HBV诱导表达的novel-miR-10对肝癌细胞恶性行为的作用与机制研究
发布时间：2018-07-22 20:58
【摘要】：【目的】肝细胞癌(hepatocellular carcinoma,HCC)是一种常见的高度恶性肿瘤。由于缺乏早期诊断方法及有效地治疗策略,预后极差。HBV感染是肝细胞癌发生的重要危险因素之一,其导致慢性肝炎肝硬化直至最终发展为肝癌是个漫长的过程并受多种因素调控,但其具体机制尚不明确。miRNAs(microRNAs)是一类约22nt大小的非编码RNA分子,在转录后水平调控基因的表达。研究表明HBV感染可导致肝细胞内多种mi RNAs表达水平的变化。miRNAs可通过调控多个癌基因和抑癌基因的表达影响肝细胞的生物学功能,包括细胞活性,增殖能力,迁移和侵袭能力等等,进而影响肝癌的发生发展。由此我们猜想,HBV感染导致肝癌发生发展可能是通过调控部分mi RNAs的表达实现的。本课题着重研究HBV感染导致的mi RNAs表达变化及其这些变化对肝癌的发生发展产生的作用。提出了HBV促进肝癌发生的新机制。【方法】首先利用深度测序技术分析HBV阳性肝癌组织和HBV阴性肝癌组织中miRNAs的表达谱并选择了差异性表达较明显的新miRNA—novel-miR-10作为研究对象;随后用qRT-PCR技术验证了HBV对novel-miR-10表达的调控作用;构建novel-miR-10的启动子报告质粒并通过双荧光素酶报告系统验证HBV以及HBx对novel-miR-10启动子的调控,同时验证了转录因子SP1和CREB1对novel-miR-10启动子活性的影响;接下来通过MTT实验、平板克隆形成实验、Transwell迁移和侵袭实验进一步确定novel-miR-10在肝癌细胞中的生物学功能;通过FACS技术和western blot实验验证了novel-miR-10对肝癌细胞凋亡和EMT过程的影响;继而通过生物信息学方法预测novel-miR-10的靶基因并通过EGFP荧光报告系统结合qRT-PCR和western blot实验进行验证;然后干扰靶基因的表达并通过MTT实验、平板克隆形成实验、Transwell迁移和侵袭实验来确定靶基因在肝癌中的生物学功能并通过FACS技术和western blot实验验证了靶基因对肝癌细胞凋亡和EMT过程的影响;最后通过一系列挽救实验证明novel-miR-10对肝癌的生物学作用是通过对靶基因的调控实现的。【结果】深度测序结果显示在HBV阳性肝癌组织中存在多个差异性表达的miRNAs,其中新发现的miRNA-novel-miR-10在HBV阳性肝癌组织中表达量高于HBV阴性肝癌组织;表达HBV1.3copy可促进肝癌细胞中novel-miR-10的表达;表达HBV1.3copy和HBx可增强novel-miR-10启动子的活性;过表达转录因子SP1抑制,CREB1促进novel-miR-10启动子的活性,并且敲降CREB1后HBV1.3copy和HBx对novel-miR-10启动子的促进作用被阻断;肝癌表型实验证实novel-miR-10可促进肝癌细胞的细胞活性、增殖能力、迁移和侵袭能力;生物信息学预测TNFRSF19和RAB43是novel-miR-10的两个直接靶基因,EGFP荧光报告载体验证novel-miR-10直接靶定TNFRSF19和RAB43的3’UTR区域并下调其表达;qRT-PCR和western blot实验证实novel-miR-10抑制内源性TNFRSF19和RAB43的表达;敲降TNFRSF19和RAB43这两个靶基因的表达促进肝癌细胞的细胞活性、增殖能力、迁移和侵袭能力;挽救实验证明敲降TNFRSF19和RAB43能逆转由降低novel-miR-10表达引起的肝癌细胞生物学功能的抑制作用。【结论】HBV可通过HBx与CREB1增强novel-miR-10启动子的活性,从而促进其表达。novel-miR-10促进肝癌细胞的细胞活性、增殖能力、迁移和侵袭能力,起到癌基因的作用,并且其癌基因作用是或至少部分是通过靶定并下调其靶基因TNFRSF19和RAB43的表达来实现的。总之,这些结果证明了HBV可通过调节miRNA表达促进肝癌发生,并阐明了其具体机制。
[Abstract]:[Objective] hepatocellular carcinoma (HCC) is a common high malignant tumor. Due to the lack of early diagnosis and effective treatment strategy, the poor prognosis of.HBV is one of the most important risk factors for the occurrence of hepatocellular carcinoma. It is a long process to lead to the hardened liver of chronic hepatitis until it is eventually developed into liver cancer. It is regulated by a variety of factors, but its specific mechanism is not yet clear that.MiRNAs (microRNAs) is a class of about 22nt size of non coded RNA molecules that regulate the expression of genes at the post transcriptional level. The study shows that HBV infection can lead to a variety of MI RNAs expression levels in the liver cells..miRNAs can regulate the expression of multiple oncogenes and tumor suppressor genes. The biological functions of hepatocytes, including cell activity, proliferation, migration and invasion, etc., and thus affect the development and development of liver cancer. Therefore, we suspect that HBV infection may lead to the development of liver cancer by regulating the expression of partial mi RNAs. This topic focuses on the changes in the expression of MI RNAs caused by HBV infection and these The effect of change on the development and development of liver cancer was proposed. A new mechanism for HBV to promote the occurrence of liver cancer was proposed. [method] first, the expression profiles of miRNAs in HBV positive liver cancer tissues and HBV negative liver cancer tissues were analyzed by deep sequencing technology, and the new miRNA novel-miR-10 was selected as the research object, and then qRT-PC was used as the research object. The R technique verified the regulation of HBV on the expression of novel-miR-10, constructed the promoter report plasmid of novel-miR-10 and verified the regulation of HBV and HBx on novel-miR-10 promoter through the double luciferase reporter system, and verified the effect of the transcription factor SP1 and CREB1 on novel-miR-10 promoter activity. Transwell migration and invasion experiments further determine the biological function of novel-miR-10 in hepatoma cells; the effects of novel-miR-10 on the apoptosis and EMT process of hepatoma cells are verified by FACS and Western blot experiments. Then, the target gene of novel-miR-10 is pretested by bioinformatics and the EGFP fluorescence report is reported. The system was verified by qRT-PCR and Western blot experiments, and then the target gene was interfered with the expression of the target gene and through the MTT experiment, the flat clone formation experiment, the Transwell migration and invasion experiment to determine the biological function of the target gene in the liver cancer. The target gene was tested for the apoptosis and EMT process of the liver cancer cells by the FACS technique and the Western blot test. Finally, through a series of rescue experiments, the biological effect of novel-miR-10 on liver cancer was achieved through the regulation of the target gene. [results] the results of deep sequencing showed that there were multiple differentially expressed miRNAs in the HBV positive liver cancer tissues, and the newly discovered miRNA-novel-miR-10 was expressed in the HBV positive liver cancer tissues. Higher than HBV negative liver cancer tissue; expression of HBV1.3copy can promote the expression of novel-miR-10 in hepatoma cells; expression of HBV1.3copy and HBx can enhance the activity of novel-miR-10 promoter; over expression of transcription factor SP1 inhibits, CREB1 promotes the activity of novel-miR-10 promoter, and HBV1.3copy and HBx to promoter promoter after knocking down CREB1 Novel-miR-10 can promote the cell activity, proliferation, migration and invasion of hepatoma cells; bioinformatics predicts that TNFRSF19 and RAB43 are the two direct target genes of novel-miR-10, and EGFP fluorescent reporter vectors verify that novel-miR-10 directly targets the 3 'UTR region of TNFRSF19 and RAB43 and down its table. QRT-PCR and Western blot experiments confirmed that novel-miR-10 inhibited the expression of endogenous TNFRSF19 and RAB43, and the expression of two target genes that knocked down TNFRSF19 and RAB43 promoted the cell activity, proliferation, migration and invasion ability of liver cancer cells; the rescue experiment showed that the knockdown TNFRSF19 and RAB43 can reverse the liver caused by decreasing novel-miR-10 expression. [Conclusion] HBV can enhance the activity of novel-miR-10 promoter through HBx and CREB1, thus promoting the expression of.Novel-miR-10 to promote the cell activity, proliferation, migration and invasion of hepatoma cells, and play the role of the oncogene, and its oncogene effect is or at least partly through the target. The expression of the target gene TNFRSF19 and RAB43 was downregulated. In conclusion, these results showed that HBV could promote the development of liver cancer by regulating the expression of miRNA and elucidate its specific mechanism.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
本文编号：2138557
[Abstract]:[Objective] hepatocellular carcinoma (HCC) is a common high malignant tumor. Due to the lack of early diagnosis and effective treatment strategy, the poor prognosis of.HBV is one of the most important risk factors for the occurrence of hepatocellular carcinoma. It is a long process to lead to the hardened liver of chronic hepatitis until it is eventually developed into liver cancer. It is regulated by a variety of factors, but its specific mechanism is not yet clear that.MiRNAs (microRNAs) is a class of about 22nt size of non coded RNA molecules that regulate the expression of genes at the post transcriptional level. The study shows that HBV infection can lead to a variety of MI RNAs expression levels in the liver cells..miRNAs can regulate the expression of multiple oncogenes and tumor suppressor genes. The biological functions of hepatocytes, including cell activity, proliferation, migration and invasion, etc., and thus affect the development and development of liver cancer. Therefore, we suspect that HBV infection may lead to the development of liver cancer by regulating the expression of partial mi RNAs. This topic focuses on the changes in the expression of MI RNAs caused by HBV infection and these The effect of change on the development and development of liver cancer was proposed. A new mechanism for HBV to promote the occurrence of liver cancer was proposed. [method] first, the expression profiles of miRNAs in HBV positive liver cancer tissues and HBV negative liver cancer tissues were analyzed by deep sequencing technology, and the new miRNA novel-miR-10 was selected as the research object, and then qRT-PC was used as the research object. The R technique verified the regulation of HBV on the expression of novel-miR-10, constructed the promoter report plasmid of novel-miR-10 and verified the regulation of HBV and HBx on novel-miR-10 promoter through the double luciferase reporter system, and verified the effect of the transcription factor SP1 and CREB1 on novel-miR-10 promoter activity. Transwell migration and invasion experiments further determine the biological function of novel-miR-10 in hepatoma cells; the effects of novel-miR-10 on the apoptosis and EMT process of hepatoma cells are verified by FACS and Western blot experiments. Then, the target gene of novel-miR-10 is pretested by bioinformatics and the EGFP fluorescence report is reported. The system was verified by qRT-PCR and Western blot experiments, and then the target gene was interfered with the expression of the target gene and through the MTT experiment, the flat clone formation experiment, the Transwell migration and invasion experiment to determine the biological function of the target gene in the liver cancer. The target gene was tested for the apoptosis and EMT process of the liver cancer cells by the FACS technique and the Western blot test. Finally, through a series of rescue experiments, the biological effect of novel-miR-10 on liver cancer was achieved through the regulation of the target gene. [results] the results of deep sequencing showed that there were multiple differentially expressed miRNAs in the HBV positive liver cancer tissues, and the newly discovered miRNA-novel-miR-10 was expressed in the HBV positive liver cancer tissues. Higher than HBV negative liver cancer tissue; expression of HBV1.3copy can promote the expression of novel-miR-10 in hepatoma cells; expression of HBV1.3copy and HBx can enhance the activity of novel-miR-10 promoter; over expression of transcription factor SP1 inhibits, CREB1 promotes the activity of novel-miR-10 promoter, and HBV1.3copy and HBx to promoter promoter after knocking down CREB1 Novel-miR-10 can promote the cell activity, proliferation, migration and invasion of hepatoma cells; bioinformatics predicts that TNFRSF19 and RAB43 are the two direct target genes of novel-miR-10, and EGFP fluorescent reporter vectors verify that novel-miR-10 directly targets the 3 'UTR region of TNFRSF19 and RAB43 and down its table. QRT-PCR and Western blot experiments confirmed that novel-miR-10 inhibited the expression of endogenous TNFRSF19 and RAB43, and the expression of two target genes that knocked down TNFRSF19 and RAB43 promoted the cell activity, proliferation, migration and invasion ability of liver cancer cells; the rescue experiment showed that the knockdown TNFRSF19 and RAB43 can reverse the liver caused by decreasing novel-miR-10 expression. [Conclusion] HBV can enhance the activity of novel-miR-10 promoter through HBx and CREB1, thus promoting the expression of.Novel-miR-10 to promote the cell activity, proliferation, migration and invasion of hepatoma cells, and play the role of the oncogene, and its oncogene effect is or at least partly through the target. The expression of the target gene TNFRSF19 and RAB43 was downregulated. In conclusion, these results showed that HBV could promote the development of liver cancer by regulating the expression of miRNA and elucidate its specific mechanism.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
【共引文献】
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