (en) | A plasma miRNA marker related to auxiliary diagnosis of nasopharyngeal carcinoma and its application | |
| CN109593852B (en) | Serum miRNA marker related to nasopharyngeal carcinoma auxiliary diagnosis and application thereof | |
| CN109593851B (en) | Plasma miRNA marker related to breast cancer auxiliary diagnosis and application thereof | |
| CN106350582B (en) | Serum miRNA marker related to lung squamous carcinoma auxiliary diagnosis and application thereof | |
| CN109022586B (en) | Plasma miRNA marker related to cervical cancer auxiliary diagnosis and application thereof | |
| CN106086178A (en) | A kind of serum miRNA marker relevant to gastric cancer auxiliary diagnosis and application thereof | |
| CN107723365B (en) | One kind blood plasma miRNA marker relevant to lung squamous cancer auxiliary diagnosis and its application |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | Effective date of registration:20250605 Address after:211500 Jiangsu Province, Nanjing City, Jiangbei New District, Changlu Street, Ningliu Road No. 606, Building B, Room 174 Patentee after:Yansi (Nanjing) Life Technology Co.,Ltd. Country or region after:China Address before:210029 Jiangsu Province, Nanjing City, Gulou District, Guangzhou Road 300 (First Affiliated Hospital of Nanjing Medical University) Patentee before:Zhu Wei Country or region before:China | |
| TR01 | Transfer of patent right |