乙型肝炎病毒相关LncRNA-DR4的功能研究

发布时间：2018-03-31 12:56

本文选题：肝癌　切入点：乙型肝炎病毒　出处：《安徽大学》2017年硕士论文

【摘要】：肝癌(HCC)是一种全球发病率较高的恶性肿瘤,全球每年的新发病例超过600000例。肝癌根据其疾病的恶性程度划分,在所有的肿瘤中排名第五,致死率居全球第三。乙型肝炎病毒(HBV)感染是肝癌形成的重要诱因。随着肝癌研究的深化以及现代技术的发展,发现肝癌在缓慢的形成的过程中,往往会造成许多分子和信号通路的改变。因此,从微观分子的角度探究肝癌发生发展的机制,尤其是HBV感染导致的分子及信号通路的改变,并将特异的分子看作抗肿瘤研究新的靶点,可以为肝癌的临床治疗提供新的思路。长链非编码RNA(LncRNA)是一类内源性RNA,从细胞中转录出来的转录本长度大于200nt,在进化上保守性较差,能够在多个水平上调控基因的表达,包括染色质修饰、转录和转录后加工。近年研究表明,大量的LncRNAs具有复杂的生物学功能,而且参与到许多癌症的形成过程。例如,结直肠癌组织与对应的非癌组织相比,HOTAIR的表达水平有明显上升,它可以通过直接与PRC2复合体的结合,影响依赖PRC2的染色质修饰,进而调节多种基因的表达。通过对比HBx转基因小鼠以及对照组小鼠肝脏中lncRNA量的差异,研究者发现LncRNA Dreh表达量在转基因小鼠中显著下调,并且这种下调与鼠龄和性别没有相关性,由HBx抑制表达的LncRNAs Dreh能够通过抑制中间纤维蛋白Vimentin的表达来调控细胞骨架的修饰状况,从而使HCC的生长、转移均处在一个抑制的状态。本课题进行了以下3部分的研究:(1)通过RNA-seq检测肝癌细胞系HepG2及整合了 HBV基因组的肝癌细胞系4D14中差异表达的LncRNAs,进而利用定量PCR确定了一条在4D14中显著下调的长链非编码RNA,命名为LncRNA-DR4作为本课题的研究对象。(2)构建了 LncRNA-DR4过表达的肝癌细胞系,通过CCK8,细胞周期等实验,发现LncRNA-DR4的过量表达能够显著抑制肝癌细胞系HepG2的细胞增殖,且这种抑制作用可能是HepG2细胞周期中S期的阻滞造成的。(3)利用定量PCR确定了 HBV特有基因HBx与LncRNA-DR4表达的负相关性,进一步的荧光素酶活性实验,发现HBx在转录水平上下调了LncRNA-DR4表达,并且这种调控可能依赖p53蛋白。总之,我们鉴定了一条在HBV阳性细胞系中下调的长链非编码RNA,命名为LncRNA-DR4。确定了 HBx蛋白对LncRNA-DR4的负调控作用,并且这种调控在转录水平上进行,这种调控作用是依赖p53的。另外,对于LncRNA-DR4的生物学功能,我们发现过表达LncRNA-DR4能显著抑制肝癌细胞的增殖,引起S期细胞阻滞。
[Abstract]:Hepatocellular carcinoma (HCC) is a malignant tumor with a high incidence worldwide, with more than 600000 new cases per year. Liver cancer ranks fifth among all tumors according to the degree of malignancy of the disease. Hepatitis B virus hepatitis B virus (HBV) infection is an important inducement for the formation of liver cancer. With the deepening of liver cancer research and the development of modern technology, it is found that liver cancer is slowly forming. Therefore, from the micromolecular perspective, to explore the mechanism of the development of liver cancer, especially the changes in the molecular and signal pathways caused by HBV infection, The specific molecules are regarded as new targets for antitumor research, which can provide a new idea for the clinical treatment of liver cancer. LncRNAs are a class of endogenous RNAs that transcribe from cells with a length larger than 200nt, which is less evolutionarily conservative. The ability to regulate gene expression at multiple levels, including chromatin modification, transcription and post-transcriptional processing. Recent studies have shown that a large number of LncRNAs have complex biological functions and are involved in the formation of many cancers. The expression level of HOTAIR in colorectal cancer tissues was significantly higher than that in the corresponding non-cancerous tissues. It could affect the chromatin modification dependent on PRC2 by directly binding with PRC2 complex. By comparing the amount of lncRNA in the liver of HBx transgenic mice and control mice, the researchers found that the expression of LncRNA Dreh was significantly down-regulated in transgenic mice. And this down-regulation is not related to age and sex of mice. LncRNAs Dreh, which is inhibited by HBx, can regulate the modification of cytoskeleton by inhibiting the expression of intermediate fibrin Vimentin, thus making HCC grow. In this study, we studied the following three parts: 1) Detection of differential expression of LncRNAsin hepatocellular carcinoma cell line HepG2 and 4D14 cell line with HBV genome by RNA-seq, and then use quantitative PCR to confirm the expression of LncRNAs. A liver cancer cell line with overexpression of LncRNA-DR4 was constructed by targeting a long chain noncoding RNAs that were significantly down-regulated in 4D14 and named LncRNA-DR4 as the research object of this study. By CCK8 and cell cycle experiments, it was found that overexpression of LncRNA-DR4 could significantly inhibit the proliferation of hepatoma cell line HepG2. The inhibitory effect may be caused by the arrest of S phase in HepG2 cell cycle. (3) quantitative PCR was used to determine the negative correlation between HBx and LncRNA-DR4 expression of HBV specific gene, and further luciferase activity experiment was carried out. We found that HBx down-regulated the expression of LncRNA-DR4 at the transcriptional level, and this regulation may depend on p53 protein. In conclusion, we identified a long chain noncoding RNAs down-regulated in HBV positive cell lines, named LncRNA-DR4. The negative regulation of HBx protein on LncRNA-DR4 was determined. In addition, we have found that overexpression of LncRNA-DR4 can significantly inhibit the proliferation of hepatoma cells and induce S phase cell arrest for the biological function of LncRNA-DR4.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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