流行性乙型脑炎病毒感染相关宿主miRNA的鉴定与功能分析

MicroRNAs (miRNAs) are recently discovered major regulatos of gene expression, which play a pivotal role in a wide spectrum of biological processes including antiviral defence.There was an intricate interaction between host and virus in the regulation of the miRNA levels. Based on gene chip assay, our previous work studied the expression profile of mRNA and miRNA of pimary cultured neurons of newborn mice fom the cerebral cortex and mice brain samples infection with the Japanese encephalitis virus, and the results indicated that there was an interaction mediated by miRNA between the encephalitis virus and the host. The present work studied about the miRNA selected from miRNA expression profile, and aimed to show the Japanese encephalitis virus would lead to neuronal dysfunction. Aslo, by analyzing the bioinformatics data, the discipline of reverse expression of miRNA and mRNA, experimental testing and so on, the work aimed to discriminate the target relationship of functional miRNAs which adjusted the target gene, so that to find miRNA connected with pathogenic encephalitis-virus infection. And by confirming the inherent connection of intented miRNA, target relationship of target gene, virus infection and abnormity of neural function through over-expression and gene silencing in vitro and vivo experiments, the work finally revealed the mechanism of virus, cell signaling pathway, transcription factor, intented miRNA, target genes downstream pathways and virus infection phenotype or neuronal dysfunction phenotype. A better understanding of host-virus interaction mediated by miRNAs would not only enable us to unravel the molecular basis of viral pathogenesis, but also enable us to develop better therapeutic strategies.

宿主和病毒在miRNA调控水平上存在着复杂的相互作用。前期工作基于基因芯片技术分别研究了乙型脑炎病毒感染鼠脑和原代神经元的mRNA和miRNA表达谱，提示乙型脑炎病毒与宿主在miRNA水平上存在互作。本研究拟以miRNA表达谱遴选出的miRNA为研究对象，以乙型脑炎病毒致神经功能异常为主要研究目标，通过生物信息学数据分析、miRNA和mRNA反向表达的规律和实验验证等分辨出功能性miRNA调控靶基因的靶向关系，获得乙型脑炎病毒感染致病相关miRNA分子；应用过表达和基因沉默等技术在离体和在体水平上确证目的miRNA→靶基因靶向关系与病毒感染及致神经功能异常之间的内在关联，最终揭示病毒→细胞信号通路→转录因子→目的miRNA→靶基因下游通路等→病毒感染表型或致神经功能异常表型的机制。研究成果不仅可为揭示乙型脑炎病毒致病机制积累学术基础，也可为乙型脑炎病毒临床治疗的突破提供些许提示。

流行性乙型脑炎是一种人畜共患的急性的中枢神经系统传染病，蚊为其主要传播媒介，感染后患者体内的病毒可通过血脑屏障进入脑内增殖，引起脑膜及脑组织发炎。miRNA可参与生命过程中的一系列重要进程，是一种新的基因表达调控网络。无论对于病毒或宿主细胞而言，miRNA都是一类快速有效的分子开关。 采用基因芯片技术确认了发现的miRNA的表达，并筛选出接种病毒后有表达差异的miRNA分子，有18个miRNA分子分子上调表达，8个miRNA分子下调表达。采用miRNA表达载体瞬时转染宿主神经细胞作为筛选模型对miRNA表达载体库进行了筛选，共获得了5条与JEV复制相关的miRNA分子。对瞬时转染后细胞的E基因和蛋白检测表明miR-146a，miR-223，miR-142a分子转染后细胞E蛋白水平升高；miR-124a ，miR-301a ，分子转染后细胞E蛋白水平下降。 通过生物信息学方法对上述5个miRNA的宿主靶基因进行了预测，结果包含数个参与细胞转录调控及重要信号通路的基因，其中已有研究表明STAT3，TGF-β2，Nos2等是JEV复制所必需的细胞调控因子。 转染了miR-146a 与STAT1 及Nos2 3’-UTR 载体的实验组同对照组相比，luciferase 活性分别下降约50%，40%；转染了miR-142与TGF-β2 3’-UTR 载体的实验组同对照组相比，luciferase 活性分别下降约54%；转染了miR-124a 与STAT3 3’-UTR载体的实验组同对照组相比，luciferase 活性下降约60%。以上结果表明预测得到的这几个靶基因能够与各自相应的miRNA 分子产生相互作用。通过细胞瞬时转染验证了合成miR-124分子（miR-124 mimics）对病毒JEV具有抑制作用，miR-124抑制剂（miR-124 inhibitor）则对JEV复制具有促进作用，证明了筛选结果的正确；采用miRNA分子靶基因预测软件（TargetScan）对Caveolin-1，APP基因序列中miR-124可能的靶位点进行了预测，结果显示Caveolin-1，APP3’-UTR含有2个miR-124可能的靶标。miR-124可以与Caveolin-1，APP 3’-UTR中2个靶位点产生作用；通过Caveolin-1，APP特异性siRNA验证了抑制C