MiR-26b通过靶标基因抑制乙型肝炎病毒转录和复制的研究

发布时间：2018-02-26 14:51

本文关键词： 乙型肝炎病毒 miR-26b 含半胱氨酸和组氨酸丰富域蛋白1　出处：《武汉大学》2014年博士论文　论文类型：学位论文

【摘要】：乙型肝炎病毒(HBV,简称乙肝病毒)能引起人类感染急性和慢性乙肝疾病,其病毒感染对全球的公共健康是一个巨大的威胁。虽然乙肝疫苗的应用使乙肝病毒的传播得到了遏制,但乙肝病毒的慢性感染依旧难以治愈。乙肝病毒在肝细胞中的表达和复制是受一系列宿主细胞因子调节的,而乙肝病毒可以通过调控这些细胞因子来增强自身的病毒增殖。细胞miRNA是高度保守的非编码RNA,它参与了多种生理过程和疾病进程,目前很多文献报道miRNA在病毒与宿主细胞的交互作用中有重要作用。在本研究中,我们报道了通过转染化学合成的miR-26b模拟物可以抑制乙肝病毒的抗原HBeAg与HBsAg表达、前基因组RNA (pgRNA)转录和衣壳DNA复制,而通过反义抑制子来降低内源性的miR-26b表达后可以增强乙肝病毒的基因表达和DNA复制。过量表达和敲除实验显示miR-26b可以显著降低乙肝病毒的增强子／启动子的活性,证明miR-26b可以通过调节病毒转录水平来实现抑制乙肝病毒。然后,我们鉴定了一系列潜在的miR-26b宿主靶标基因,它们可能与HBV复制的调控相关,其中主要有含半胱氨酸和组氨酸丰富域蛋白1(CHORDCl)。通过miR-26b与CHORDCl信使RNA的3'UTR互补序列特异性地碱基配对,miR-26b可以明显降低CHORDCl的蛋白表达。过表达和敲除实验显示CHORDCl可以通过促进增强子／启动子活性来增加乙肝病毒病毒抗原表达、pgRNA转录和DNA复制。我们同时还发现,乙肝病毒的感染可以使人外周血单核细胞和肝细胞中的miR-26b的表达水平明显下调。另外,我们还证实了miR-26a,另外一条成熟的miR-26家族miRNA,也能降低CHORDCl蛋白的表达,从而也能抑制乙肝病毒的转录和复制。我们的实验结果表明,在肝癌细胞中miR-26b可以下调包括CHORDCl在内的多个靶标基因的表达,降低乙肝病毒的增强子／启动子活性,从而抑制病毒转录、蛋白表达和病毒复制。乙肝病毒感染导致的miR-26b表达下调,表明乙肝病毒可以通过抑制miR-26b的表达水平促进自身的病毒增值。同时,由于miR-26b具有潜在抑癌作用,乙肝病毒诱导的miR-26b水平下降可能在相关肝癌的发生也有重要的作用。我们的发现为miR-26b用于潜在的临床治疗乙肝病毒感染的应用提供实验依据。
[Abstract]:Hepatitis B virus (HBV) can cause acute and chronic hepatitis B diseases in human beings, and its infection is a great threat to global public health. Although the application of hepatitis B vaccine has prevented the spread of hepatitis B virus, However, chronic hepatitis B virus infection is still difficult to cure. The expression and replication of hepatitis B virus in liver cells are regulated by a series of host cytokines. Hepatitis B virus can enhance its virus proliferation by regulating these cytokines. Cell miRNA is a highly conserved noncoding RNAs that participate in many physiological processes and disease processes. At present, many literatures have reported that miRNA plays an important role in the interaction between virus and host cells. In this study, we reported that miR-26b mimics could inhibit the expression of hepatitis B virus antigen HBeAg and HBsAg, pregenomic RNA pgRNAs transcription and capsid DNA replication. The gene expression and DNA replication of hepatitis B virus could be enhanced by reducing endogenous miR-26b expression by antisense suppressor. Overexpression and knockout experiments showed that miR-26b could significantly reduce the activity of enhancer / promoter of hepatitis B virus. We then identified a series of potential miR-26b host target genes, which may be related to the regulation of HBV replication. Among them, there are mainly cysteine and histidine rich domain proteins (1) CHORDCl1. The protein expression of CHORDCl can be significantly reduced by the complementary sequence of miR-26b and CHORDCl messenger RNA. The overexpression and knockout experiments show that CHORDCl can pass through. We also found that, by increasing the activity of promoter / promoter to increase the expression of hepatitis B virus antigen, pgRNA transcription and DNA replication, Hepatitis B virus infection can significantly down-regulate the expression of miR-26b in human peripheral blood monocytes and hepatocytes. In addition, we have also confirmed that miR-26a, another mature miR-26 family, can also reduce the expression of CHORDCl protein. It can also inhibit the transcription and replication of hepatitis B virus. Our results show that miR-26b can down-regulate the expression of multiple target genes, including CHORDCl, and reduce the activity of enhancers / promoters of hepatitis B virus in hepatoma cells, thus inhibiting the transcription of the virus. Protein expression and viral replication. The down-regulation of miR-26b expression caused by HBV infection indicates that HBV can promote the proliferation of its own virus by inhibiting the expression level of miR-26b. At the same time, because miR-26b has a potential anti-cancer effect, The decrease of miR-26b level induced by hepatitis B virus may also play an important role in the occurrence of liver cancer. Our findings provide experimental evidence for the application of miR-26b in the potential clinical treatment of hepatitis B virus infection.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.62;R373

【相似文献】