miR-122对HBV调节作用的研究

发布时间：2019-02-22 19:15

【摘要】： 最近几年,miRNA已成为生命科学领域研究的热点,它的发现不仅为生物学开辟了新的领域,更为其生物学原理应用于疾病治疗的转译研究提供了新的武器,曾一度被认为是“垃圾”RNA的小分子近十年先后7次被美国《SCIENCE》评选为“年度全球十大科技突破”。到目前为止,人们已经发现了三千余种miRNAs,被认为是最大的一类基因表达调控因子,它通过与靶基因的结合调节细胞分裂、增殖、分化和多种生物学信号通路,也可以起到肿瘤抑制基因和癌基因的作用,以及应用在疾病诊断和基因功能研究中。多项研究结果表明miR-122是肝脏特异的miRNA,调节脂肪酸、胆固醇的代谢和肝脏的分化,上调嗜肝病毒HCV的复制。为了研究miR-122与HBV的复制关系,我们利用生物信息学预测在HBV上1689nt-1711nt区域存在miR-122的可能靶序列。首先利用miRNA的生物学原理,将miR-122的编码基因hcr构建在包含绿色荧光蛋白的载体上,将此重组质粒转染到细胞内,hcr基因在细胞内表达,继而在宿主细胞内发展为成熟的miR-122,实验中利用RT-PCR、T-A克隆技术对于细胞内的miR-122的表达进行检测,证实获得能稳定表达miR-122的真核表达细胞系。为了验证miR-122作用HBV的靶点,首先,从HepG2215基因组DNA扩增miR-15靶序列—BCL2,插入荧光素酶报告基因(luciferase)下游的Xba1酶切位点,构建了阳性的miR-15荧光素酶报告基因系统,用于验证miR-122靶点的阳性对照系统。已有研究证明HBV在1689nt-1711nt位置有四种转录本,根据miRNA的生物学原理推测miR-122可能对这四种mRNA都有作用,本实验选用其中之一的HBx来验证1689nt-1711nt序列与miR-122之间是否为靶向的关系。本研究构建了包含HBx序列的荧光素酶报告基因系统及HBx蛋白的表达系统,与miR-122共转染到自身低表达miR-122的细胞系中,在过表达mirR-122、锁定miR-122和突变miR-122三个层面上,用荧光素酶报告基因的检测及western blot实验方法,证实miR-122通过作用靶序列HBx对HBV复制进行负向调控,并在翻译水平抑制HBX蛋白的表达。然后将表达HBV1.1倍体的质粒和miR-122共转染细胞,提取细胞内的乙肝病毒复制中间体DNA,做southern blot检测,结果发现与miR-122共转染之后HBV复制中间体DNA的含量明显减少,确定miR-122与HBV的复制的降低有关。
[Abstract]:In recent years, miRNA has become a hot spot in the field of life sciences. Its discovery has not only opened up a new field for biology, but also provided a new weapon for the translation of its biological principles in the study of disease therapy. Small molecules once known as "garbage" RNA have been named "Top Ten technological breakthroughs of the year" by the United States'< SCIENCE > for seven times in the past decade. So far, more than 3, 000 species of miRNAs, have been found to be the largest class of gene expression regulators, which regulate cell division, proliferation, differentiation and a variety of biological signaling pathways by binding to target genes. It can also play the role of tumor suppressor gene and oncogene, and be used in disease diagnosis and gene function research. Many studies have shown that miR-122 is a liver-specific miRNA, that regulates fatty acids, cholesterol metabolism and liver differentiation, and up-regulates the replication of hepatoviral HCV. In order to study the replication relationship between miR-122 and HBV, we used bioinformatics to predict the possible target sequences of miR-122 in the 1689nt-1711nt region on HBV. Firstly, based on the biological principle of miRNA, the encoding gene hcr of miR-122 was constructed on the vector containing green fluorescent protein. The recombinant plasmid was transfected into the cells and the hcr gene was expressed in the cells. Then the expression of miR-122 in host cells was detected by RT-PCR,T-A cloning in the mature miR-122, experiment, and the eukaryotic expression cell lines which could stably express miR-122 were obtained. In order to verify the target of miR-122 acting on HBV, firstly, the miR-15 target sequence-BCL2, was amplified from HepG2215 genome DNA and inserted into the downstream Xba1 cleavage site of luciferase reporter gene (luciferase), and a positive miR-15 luciferase reporter gene system was constructed. A positive control system used to validate miR-122 targets. Studies have shown that HBV has four transcripts at the 1689nt-1711nt site, and it is speculated that miR-122 may play a role in all four mRNA based on the biological principles of miRNA. In this study, one of the HBx was selected to verify the relationship between 1689nt-1711nt sequence and miR-122. In this study, the luciferase reporter gene system containing HBx sequence and the expression system of HBx protein were constructed. The system co-transfected with miR-122 into a cell line with low expression of miR-122 and overexpressed mirR-122,. By using luciferase reporter gene detection and western blot assay, it was confirmed that miR-122 negatively regulated HBV replication by acting on target sequence HBx, and inhibited the expression of HBX protein at the translation level on the three levels of locking miR-122 and mutated miR-122, and the expression of HBX protein was inhibited at the translation level by luciferase reporter gene detection and western blot assay. Then the plasmid expressing HBV1.1 was co-transfected with miR-122 to extract the DNA, of HBV replication intermediates in the cells for southern blot detection. The results showed that the content of DNA, the intermediate of HBV replication, was significantly decreased after co-transfection with miR-122. Determine that miR-122 is associated with reduced replication of HBV.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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