基于TCGA数据库的肝癌组织microRNA表达谱分析及miR-589-5p在肝癌中作用的初步探究

发布时间：2018-06-02 20:14

本文选题：肝癌 + microRNA　；参考：《吉林大学》2017年硕士论文

【摘要】：研究背景:肝细胞癌(Hepatocellular carcinoma,HCC)是成年人最常见的肝脏原发性恶性肿瘤,占原发性肝癌的80-90%。在全球范围内,其发病率在人体恶性肿瘤中居第五位,是癌症相关死亡的第三大主因。据统计,HCC发生率每年增加3-9%,新增病例约78万,其中50%发生在我国,由乙肝导致肝硬化进而发展为HCC是其发病率逐年递增的主要原因。HCC的发生是一个多因素、多阶段的过程,但由于其发病隐匿,转移迅速,侵袭性强,且缺乏灵敏的早期诊断分子标志物,诊断时多已发展到晚期。尽管有肝移植、肿瘤切除、栓塞、化疗等多种方案适用于HCC的治疗,其预后依然很差,5年生存率不到5%,故寻求高灵敏度、高特异性的肿瘤标志物对提高肝细胞癌患者的生存质量和生存时间有着极为重要的意义。Micro RNA(miRNA)是一类内生的、长度约为18-25个核苷酸的非编码小分子RNA,常通过结合靶标信使RNA(m RNAs)的3’非翻译区(3’UTR)抑制其翻译或加速其降解这两种主要机制调控m RNA的表达,从而在生物体发育,疾病的发生发展等方面发挥重要作用,其在肿瘤中的作用也越来越被人们所重视。研究表明多种miRNA在HCC组织中表达异常,对肝癌细胞的增殖、凋亡、侵袭、转移等发挥重要的调控作用,因此分析miRNA在肝癌中的表达特点,阐明miRNA在肝癌中的作用对HCC诊断和治疗具有重要意义。研究材料和方法:本研究通过对TCGA数据库中肝癌组织和癌旁组织miRNA测序数据进行分析,筛选出肝癌组织中差异表达的miRNA并且结合患者的临床数据确定出与肝癌患者临床特征相关的差异表达miRNA。另外对与肝癌中明显异常表达的miRNA进行GO和KEGG Pathway分析。其次,我们挑选出在肝癌组织中明显高表达的miR-589-5p对其在肝癌中的功能进行研究,通过QPCR检测其在肝癌细胞系中的表达量;在肝癌细胞中转染miR-589-5p抑制剂后通过MTT、克隆形成以及流式细胞周期检测研究其对肝癌细胞增殖的影响;通过生物信息学方法预测出miR-589-5p可能的靶基因并通过QPCR及Western Blot进行验证。研究结果:通过对TCGA数据库中肝癌相关数据的分析,我们总共筛选出49个在肝癌组织中异常表达的micro RNA,其中有14个在肝癌组织中高表达,33个在肝癌组织中低表达。结合患者临床特征数据我们发现其中有21个micro RNA与肝癌患者的临床特征有关。从这些异常表达的micro RNA中我们挑选出miR-589-5p研究其在肝癌中的功能,micro RNA测序数据表明,miR-589-5p在肝癌组织中表达量是在癌旁组织中的1.93倍(P0.001),并且其高表达与肝癌患者的低生存时间相关(P=0.0477);QPCR实验验证表明miR-589-5p在肝癌细胞系HepG2和Huh-7中比在肝正常细胞系L-O2中明显高表达;抑制HepG2和Huh7中miR-589-5p的表达后,细胞的增殖能力降低;通过生物信息学方法预测出miR-589-5p可能的靶基因MIG-6,用PCR和Western Blot实验进行验证,发现HepG2细胞导入miR-589-5p抑制剂后,MIG-6的表达量明显升高。研究结论:1.肝癌组织中存在大量异常表的的micro RNA,其中有些micro RNA的表与患者的临床特征有关,并且这些micro RNA可能在肝癌中发挥重要作用。2.MiR-589-5p在肝癌组织和肝癌细胞中高表达,并且其表达量与肝癌患者的生存时间有关,抑制miR-589-5p的表达后肝癌细胞的增殖能力下降。3.MiR-589-5p可能是通过靶向抑制MIG-6的表达发挥作用。
[Abstract]:Background: Hepatocellular carcinoma (HCC) is the most common primary liver malignant tumor in adults, which accounts for the 80-90%. of primary liver cancer in the world. Its incidence is the fifth largest in human malignant tumor. It is the third major cause of cancer related death. According to statistics, the incidence of HCC is increased by 3-9%, and the number of new cases is about 78. Ten thousand, 50% of them occur in China. The main cause of the increase of the incidence of liver cirrhosis, which is caused by hepatitis B, is the main cause of its incidence year by year. The occurrence of.HCC is a multi factor, multi stage process. But because of its concealment, rapid metastasis, strong invasiveness, and lack of sensitive early diagnostic markers, the diagnosis of HCC has developed to a late stage. Liver transplantation, tumor resection, embolization, chemotherapy and so on are suitable for the treatment of HCC. The prognosis is still poor and the 5 year survival rate is less than 5%. Therefore, the search for high sensitivity and highly specific tumor markers is of great importance to the improvement of the survival quality and survival time of the patients with hepatocellular carcinoma (.Micro RNA (miRNA) is a kind of endogenous length. RNA, a non coding small molecule of about 18-25 nucleotides, often plays an important role in the development of organisms, the development of the disease and the development of the disease by inhibiting the translation or accelerating its degradation by combining the 3 'non translation zone (3' UTR) of the target messenger RNA (m RNAs) and accelerating its degradation, and thus plays an important role in the development of the organism and the development of the disease, and its role in the tumor is becoming more and more important. The study shows that the expression of a variety of miRNA in HCC tissues plays an important role in regulating the proliferation, apoptosis, invasion and metastasis of hepatoma cells. Therefore, it is important to analyze the expression of miRNA in the liver cancer and to clarify the role of miRNA in the diagnosis and treatment of HCC. After analyzing the miRNA sequencing data of liver cancer tissue and para cancer tissue in the TCGA database, the differential expression of miRNA in the liver cancer tissues was screened and the difference expression related to the clinical features of the patients with liver cancer was determined by combining the clinical data of the patients with miRNA., and the GO and KEGG Pathway analysis of miRNA, which was obviously abnormal in the liver cancer, was analyzed. At the same time, we selected miR-589-5p, which was highly expressed in the liver cancer tissue, to study its function in liver cancer, and to detect its expression in the liver cancer cell line by QPCR. After transfection of miR-589-5p inhibitor in the hepatoma cells, the effects of the cloning and flow cytometry on the proliferation of hepatoma cells were studied. Bioinformatics methods have predicted the possible target genes of miR-589-5p and verified by QPCR and Western Blot. Results: through the analysis of the liver cancer related data in the TCGA database, we screened out 49 abnormal expressions of micro RNA in the liver cancer tissues, of which 14 were highly expressed in the liver cancer tissues and 33 were in the liver cancer tissue. We found that 21 of micro RNA were associated with the clinical characteristics of the patients with liver cancer. We selected miR-589-5p to study the function of miR-589-5p in liver cancer from these abnormal micro RNA. The micro RNA sequencing data showed that the expression of miR-589-5p in the liver cancer tissues was 1.9 in the para cancer tissues. 3 times (P0.001), and its high expression was associated with low survival time of liver cancer patients (P=0.0477); QPCR test showed that miR-589-5p was significantly higher in HepG2 and Huh-7 of liver cancer cell lines than in the normal liver cell line L-O2; the proliferation ability of the cells decreased after inhibiting the expression of miR-589-5p in HepG2 and Huh7; predicted by bioinformatics methods. MIG-6, a possible target gene for miR-589-5p, was verified by PCR and Western Blot experiments. It was found that the expression of MIG-6 was significantly increased after HepG2 cells were introduced into miR-589-5p inhibitors. Conclusions: there are a large number of abnormal tables in micro RNA in 1. liver cancer tissues, and some micro RNA tables are related to the clinical characteristics of the patients. It may play an important role in liver cancer,.2.MiR-589-5p is highly expressed in liver cancer tissues and hepatoma cells, and its expression is related to the survival time of the liver cancer patients. The decrease of the proliferation ability of hepatoma cells after the inhibition of miR-589-5p expression may play a role in the expression of target inhibition of the expression of MIG-6.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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