【作者】 章广玲；

【导师】 汤华；

【作者基本信息】 天津医科大学 ， 病原生物学， 2008， 博士

【摘要】 目的：在我国乙型肝炎病毒(HBV)相关性肝细胞癌(hepatocellular carcinoma, HCC)发病率、死亡率均排名于疾病谱前列,严重危害人民健康,目前尚无满意的治疗方法。为此寻找新的抗HBV治疗方法成为当前急需解决的重要问题。近年发现的microRNA (miRNA),是一种广泛存在于真核生物中、大小约18-25个核苷酸(nt)的内源性单链小分子RNA,参与发育、增殖、分化、凋亡等多种生物学过程。越来越多的研究表明,miRNA也能够调控RNA和DNA病毒的复制。那么,miRNA是否也参与了HBV的复制呢?为此,我们首次研究了靶定于HBV基因组和宿主细胞基因组的人类的miRNA,为研究HBV和miRNA之间的相互作用及其机制奠定初步的实验依据,为今后抗HBV增殖药物的开发提供新的手段和分子基础。方法：1.分别用328种miRNA的反义链转染HepG22.2.15细胞,72小时收获上清,MTS检测细胞的增殖活性,ELISA检测表面抗原和e抗原的表达水平2.将筛选到的对表面抗原产生有抑制作用和促进作用的miRNA的反义链梯度转染HepG22.2.15细胞,72小时收获上清,实时定量PCR检测其中HBV的拷贝数,ELISA检测表面抗原和e抗原的表达水平。3.用miRanda, TargetScan, PicTar, miRDB和ViTa五个网站预测筛选miRNA在HBV基因组上的靶基因,对于在HBV基因组上没有靶位点的miRNA,网站预测的结果联合mRNA表达谱芯片和RT-PCR技术,对其靶基因做进一步预测。4.分别构建筛选到的miRNA的过表达载体,包含靶位点的EGFP报告载体和靶位点突变的EGFP报告载体。并且分别在HEK293和HepG22.2.15细胞中进行荧光报告载体实验验证候选的靶基因。结果：1.MTS和ELISA的结果显示,在没有对细胞的增殖活性产生影响的情况下,miR-199a-3p, miR-210和miR-185抑制了HBV表面抗原的表达,而miR-370促进了病毒表面抗原的表达。2.实时定量PCR的结果显示,miR-199a-3p, miR-210和miR-185抑制了HBV的复制,这种抑制作用具有剂量依赖性。3.网站预测了miR-199a-3p, miR-210, miR-185和miR-370的靶基因,miR-199a-3p, miR-210分别靶定于HBV的表面抗原和表面抗原的启动子2。而在HBV上没有发现miR-185和miR-370的靶位点,通过mRNA表达谱芯片结合生物信息学的方法,我们筛选到miR-185可能的靶基因有PRKCH,ATP5B和NR1D1, miR-370可能的靶基因有FUS和TPM4。并且通过RT-PCR和芯片验证,PRKCH, ATP5B的RT-PCR结果和芯片结果相符。4.在HEK293和HepG22.2.15两种细胞中,利用荧光报告载体实验验证了miR-199a-3p, miR-210和miR-185与候选基因的直接作用关系,结果显示,miR-199a-3p, miR-210, miR-185分别通过靶定HBsAg, HBSP2和PRKCH,从而抑制了HBV表面抗原的产生以及HBV的复制。结论：本研究首次发现了通过直接靶定HBV基因组或者宿主细胞基因,从而抑制病毒表面抗原产生和病毒复制的hsa-miRNA。miRNA作为病毒与宿主细胞相互作用中的调节因子(激活因子或抑制因子),在HBV的生命活动周期中具有重要作用。该研究对于阐明细胞miRNA和HBV之间错综复杂的相互作用提供了依据,对于我们理解宿主和病原体之间内在的相互调控提供很大的帮助,可能为解释病毒的组织嗜性,潜伏感染,和癌原性提供新的理论依据,而且,病毒感染相关miRNA的研究有望成为抗病毒治疗新的重要靶点。更多还原

【Abstract】 Objective:Chronic hepatitis B virus(HBV) infection is one of the leading cause of liver cirrhosis and hepatocellular carcinoma(HCC).Current strategies of HBV infection have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. MicroRNAs(miRNAs) are single-stranded noncoding RNAs of18to25nucleotides that play critical roles in a wide spectrum of biological processes. Accumulating evidence suggests that the miRNAs pathway also controls the replication of both RNA and DNA viruses. To address whether the miRNAs-silencing machinery influences HBV replication and antigen expression, miRNAs which can target crucial HBV genes including HBsAg or SP2genes or target cell genes including PRKCH and ATP5B genes, which play important roles in HBV infection, were studied.Methods:To address which miRNAs influences HBsAg expression, HBeAg expression or the proliferation of HBV producing HepG22.2.15cells, antisense oligonucleotide of special miRNAs was used to reduce the expression of endogenous miRNAs. On the basis of enzyme linked immunosorbent assay (ELISA) and CellTiter96(?) Aqueous Non-Radioactive Cell Proliferation Assay (MTS) performed on culture supernatant harvested from HepG22.2.15cells and HepG22.2.15cells72hours after transfection with miRNA ASOs, three miRNAs that inhibited HBsAg expression and one that enhanced HBsAg expression were selected.To understand the function of these miRNAs in HBV replication, culture supernatant harvested from HepG22.2.15cells transfected with miRNA ASOs and control oligo, respectively, HBV replication was tested by quantification of Hepatitis B virus DNA.To further analyze the mechanisms of miRNAs in controlling HBsAg expression and HBV replication, we used five well-established microRNA target prediction software-miRanda, TargetScan, PicTar, miRDB and ViTa were used to predict targets for the four miRNAs. Sequence analysis indicates that two of them do not directly target the HBV genome, which suggests that they might affect HBV replication by targeting cellular protein(s).Then, combined mRNA microarray with semi-quantitative RT-PCR was used to predict the target genes of these two miRNAs.These prediction results were conformed by using reporter vectors with the intact putative miRNAs recognition sequence from HBV or the3’-UTR of predicted gene (pcDNA3/EGFP-UTR) or randon mutations (pcDNA3/EGFP-UTRmut) cloned downstream of the EGFP gene, respectively. HEK293and HepG22.2.15were cotransfected with pcDNA3/EGFP-UTR with or without precursor miRNAs, with or without miRNA ASOs, respectively. So did the pcDNA3/EGFP-UTRmut. GFP and RFP activity were measured with a fluorescence spectrophotometer (HITACHI F4500)48hours after transfection. Results were represented as normalized ratio of EGFP to RFP.Results:First, those miRNAs that influenced HBsAg expression by ELISA and MTS were screened. There are three miRNAs, miR-199a-3p, miR-210and miR-185that inhibited the expression of HBsAg and miR-370enhanced HBsAg expression.Second, quantification of Hepatitis B virus DNA in culture supersnatant of HBV producing HepG22.2.15cells transfected with ASO indicates that miR-199a-3p, miR-210, miR-185reduce HBV production, compared with control ASO(Lacz) transfected cells.Third, microRNA target prediction software was used to screen for potential targets of miR-199a-3p, miR-210,miR-185and miR-370. Sequence analysis indicates that miR-199a-3p and miR-210might target HBsAg and SP2, respectively. Nevertheless, sequence analysis indicates that miR-185and miR-370do not directly target the viral genome, which suggests that they might affect HBV replication by targeting cellular protein(s). So, a7,267-element human cDNA microarray was used to screen up-regulated genes owning to miR-185or miR-370being blocked down. Combining target prediction and expression profiling, three candidate miR-185-targeted genes, PRKCH, ATP5B and NR1D1and two putative target genes including FUS and TPM4for miR-370were identified.Fourth, HBsAg, SP2and PRKCH were verified as targets for miR-199a-3p,miR-210and miR-185by EGFP reporter contructs, respectively.Conclusion:Up to date, this is the first report about human miRNAs that can inhibit HBV replication by targeting important HBV genes or cellular proteins. Evidence for an intricate physiological interplay between the cell’s miRNAs and HBV was provided. It offers an immense opportunity not only to understand the intricacies of host-pathogen interactions, and possible explanations to viral tropism, lantency and oncogenesis, but also develop novel biomarkers and therapeutics.更多还原