HBV编码的miRNA对病毒复制的作用及其机制研究

发布时间：2018-03-18 03:19

本文选题：微小RNA　切入点：乙肝病毒　出处：《天津医科大学》2017年博士论文　论文类型：学位论文

【摘要】：【目的】乙型肝炎病毒(HBV)是一种包膜的DNA病毒,包括较少的完整病毒颗粒以及大量缺少核心蛋白的亚病毒颗粒。HBV基因组是部分双链DNA,基因组3.2 kb,编码产生四个转录本,即3.5 kb mRNA,2.4 kb mRNA,2.1 kb mRNA以及0.7 kb mRNA。有四个重叠的开放阅读框(ORF):S、P、C、X,分别编码乙型肝炎病毒表面抗原(HBs Ag)、DNA聚合酶(HBV DNA pol)、核心蛋白(HBc Ag和HBe Ag),以及X蛋白(HBx)。micro RNA(miRNA)是一类长约22 nt的单链非编码RNA,主要通过调控与之互补的靶基因的表达发挥其生物学功能。越来越多的人和动物的病毒被确定编码miRNA,病毒miRNA可影响宿主功能以及体内一些病毒感染的自然进程。然而HBV是否编码miRNA缺乏实验依据以及HBV编码的非编码RNA是否影响其在感染过程中的自我复制仍然是一个值得研究的问题。前期研究中我们利用深度测序在HBV阳性的肝癌组织中获得了HBV编码的小非编码RNA。本课题着重探究HBV编码的HBV-miR-3对HBV复制的作用以及其机制研究。【方法】首先我们通过生物信息学网站预测了HBV-miR-3的二级结构,分析其在HBV不同基因型中的保守性。通过Northern blot及RT-q PCR实验验证其存在。通过RT-q PCR实验验证HBV-miR-3由HBV哪个转录本编码,检测HBV阳性及阴性肝癌组织,HBV阳性及阴性肝细胞系及非肝细胞系,HBV感染病人血清中HBV-miR-3的表达情况。通过Western blot实验检测HBV-miR-3的生成是否是经典的miRNA生成过程,RNA免疫共沉淀实验研究HBV-miR-3在RISC-Ago2复合物中的表达情况,蔗糖密度梯度离心实验及RNA免疫共沉淀实验研究HBV-miR-3是通过何种方式分泌到细胞胞外。同时,我们通过ELISA实验研究HBV-miR-3对HBs Ag,HBe Ag分泌的影响,RT-q PCR实验研究HBV-miR-3对上清HBV DNA水平、HBV完整病毒颗粒,HBV前基因组RNA、总RNA水平的影响,Southern blot实验检测HBV-miR-3对HBV复制中间体的影响,再次我们通过生物信息学的方法预测,并通过Western blot、RT-q PCR以及EGFP荧光报告载体实验验证HBV-miR-3的靶基因,并进一步研究了靶基因的功能。最后我们通过双荧光素酶实验研究HBV-miR-3及靶基因对HBV启动子的影响。【结果】Northern blot及RT-q PCR实验确证HBV编码HBV-miR-3,HBV-miR-3来自乙肝病毒三个转录本3.5 kb mRNA,2.4 kb mRNA,2.1 kb mRNA。HBV-miR-3存在于HBV阳性的肝癌组织及细胞系中,HBV急性感染病人血清中HBV-miR-3的表达量较恢复期的高。HBV-miR-3的生成是依赖Drosha,Dicer的经典的miRNA生成途径,存在于RISC-Ago2复合物中,通过外泌体和完整病毒颗粒的方式分泌到细胞外。HBV-miR-3抑制HBs Ag,HBe Ag分泌,上清HBV DNA水平,完整病毒体的分泌,HBV复制中间体,HBV前基因组RNA及总RNA的表达。HBV-miR-3靶定HBV 3.5 kb转录本,抑制HBV核心蛋白的表达,但不影响HBV DNA聚合酶的表达。HBV-miR-3通过靶定宿主因子STAT3抑制HBV Enh I/Xp的活性。【结论】HBV编码miRNA,即HBV-miR-3。HBV-miR-3通过两个途径抑制HBV复制,一种途径是通过靶定下调HBV 3.5 kb转录本,抑制HBc蛋白的产生,另一种途径是通过靶定并下调宿主因子STAT3抑制HBV Enh I/Xp启动子活性,进而抑制HBV复制转录。
[Abstract]:[Objective] hepatitis B virus (HBV) is an enveloped DNA virus, including complete virus particles and less subviral particles lack core protein.HBV genome is part of double stranded DNA genome 3.2 KB, encoding four transcripts, 3.5 KB mRNA, 2.4 KB mRNA, 2.1 KB and mRNA 0.7 KB mRNA. has four overlapping open reading frames (ORF): S, P, C, X respectively, encoding hepatitis B virus surface antigen (HBs Ag), DNA polymerase (HBV DNA POL), core protein (HBc Ag and HBe Ag) and X protein (HBx),.Micro RNA (miRNA) is a kind of long chain of about 22 NT encoding RNA, mainly through the expression of target gene regulation and complementary play its biological functions. More and more people and animal viruses were identified encoding miRNA, miRNA virus can affect the host function and the body of some virus infection natural processes. However whether HBV encoding miRNA. The lack of experimental basis and HBV encoding the non RNA encoding effect of the infection in the process of self replication is still a problem worthy of study. In previous research, we use deep sequencing in HBV positive hepatocellular carcinoma received non RNA. encoding HBV encoding this paper focuses on the research of HBV encoding HBV-miR-3 on HBV replication the role and its mechanism study. [Methods] we first through the website of bioinformatics prediction two level structure of HBV-miR-3, analysis of the different genotypes of HBV in Northern blot and RT-q. Through the conservative PCR experiments to verify its existence. Through the RT-q PCR experiment by HBV HBV-miR-3 which transcripts encoding, and detection of HBV positive negative hepatocellular carcinoma, HBV positive and negative liver cells and non liver cells, the expression of HBV-miR-3 in serum of patients with HBV infection by Western blot assay of HBV-miR-3 Whether miRNA is the generation process of the classic RNA, CO immunoprecipitation experiments of HBV-miR-3 expression in the RISC-Ago2 complex, sucrose density gradient centrifugation experiment and RNA immunoprecipitation experiments of HBV-miR-3 is the means by which secreted into the extracellular. At the same time, we through experimental study on HBV-miR-3 HBs ELISA Ag, Ag HBe the supernatant of HBV secretion, DNA level RT-q PCR experimental study of HBV-miR-3, HBV complete virus particles, HBV genomic RNA, total RNA levels, Southern blot assay of HBV-miR-3 effect on HBV replication intermediates, we again through the method of bioinformatics prediction, and by Western blot, RT-q PCR and EGFP fluorescent reporter vector experimental verification of HBV-miR-3 target genes, and further study the target gene function. Finally we by dual luciferase activity assay of HBV-miR-3 and target gene of HBV promoter The influence of. [result] Northern blot and RT-q PCR experiments confirmed HBV encoding HBV-miR-3, HBV-miR-3 from three to 3.5 KB of the hepatitis B virus transcription mRNA, 2.4 KB mRNA, there are liver cancer tissues and cells in the HBV positive in 2.1 KB mRNA.HBV-miR-3,.HBV-miR-3 high expression of HBV-miR-3 generation of serum HBV in a patient with acute infection the recovery period is dependent on Drosha, Dicer classic miRNA generating ways, exist in the RISC-Ago2 complex, the exosomes and the complete virus particles are secreted into the extracellular.HBV-miR-3 mode of inhibition of HBs Ag, HBe Ag HBV DNA level secreted supernatant, secretion of intact virions, HBV replication intermediates, HBV expression of.HBV-miR-3 the target of genomic RNA and total RNA HBV 3.5 KB transcripts, inhibiting the expression of HBV core protein, but does not affect the expression of.HBV-miR-3 HBV DNA polymerase by targeting host factor STAT3 inhibition of HBV Enh I/Xp Activity. [Conclusion] HBV encoding miRNA, HBV-miR-3.HBV-miR-3 through two ways to inhibit the replication of HBV, one way is by targeting the downregulation of HBV 3.5 KB transcripts, inhibit HBc expression, another way is by targeting and down-regulation of host factor STAT3 inhibits HBV Enh I/Xp promoter activity, thereby inhibiting HBV replication transcription.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R373

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