技术领域technical field
本发明属于基因工程及肿瘤学领域，涉及一种新的食管癌血液miRNA标志物及其应用。The invention belongs to the fields of genetic engineering and oncology, and relates to a new blood miRNA marker for esophageal cancer and its application.
背景技术Background technique
食管癌是世界上最常见的六大恶性肿瘤之一(发病率和死亡率占第6位)，中国是食管癌发病率和死亡率最高的国家(全国发病率居第6位，死亡率居第4)。全球每年新增50万例食管癌患者中，一半以上发生在中国。与西方人群明显不同，中国人食管癌突出的流行病学特征是显著的地域性分布差异，形成明显的高低发区(发病率相差100倍)。河南、河北和山西三省交界的太行山地区，特别是河南的林县、安阳、辉县等地是中国，也是世界上食管癌发病率和死亡率最高的地区。癌预后极差，中晚期患者5年生存率仅15％左右。尽管早期癌(T1N0M0)5年生存率明显高于中晚期，但临床上首次确诊的患者中，早期癌仅占5％左右，其主要原因是患者早期无特异症状，缺乏高危人群预警筛查和早期发现的有效指标和方法。很显然，建立高危人群分子分型和早期发现分子标志物筛查体系，提高早期食管癌发现率，是降低食管癌死亡率的关键。Esophageal cancer is one of the six most common malignant tumors in the world (ranking 6th in incidence and mortality), and China is the country with the highest incidence and mortality of esophageal cancer (ranking 6th in incidence and 1st in mortality) No. 4). Of the 500,000 new cases of esophageal cancer each year worldwide, more than half occur in China. Significantly different from the Western population, the prominent epidemiological feature of Chinese esophageal cancer is the significant regional distribution difference, forming an obvious high and low incidence area (100-fold difference in incidence). The Taihang Mountain area at the junction of Henan, Hebei, and Shanxi provinces, especially Lin County, Anyang, and Hui County in Henan, is the area with the highest incidence and mortality of esophageal cancer in China and the world. The prognosis of cancer is extremely poor, and the 5-year survival rate of advanced patients is only about 15%. Although the 5-year survival rate of early-stage cancer (T1N0M0) is significantly higher than that of middle-advanced cancer, early-stage cancer only accounts for about 5% of patients diagnosed for the first time clinically. Effective indicators and methods for early detection. Obviously, the key to reducing the mortality rate of esophageal cancer is to establish a screening system for molecular typing and early detection of molecular markers in high-risk groups and to improve the detection rate of early esophageal cancer.
食管癌一般依靠临床表现进行诊断。早期，沈琼教授等发明了脱落细胞学检查，为当代食管癌患者带来了福音，提高了临床上食管癌的检出率，使一大批食管癌患者获益。随着技术的发展，X线钡餐检查、B型超声检查、CT、磁共振成像和内镜检查等方法相继产生。但是，这些方法在早期诊断上都存在一定程度的缺陷：影像学学检查对食管癌的诊断具有一定的局限性，内镜检查和脱落细胞学检查取材不当或人为经验不足等都会导致误诊或漏诊。尽管已发现一些肿瘤标志物，如癌胚抗原(CEA)和糖类抗原(如CA19-9、CA74-2等)等，但对食管癌诊断的敏感度和特异度均有限，尤其在发现早期食管癌和鉴别侵袭性无痛性肿瘤方面。因此，寻找新的特异性高和敏感性好的标志物越来越受到国内外的重视。Esophageal cancer is generally diagnosed based on clinical manifestations. In the early days, Professor Shen Qiong and others invented exfoliative cytology examination, which brought good news to contemporary esophageal cancer patients, improved the clinical detection rate of esophageal cancer, and benefited a large number of esophageal cancer patients. With the development of technology, methods such as X-ray barium meal examination, B-mode ultrasonography, CT, magnetic resonance imaging and endoscopy have been produced one after another. However, these methods have a certain degree of defects in early diagnosis: imaging examinations have certain limitations in the diagnosis of esophageal cancer, endoscopic examination and exfoliative cytology examination are improperly drawn or human experience is insufficient, etc. will lead to misdiagnosis or missed diagnosis . Although some tumor markers have been found, such as carcinoembryonic antigen (CEA) and carbohydrate antigens (such as CA19-9, CA74-2, etc.), the sensitivity and specificity for the diagnosis of esophageal cancer are limited, especially in the early stage of detection Esophageal Cancer and Differentiation of Aggressive Painless Tumors. Therefore, finding new markers with high specificity and good sensitivity has attracted more and more attention at home and abroad.
近年来，MicroRNAs(即miRNAs)成为肿瘤分子生物学领域的一个热点，它是一类进化上具有极高保守性的、约有20-25个核苷酸的组成的小单链RNA分子。miRNA本身不具有开放阅读框(ORF)，是一组不编码蛋白质的短序列RNA，并且广泛存在于真核生物中。miRNA具有高度的时序性、保守性和组织特异性，通过与靶mRNA的3′端非编码区结合，降解或者抑制mRNA的翻译导致靶基因的转录后沉默，从而调节生物体内在的与机体生长、发育、疾病发生过程有关的基因的表达。自从参与调控线虫时序发育的lin-4与let-7被发现以来，miRNA分别在2002年和2003年两度入选Science杂志年度十大科技突破。2005年预测miRNAs至少能调控5300个人类基因，即所有基因的30％。随着研究的深入，越来越多的miRNAs不断被发现。成为人们焦点的miRNA已经初步摆脱了DNA光芒的掩盖，从“配角”变成“主角”，并且给DNA的中心地位带来了新的挑战。近年来，miRNA与各种肿瘤的关系已经成为科学家研究的热点和重点，已经发现miRNA通过负调控基因的表达与肺癌，乳腺癌，胃癌，胰腺癌等的发病高度相关。In recent years, MicroRNAs (namely miRNAs) have become a hotspot in the field of tumor molecular biology. It is a class of small single-stranded RNA molecules with a highly conserved evolution and a composition of about 20-25 nucleotides. miRNA itself does not have an open reading frame (ORF), is a group of short-sequence RNAs that do not encode proteins, and widely exists in eukaryotes. miRNA has a high degree of timing, conservation and tissue specificity. By binding to the 3' non-coding region of the target mRNA, it degrades or inhibits the translation of the mRNA, resulting in post-transcriptional silencing of the target gene, thereby regulating the internal and biological growth of the organism. , development, and the expression of genes involved in the process of disease occurrence. Since the discovery of lin-4 and let-7, which are involved in regulating the timing development of nematodes, miRNA has been selected as one of the top ten scientific and technological breakthroughs of the Science magazine in 2002 and 2003 respectively. In 2005, it was predicted that miRNAs could regulate at least 5300 human genes, ie 30% of all genes. With the deepening of research, more and more miRNAs have been discovered. miRNA, which has become the focus of people, has initially got rid of the cover of the light of DNA, changed from a "supporting role" to a "leading role", and brought new challenges to the central position of DNA. In recent years, the relationship between miRNA and various tumors has become the focus and focus of scientists' research. It has been found that miRNA is highly related to the pathogenesis of lung cancer, breast cancer, gastric cancer, pancreatic cancer, etc. through negative regulation of gene expression.
研究发现，血清/血浆miRNA非常稳定，酸碱环境、煮沸、长期保存、反复冻融等均不会造成血清/血浆miRNA的损失，这些小分子RNAs含量丰富、易于定量检测，而且miRNA在外周血中的表达同样具有肿瘤相关性和组织特异性。血清/血浆miRNA作为肿瘤诊断和预后分子标志物不仅具有创伤小、灵敏度高、可重复性好、费用低、便捷的优势，而且还可改进疾病诊断、肿瘤分类、预后估计、疗效及复发预测的精度，其作为肿瘤标志物具有良好的临床应用前景。近年来，已有研究发现了一些在食管癌患者血清/血浆中异常表达的miRNAs，其中有7种血清miRNA有潜能成为诊断食管癌的非侵袭性生物标志物,这使血清游离miRNA作为食管癌特异性肿瘤标志物的设想成为可能，这些miRNAs在血清/血浆中表达情况与在组织中的表达情况是否一致，是否能以此作为食管癌的特异性标志物用于食管癌的诊断还有待进一步研究。Studies have found that serum/plasma miRNA is very stable, acid-base environment, boiling, long-term storage, repeated freezing and thawing will not cause the loss of serum/plasma miRNA, these small molecule RNAs are abundant and easy to quantitatively detect, and miRNA in peripheral blood The expression in is also tumor-associated and tissue-specific. As a molecular marker for tumor diagnosis and prognosis, serum/plasma miRNA not only has the advantages of less trauma, high sensitivity, good repeatability, low cost, and convenience, but also can improve the methods of disease diagnosis, tumor classification, prognosis estimation, curative effect and recurrence prediction. Accuracy, it has a good clinical application prospect as a tumor marker. In recent years, some studies have found some abnormally expressed miRNAs in the serum/plasma of patients with esophageal cancer, among which 7 serum miRNAs have the potential to become non-invasive biomarkers for the diagnosis of esophageal cancer, which makes serum free miRNAs a useful tool for esophageal cancer. It is possible to conceive specific tumor markers. Whether the expression of these miRNAs in serum/plasma is consistent with that in tissues, and whether they can be used as specific markers for esophageal cancer in the diagnosis of esophageal cancer remains to be seen. Research.
目前还没有用于食管癌诊断的较为稳定的生物标志物的报道，若能筛选出食管癌异常表达的血清/血浆miRNAs作为生物标志物，并开发出相应的诊断试剂盒，将会有力的推动我国食管癌的诊断。At present, there are no reports of relatively stable biomarkers for the diagnosis of esophageal cancer. If the abnormally expressed serum/plasma miRNAs of esophageal cancer can be screened out as biomarkers and corresponding diagnostic kits can be developed, it will be a strong impetus. Diagnosis of esophageal cancer in my country.
发明内容Contents of the invention
针对现有技术存在的问题和不足，发明人通过分离和研究食管癌患者以及与食管癌患者年龄、性别匹配的健康人对照血清/血浆中miRNAs，寻找一组与食管癌高度相关的高特异性和敏感性的miRNAs，并研制出可便于临床应用的食管癌诊断试剂盒，为食管癌的筛查和诊断提供数据支持，为发现具有潜在治疗价值的新型小分子药物提供数据支持。Aiming at the problems and deficiencies in the prior art, the inventors searched for a group of highly specific miRNAs highly related to esophageal cancer by isolating and studying the miRNAs in the serum/plasma of esophageal cancer patients and age- and gender-matched healthy controls of esophageal cancer patients. and sensitive miRNAs, and developed a diagnostic kit for esophageal cancer that is convenient for clinical application, providing data support for the screening and diagnosis of esophageal cancer, and providing data support for the discovery of new small molecule drugs with potential therapeutic value.
为实现发明目的，本发明采用的技术方案如下：For realizing the purpose of the invention, the technical scheme adopted in the present invention is as follows:
本发明提供了一种用于食管癌辅助诊断的血液miRNA标志物，所述血液miRNA标志物为miR-30d和miR-10的组合，所述miR-30d的序列如SEQ ID NO.8所示，所述miR-10的序列如SEQ ID NO.9所示。The present invention provides a blood miRNA marker for auxiliary diagnosis of esophageal cancer, the blood miRNA marker is a combination of miR-30d and miR-10, and the sequence of miR-30d is shown in SEQ ID NO.8 , the sequence of the miR-10 is shown in SEQ ID NO.9.
本发明还提供了一种上述血液miRNA标志物在制备食管癌辅助诊断试剂盒或试剂中的应用。The present invention also provides an application of the above-mentioned blood miRNA marker in the preparation of an auxiliary diagnostic kit or reagent for esophageal cancer.
根据上述的应用，优选地，所述试剂盒包含检测miR-30d和miR-10的检测试剂。According to the above application, preferably, the kit includes detection reagents for detecting miR-30d and miR-10.
本发明还提供了一种miR-30d和miR-10的检测试剂在制备食管癌辅助诊断试剂或试剂盒中的应用。The present invention also provides the application of a detection reagent of miR-30d and miR-10 in the preparation of an auxiliary diagnostic reagent or kit for esophageal cancer.
根据上述的应用，优选地，所述检测试剂包括RNA反转录引物和/或DNA扩增引物。According to the above application, preferably, the detection reagent includes RNA reverse transcription primers and/or DNA amplification primers.
根据上述的应用，优选地，所述RNA反转录引物为Oligo(dt)引物；用于检测miR-30d的DNA扩增引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示，用于检测miR-10的DNA扩增引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。According to the above application, preferably, the RNA reverse transcription primer is an Oligo (dt) primer; the nucleotide sequence of the DNA amplification primer used to detect miR-30d is as SEQ ID NO.1 and SEQ ID NO.2 As shown, the nucleotide sequences of the DNA amplification primers used to detect miR-10 are shown in SEQ ID NO.3 and SEQ ID NO.4.
本发明还提供了一种用于食管癌辅助诊断的试剂盒，所述试剂盒用于检测血液中的miR-30d和miR-10。The present invention also provides a kit for auxiliary diagnosis of esophageal cancer, the kit is used for detecting miR-30d and miR-10 in blood.
根据上述的试剂盒，优选地，所述试剂盒包含检测miR-30d和miR-10的检测试剂。According to the kit above, preferably, the kit includes detection reagents for detecting miR-30d and miR-10.
根据上述的试剂盒，优选地，所述检测试剂包括QPCR实验中使用的RNA反转录引物和/或DNA扩增引物，所述RNA反转录引物为Oligo(dt)引物；用于检测miR-30d的DNA扩增引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示，用于检测miR-10的DNA扩增引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。According to the above-mentioned kit, preferably, the detection reagent includes RNA reverse transcription primers and/or DNA amplification primers used in QPCR experiments, and the RNA reverse transcription primers are Oligo (dt) primers; used to detect miR The nucleotide sequence of the DNA amplification primer of -30d is as shown in SEQ ID NO.1 and SEQ ID NO.2, is used for detecting the nucleotide sequence of the DNA amplification primer of miR-10 such as SEQ ID NO.3 and Shown in SEQ ID NO.4.
根据上述的试剂盒，优选地，所述试剂盒还包含检测内参基因的检测试剂；所述内参基因为SnRNA U6，所述内参基因的检测试剂包括QPCR实验中使用的RNA反转录引物和/或DNA扩增引物，所述RNA反转录引物的核苷酸序列如为SEQ ID NO.5所示，用于检测SnRNA U6的DNA扩增引物的核苷酸序列如SEQ ID NO.6和SEQ ID NO.7所示。According to the kit above, preferably, the kit also includes a detection reagent for detecting an internal reference gene; the internal reference gene is SnRNA U6, and the detection reagent for the internal reference gene includes RNA reverse transcription primers and/or used in QPCR experiments Or DNA amplification primer, the nucleotide sequence of described RNA reverse transcription primer is as shown in SEQ ID NO.5, is used to detect the nucleotide sequence of the DNA amplification primer of SnRNA U6 such as SEQ ID NO.6 and Shown in SEQ ID NO.7.
根据上述的试剂盒，优选地，所述试剂盒还包含反转录反应试剂和PCR扩增试剂，如反转录酶、缓冲液、dNTPs，MgCl2，DEPC水和Taq酶等；还可以含有标准品和/或对照品。According to the above kit, preferably, the kit also includes reverse transcription reaction reagents and PCR amplification reagents, such as reverse transcriptase, buffer, dNTPs, MgCl2 , DEPC water and Taq enzyme, etc.; Standards and/or controls.
具体地说，本发明解决问题的技术方案包括：Specifically, the technical scheme for solving problems of the present invention includes:
(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。(1) Establish a unified standard specimen library and database: collect standard operating procedures (SOP) to collect standard blood samples, and systematically collect complete demographic and clinical data.
(2)血液miRNA差异表达谱分析：选择食管癌病例，与食管癌病例年龄、性别匹配的健康人对照，检测其血清/血浆miRNA表达谱及含量，分析食管癌病例和健康人对照间血清/血浆miRNA的共性和特性，筛选差异表达miRNAs；采用筛选的差异表达miRNAs进行进一步验证，确定食管癌发病相关血清/血浆miRNAs。(2) Differential expression profile analysis of blood miRNA: select esophageal cancer cases, and healthy controls matched with age and sex of esophageal cancer cases, detect their serum/plasma miRNA expression profile and content, and analyze the serum/plasma miRNA expression profile between esophageal cancer cases and healthy controls. The commonality and characteristics of plasma miRNAs were screened for differentially expressed miRNAs; the screened differentially expressed miRNAs were used for further verification to determine the serum/plasma miRNAs related to the pathogenesis of esophageal cancer.
(3)血清/血浆miRNA筛查和诊断试剂盒的研制：根据食管癌病例和健康人对照的特异血清/血浆miRNA开发miRNAs诊断试剂盒。(3) Development of serum/plasma miRNA screening and diagnostic kits: develop miRNAs diagnostic kits based on specific serum/plasma miRNAs from esophageal cancer cases and healthy controls.
上述步骤(2)中血清/血浆miRNA定量分析可以采用RT-PCR、QPCR、Solexa测序技术、Tapman low densityarray(TLDA)芯片检测等来完成。在本发明的具体的实施方案中，采用QPCR进行验证。Quantitative analysis of serum/plasma miRNA in the above step (2) can be accomplished by RT-PCR, QPCR, Solexa sequencing technology, Tapman low density array (TLDA) chip detection and the like. In a specific embodiment of the present invention, QPCR is used for verification.
采用QPCR进行差异表达miRNA的验证，具体的操作步骤为：Using QPCR to verify the differentially expressed miRNA, the specific steps are as follows:
(1)提取样本总RNA；(1) Extract the total RNA of the sample;
(2)将步骤(1)获得的RNA逆转录成cDNA；(2) Reverse transcribing the RNA obtained in step (1) into cDNA;
(3)在荧光实时定量PCR仪上将miRNA和参照基因进行扩增检测；(3) On the fluorescent real-time quantitative PCR instrument, the miRNA and the reference gene are amplified and detected;
(4)通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量。(4) Determine the target band by melting curve analysis and electrophoresis, and perform relative quantification by ΔΔCT method.
与现有技术相比，本发明取得的积极有益效果为：Compared with the prior art, the positive beneficial effect that the present invention obtains is:
(1)本发明采用miRNAs芯片对食管癌病例和健康人对照的血清/血浆miRNA表达谱进行检测分析，获取在食管癌病例和健康人对照间血清/血浆中差异表达miRNAs，并将这些差异表达miRNAs在大样本人群中进行验证，筛选得到了与食管癌稳定关联的血液miRNA标志物组合(miR-30d和miR-10的组合)，该血液miRNA标志物组合对食管癌的诊断灵敏度达到了90％，特异度达到了81％，远高于现有食管癌的筛查诊断方法，因此，本发明的血液miRNA标志物组合可用于食管癌的临床筛查诊断，有助于反映食管癌无症状高危人群的疾病状态，为临床医生快速准确掌握患者病情、及时采取更具个性化的防治方案提供支持。(1) The present invention uses miRNAs chips to detect and analyze the serum/plasma miRNA expression profiles of esophageal cancer cases and healthy controls, obtain differentially expressed miRNAs in serum/plasma between esophageal cancer cases and healthy controls, and express these differentially miRNAs were verified in a large sample population, and a blood miRNA marker combination (combination of miR-30d and miR-10) stably associated with esophageal cancer was screened. The diagnostic sensitivity of this blood miRNA marker combination for esophageal cancer reached 90%. %, the specificity has reached 81%, which is much higher than the existing screening and diagnosis methods for esophageal cancer. Therefore, the blood miRNA marker combination of the present invention can be used for clinical screening and diagnosis of esophageal cancer, which helps to reflect the asymptomatic of esophageal cancer The disease status of high-risk groups provides support for clinicians to quickly and accurately grasp the patient's condition and take more personalized prevention and treatment plans in a timely manner.
(2)血液miRNAs是一种新型生物标志物，不仅稳定、微创、易于检测，且定量精确，因此，本发明通过检测血清/血浆中miR-30d和miR-10表达将大大提高食管癌诊断的敏感性和特异性，为食管癌的诊断和治疗开创全新局面，同时为其他疾病生物标志物的研制提供借鉴。(2) Blood miRNAs are a new type of biomarker, which is not only stable, minimally invasive, easy to detect, but also quantitatively accurate. Therefore, the present invention will greatly improve the diagnosis of esophageal cancer by detecting the expression of miR-30d and miR-10 in serum/plasma The sensitivity and specificity of this method will create a new situation for the diagnosis and treatment of esophageal cancer, and provide reference for the development of other disease biomarkers.
(3)本发明以检测与食管癌辅助诊断相关的血液miRNA标志物组合(miR-30d和miR-10的组合)的RNA反转录引物和DNA扩增引物来制备试剂盒，该试剂盒的灵敏度高，特异性强，操作简单，使用方便，可用于食管癌的辅助诊断和食管癌无症状高危人群的筛查，使得食管癌的诊断更加方便易行，为临床医生快速准确对食管癌无症状高危人群进行筛查诊断，为临床治疗效果评价奠定基础，并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。(3) The present invention uses RNA reverse transcription primers and DNA amplification primers to detect the blood miRNA marker combination (combination of miR-30d and miR-10) related to the auxiliary diagnosis of esophageal cancer to prepare a kit. High sensitivity, strong specificity, simple operation, easy to use, can be used for auxiliary diagnosis of esophageal cancer and screening of asymptomatic high-risk groups of esophageal cancer, making the diagnosis of esophageal cancer more convenient and easy, and providing clinicians with fast and accurate diagnosis of esophageal cancer. Screening and diagnosis of high-risk groups of symptoms will lay the foundation for the evaluation of clinical treatment effects and help to discover new small molecule drug targets with potential therapeutic value.
附图说明Description of drawings
图1为显示食管癌病例组和健康人对照组miRNA的表达箱线图。Figure 1 is a boxplot showing the expression of miRNAs in the esophageal cancer case group and the healthy control group.
图2为显示食管癌病例组对健康人对照组为参照的ROC曲线。Fig. 2 is a ROC curve showing that the esophageal cancer case group is compared with the healthy control group.
具体实施方式Detailed ways
以下通过具体的实施例对本发明作进一步详细说明，但并不限制本发明的范围。The present invention will be described in further detail below through specific examples, but the scope of the present invention is not limited.
下列实施例中未注明具体条件的实验方法，均采用本技术领域常规技术，或按照生产厂商所建议的条件；所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The experimental methods that do not indicate specific conditions in the following examples all adopt conventional techniques in this technical field, or according to the conditions suggested by the manufacturer; the reagents or instruments used that do not indicate the manufacturer are all commercially available. product.
实施例一：与人类食管癌相关的miRNA的筛选Example 1: Screening of miRNAs associated with human esophageal cancer
1、样本的收集和样本资料的整理1. Collection of samples and collation of sample data
病例为郑州大学第一附属医院河南省食管癌重点开放实验室(1973-2018年)收集的新发食管癌病例，均经病理组织学确诊。对照为同期进行社区疾病筛查的健康个体，按性别和年龄(±5岁)与病例进行频数匹配。用于研究的样本为同期收取，采样、分装、保存条件均一。通过对样品资料的整理，发明人从中选择了200例符合标准的样本(食管癌患者组100例，健康对照组100例)作为miRNA芯片检测的实验样品，并系统采集了这些样本的人口学资料和临床资料等情况。其中，样本的选取标准为：The cases were new cases of esophageal cancer collected by the Henan Key Open Laboratory of Esophageal Cancer (1973-2018), the First Affiliated Hospital of Zhengzhou University, all of which were confirmed by histopathology. Controls were healthy individuals undergoing community disease screening over the same period, and were frequency-matched with cases by sex and age (±5 years). The samples used for the research were collected at the same period, and the conditions of sampling, packaging and storage were uniform. Through sorting out the sample data, the inventor selected 200 samples that met the criteria (100 cases in the esophageal cancer patient group and 100 cases in the healthy control group) as experimental samples for miRNA chip detection, and systematically collected the demographic data of these samples and clinical data. Among them, the sample selection criteria are:
(1)新发食管癌病例；(1) new cases of esophageal cancer;
(2)采血前未经过手术和放化疗，无手术前放化疗；(2) No surgery, radiotherapy and chemotherapy before blood collection, no preoperative radiotherapy and chemotherapy;
(3)与病例年龄、性别匹配的健康人对照。(3) Healthy subjects matched with the age and sex of the cases as controls.
2、miRNA芯片检测2. miRNA chip detection
(1)外周血总RNA的提取：(1) Extraction of total RNA from peripheral blood:
对上述选取的200例样本的血液进行总RNA提取，其具体步骤为：Total RNA was extracted from the blood of the 200 samples selected above, and the specific steps were as follows:
①取冻存的血液并充分解冻，加入3倍体积红细胞裂解液，混匀后室温放置10分钟，10000rmp离心1分钟；① Take the frozen blood and thaw it fully, add 3 times the volume of red blood cell lysate, mix well, let it stand at room temperature for 10 minutes, and centrifuge at 10,000rmp for 1 minute;
②彻底吸弃上清，收集白细胞沉淀；② Discard the supernatant thoroughly and collect the white blood cell pellet;
③每100-200μl血液收集的白细胞沉淀加入1ml Trizol。室温放置5min，使样品充分裂解。③Add 1ml Trizol to every 100-200μl blood collected leukocyte precipitate. Place at room temperature for 5 minutes to fully lyse the sample.
④4℃下12000rpm离心10min，取上清；④Centrifuge at 12000rpm for 10min at 4°C, and take the supernatant;
⑤加入Trizol，室温保存5min；⑤ Add Trizol and store at room temperature for 5 minutes;
⑥加氯仿0.2ml，用力振荡离心管，充分混匀，室温下放置5min-10min；⑥ Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5min-10min;
⑦12000rpm高速离心15min后吸取上层水相(吸70％)到另一新离心管中,注意不要吸到两层水相之间的蛋白物质。移入新管，加入等体积的-20℃预冷异丙醇，充分颠倒混匀，置于冰上10min；⑦After centrifuging at 12000rpm for 15 minutes, suck the upper aqueous phase (absorb 70%) into another new centrifuge tube, be careful not to suck the protein material between the two aqueous phases. Transfer to a new tube, add an equal volume of -20°C pre-cooled isopropanol, mix thoroughly by inverting, and place on ice for 10 minutes;
⑧12000rpm高速离15min后小心弃掉上清液，按1ml/ml Trizol的比例加入75％DEPC乙醇洗涤沉淀(4℃保存)，洗涤沉淀物，振荡混合，4℃下12000rpm高速离心5min；⑧Centrifuge at 12000rpm for 15min at high speed, carefully discard the supernatant, add 75% DEPC ethanol at a ratio of 1ml/ml Trizol to wash the precipitate (store at 4°C), wash the precipitate, shake and mix, and centrifuge at 12000rpm at 4°C for 5min at high speed;
⑨弃去乙醇液体，室温下放置5min以充分晾干沉淀，加入DEPC处理过的水溶解沉淀；⑨ Discard the ethanol liquid, place it at room temperature for 5 minutes to fully dry the precipitate, and add DEPC-treated water to dissolve the precipitate;
⑩用Nanodrop2000紫外分光光度计测量RNA纯度及浓度，冻存于-70℃。⑩Use a Nanodrop2000 UV spectrophotometer to measure the RNA purity and concentration, and freeze it at -70°C.
(2)miRNA芯片操作：(2) miRNA chip operation:
miRNA芯片购自ABI公司，按照miRNA芯片说明书的指示进行miRNA表达谱的检测。The miRNA chip was purchased from ABI Company, and the miRNA expression profile was detected according to the instructions of the miRNA chip manual.
(3)结果：(3) Results:
根据miRNA芯片的检测结果，发现食管癌患者组血液中miR-30d、miR-10的表达显著高于健康对照组。According to the detection results of the miRNA chip, it was found that the expression of miR-30d and miR-10 in the blood of the esophageal cancer patient group was significantly higher than that of the healthy control group.
实施例二：QPCR验证差异表达的miRNAExample 2: QPCR verification of differentially expressed miRNAs
根据实施例一中miRNA芯片的检测结果，选择miR-30d和miR-10进行进一步的QPCR验证。按照实施例一中的样本收集和样本资料整理的方式选择食管癌组和对照组各100例样本。According to the detection results of the miRNA chip in Example 1, miR-30d and miR-10 were selected for further QPCR verification. According to the method of sample collection and sample data collation in Example 1, 100 samples were selected from each of the esophageal cancer group and the control group.
1、外周血总RNA的提取1. Extraction of total RNA from peripheral blood
RNA的提取过程的具体操作步骤与实施例一相同，在此不再赘述。The specific operation steps of the RNA extraction process are the same as those in Example 1, and will not be repeated here.
2、逆转录2. Reverse transcription
将上述步骤1提取的总RNA模板(10pg-1μg)与2μl 10×缓冲液、2μl dATP(10mM)、0.5μl polyA多聚酶、0.5μl核糖核酸酶(RNase)抑制剂和无核糖核酸酶水(RNase freewater)混合，总体积为20μl，37℃孵育1h。然后向反应管中加入1μl 0.5μg/μl Oligo(dT)RT引物，70℃孵育5min后立刻冰上孵育至少2min，打断RNA和引物的二级结构。最后，将上述20μl反应混合物与4μl 5×缓冲液、1μl dNTP(10mM)，0.5μl M-MLV逆转录酶，0.5μl核糖核酸酶(RNase)抑制剂，10μl polyA反应混合液和4μl无核糖核酸酶水(RNase free water)混合，42℃孵育1h。Mix the total RNA template (10 pg-1 μg) extracted in the above step 1 with 2 μl 10× buffer, 2 μl dATP (10 mM), 0.5 μl polyA polymerase, 0.5 μl ribonuclease (RNase) inhibitor and ribonuclease-free water (RNase freewater) in a total volume of 20 μl and incubated at 37°C for 1 h. Then add 1 μl of 0.5 μg/μl Oligo(dT)RT primer to the reaction tube, incubate at 70°C for 5 minutes and immediately incubate on ice for at least 2 minutes to break the secondary structure of RNA and primers. Finally, mix the above 20 μl reaction mixture with 4 μl 5× buffer, 1 μl dNTP (10 mM), 0.5 μl M-MLV reverse transcriptase, 0.5 μl ribonuclease (RNase) inhibitor, 10 μl polyA reaction mix and 4 μl RNA-free Enzyme water (RNase free water) was mixed and incubated at 42°C for 1h.
3、QPCR反应3. QPCR reaction
采用25μl反应体系，每个样本设置3个平行管，所有扩增反应均重复三次以上以保证结果的可靠性。QPCR的反应体系为：SYBR Green Realtime PCR Master Mix 12.5μl，正向引物(5μM/μl)1μl，反向引物(5μM/μl)1μl，模板cDNA 2.0μl，无酶水8.5μl。QPCR的扩增程序为：95℃10min；95℃15s，60℃60s，45个循环。以SYBR Green作为荧光标记物，在LightCycler荧光实时定量PCR仪上进行PCR反应。检测miR-30d的正向引物序列如SEQ ID NO.1所示，反向引物序列如SEQ ID NO.2所示；检测miR-10的正向引物序列如SEQ ID NO.3所示，反向引物序列如SEQ ID NO.4所示。以snRNA U6作为内参基因，检测内参基因snRNA U6的正向引物序列如SEQ ID NO.6所示；反向引物序列如SEQ ID NO.7所示。通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量，结果如图1所示。由图1可知，与食管癌患者组相比，miR-30d和miR-10在健康对照者血液中的含量较低。而且该检测结果同miRNA芯片的检测结果一致。A 25 μl reaction system was used, three parallel tubes were set up for each sample, and all amplification reactions were repeated more than three times to ensure the reliability of the results. The reaction system of QPCR is: SYBR Green Realtime PCR Master Mix 12.5 μl, forward primer (5 μM/μl) 1 μl, reverse primer (5 μM/μl) 1 μl, template cDNA 2.0 μl, enzyme-free water 8.5 μl. The amplification program of QPCR is: 95°C 10min; 95°C 15s, 60°C 60s, 45 cycles. Using SYBR Green as a fluorescent marker, the PCR reaction was performed on a LightCycler fluorescent real-time quantitative PCR instrument. The forward primer sequence for detecting miR-30d is shown in SEQ ID NO.1, the reverse primer sequence is shown in SEQ ID NO.2; the forward primer sequence for detecting miR-10 is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.3. The sequence of the primer is shown in SEQ ID NO.4. Using snRNA U6 as an internal reference gene, the forward primer sequence for detecting the internal reference gene snRNA U6 is shown in SEQ ID NO.6; the reverse primer sequence is shown in SEQ ID NO.7. The target band was determined by melting curve analysis and electrophoresis, and the ΔΔCT method was used for relative quantification. The results are shown in Figure 1. It can be seen from Figure 1 that compared with the esophageal cancer patient group, the levels of miR-30d and miR-10 in the blood of healthy controls were lower. Moreover, the detection result is consistent with the detection result of the miRNA chip.
实施例三：分析miRNA对食管癌的诊断价值Example 3: Analysis of the diagnostic value of miRNA for esophageal cancer
根据实施例二中QPCR的结果，对2组血浆样品(食管癌患者组和健康对照组)miR-30d、miR-10表达水平的分析，以健康对照组miR-30d、miR-10表达量的单侧95％参考值范围为标准，利用SPSS 21.0软件绘制ROC曲线来评估预测的灵敏性和特异性，进而评估这2种miRNAs对食管癌发病的判断能力，其结果见图2。由图2可知，单独使用miRNA-30d为指标区分食管癌患者组和健康对照组时，其AUC值为0.676，最佳临界点的敏感性为71％、特异性为74％。单独使用miRNA-10区分食管癌患者组和健康对照组时，其AUC值为0.724，最佳临界点的敏感性为56％、特异性为70％。According to the result of QPCR in embodiment two, to the analysis of miR-30d, miR-10 expression level of 2 groups of plasma samples (esophagus cancer patient group and healthy control group), with the healthy control group miR-30d, miR-10 expression level The one-sided 95% reference value range was used as the standard, and SPSS 21.0 software was used to draw ROC curves to evaluate the sensitivity and specificity of prediction, and then evaluate the ability of these two miRNAs to determine the incidence of esophageal cancer. The results are shown in Figure 2. It can be seen from Figure 2 that when using miRNA-30d alone as an indicator to distinguish the esophageal cancer patient group from the healthy control group, the AUC value was 0.676, the sensitivity of the optimal cut-off point was 71%, and the specificity was 74%. When using miRNA-10 alone to distinguish the esophageal cancer patient group from the healthy control group, its AUC value was 0.724, the sensitivity of the optimal cut-off point was 56%, and the specificity was 70%.
基于单个miRNA的特异性较差，我们把两种miRNA联合作为一个miRNA panle，根据单个指标的cut-off值对食管癌病人及健康对照组重新进行风险预估，得到两种miRNA联合后的复合指标，利用spss软件制作ROC曲线图(参见图2)。由图2可知，利用miRNA panle区分食管癌组和正常组时，其AUC值为0.804，即miR-30d、miR-10联合作为一个miRNA panle能够以80.4％的AUC将食管癌患者组和健康对照组分开，最佳临界点的敏感性为90％、特异性为81％，因此，miR-30d和miR-10联合使用能够很好地将健康对照和食管癌患者区分，而且诊断食管癌的敏感性和特异性均高于单独使用其中任何一种miRNA。Based on the poor specificity of a single miRNA, we combined the two miRNAs as a miRNA panle, and re-estimated the risk of esophageal cancer patients and healthy controls based on the cut-off value of a single indicator, and obtained the composite of the two miRNAs. Index, using spss software to make ROC curve (see Figure 2). It can be seen from Figure 2 that when miRNA panle is used to distinguish the esophageal cancer group from the normal group, its AUC value is 0.804, that is, the combination of miR-30d and miR-10 as a miRNA panle can separate the esophageal cancer patient group from the healthy control group with an AUC of 80.4%. The sensitivity of the optimal cut-off point was 90% and the specificity was 81%. Therefore, the combined use of miR-30d and miR-10 can well distinguish healthy controls from patients with esophageal cancer, and the sensitivity of diagnosing esophageal cancer The performance and specificity were higher than using any one of these miRNAs alone.
实施例四：食管癌辅助诊断试剂盒的制作Example 4: Production of an auxiliary diagnostic kit for esophageal cancer
食管癌辅助诊断试剂盒的制作和操作流程是基于miRNA芯片检测和QPCR定量检测技术。该食管癌辅助诊断试剂盒用于检测血液中的miR-30d和miR-10；该试剂盒包含检测miR-30d和miR-10的检测试剂；所述检测试剂包括QPCR实验中使用的RNA反转录引物和DNA扩增引物，所述RNA反转录引物为Oligo(dt)引物；用于检测miR-30d的DNA扩增引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示，用于检测miR-10的DNA扩增引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。进一步地，所述试剂盒还包含检测内参基因SnRNA U6的检测试剂，所述内参基因SnRNA U6的检测试剂包括QPCR实验中使用的RNA反转录引物和DNA扩增引物，所述RNA反转录引物的核苷酸序列如SEQ ID NO.5所示，用于检测SnRNA U6的DNA扩增引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示。更进一步地，所述试剂盒还包含常用的反转录反应试剂和PCR扩增试剂，如反转录酶、dNTPs，MgCl2，双蒸水，Taq酶等，这些常用试剂都是本领域技术人员熟知的，另外还可以含有有标准品和对照。The production and operation process of the auxiliary diagnostic kit for esophageal cancer are based on miRNA chip detection and QPCR quantitative detection technology. The esophageal cancer auxiliary diagnostic kit is used to detect miR-30d and miR-10 in the blood; the kit contains detection reagents for detecting miR-30d and miR-10; the detection reagents include RNA inversion used in QPCR experiments Recording primer and DNA amplification primer, described RNA reverse transcription primer is Oligo (dt) primer; For detecting the nucleotide sequence of the DNA amplification primer of miR-30d such as SEQ ID NO.1 and SEQ ID NO.2 As shown, the nucleotide sequences of the DNA amplification primers used to detect miR-10 are shown in SEQ ID NO.3 and SEQ ID NO.4. Further, the kit also includes detection reagents for detecting the internal reference gene SnRNA U6, the detection reagents for the internal reference gene SnRNA U6 include RNA reverse transcription primers and DNA amplification primers used in QPCR experiments, and the RNA reverse transcription The nucleotide sequence of the primer is shown in SEQ ID NO.5, and the nucleotide sequence of the DNA amplification primer used to detect SnRNA U6 is shown in SEQ ID NO.5 and SEQ ID NO.6. Furthermore, the kit also contains commonly used reverse transcription reaction reagents and PCR amplification reagents, such as reverse transcriptase, dNTPs, MgCl2 , double distilled water, Taq enzyme, etc., these commonly used reagents are skilled in the art Standards and controls may additionally be included, as is well known.
该试剂盒的检测方法包括：(1)提取样本总RNA；(2)将提取的RNA逆转录成cDNA；(3)在荧光实时定量PCR仪上将miRNA和参照基因进行扩增检测；(4)通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量等步骤；每一个步骤的具体操作参见实施例三。The detection method of the kit includes: (1) extraction of total RNA from the sample; (2) reverse transcription of the extracted RNA into cDNA; (3) amplification and detection of miRNA and reference genes on a fluorescent real-time quantitative PCR instrument; (4) ) Determination of the target band by melting curve analysis and electrophoresis, and relative quantification by ΔΔCT method; see Example 3 for the specific operation of each step.
该试剂盒的价值在于只需要外周血而不需要其它组织样品，通过最精简和特异的引物检测miR-30d和miR-10的表达辅助判断食管癌，不仅稳定，检测方便，而且定量精确，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，可以帮助指导临床准确做出诊断。The value of this kit is that only peripheral blood is needed and no other tissue samples are needed, and the expression of miR-30d and miR-10 can be detected by the most streamlined and specific primers to assist in the judgment of esophageal cancer. Improve the sensitivity and specificity of disease diagnosis, so putting this kit into practice can help guide clinical diagnosis accurately.
以上所述仅为本发明的较佳实施例而已，但不仅限于上述实例，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention, but not limited to the above examples, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.
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| CN112481373A (en)* | 2020-12-21 | 2021-03-12 | 华北理工大学 | circRNA detection kit for auxiliary diagnosis of autism |
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