人La蛋白新型抑制剂H11调控PI3K/Akt/mTOR信号通路抗乙肝病毒的分子机制

项目名称： 人La蛋白新型抑制剂H11调控PI3K/Akt/mTOR信号通路抗乙肝病毒的分子机制

项目编号： No.81470852

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 汤静

作者单位： 复旦大学

项目金额： 73万元

中文摘要： 人La蛋白是乙肝病毒（HBV）在肝脏复制的重要保护伞，项目组发现La蛋白基因突变、表达沉默、去磷酸化下调HBV复制与蛋白表达，前一项目筛选出新化合物H11，能与La特异结合并抑制La表达，具抗HBV活性但确切机制尚未阐明。文献报道PI3K/Akt/mTOR通路参与HBV复制，而La受Akt调控，我们前期基因芯片分析发现H11与La结合可显著下调mTOR上游调控因子RSK和REDD1表达，因此推测La抑制剂H11通过下调PI3K/Akt/ mTOR通路抑制HBV感染。本项目拟在体外转录翻译体系、稳定表达HBV的HepG2.2.15细胞株及HBV转基因鼠三个水平对关键基因进行功能检验，并用PI3K/Akt/mTOR抑制剂或siRNA预处理后观察H11干扰HBV复制及蛋白表达，验证PI3K/Akt/mTOR通路在H11抗HBV感染的作用，为开发靶向La的新型非核苷类抗HBV药物提供理论依据。

中文关键词： 乙型肝炎病毒；人La蛋白；新型抑制剂；PI3K/Akt/mTOR；分子机制

英文摘要： Human La protein, also named as Sj?gren syndrome type B autoantigen (SSB), forms a stabilized complex with hepatitis B virus (HBV) RNA ribonucleoprotein to promote HBV replication, and thus is a potential target for the prevention and therapy of HBV infection. In our previous study we synthesized a pyridine and pyrazole compound H11 by virtual screening, and showed that it exhibited potent anti-HBV activity. However, the molecular mechanism by which H11 achieves anti-HBV activity is unclear. In our preliminary experiments we employed lncRNA (long non-coding RNAs) gene chip assay to show that H11 treatment led to the downregulation of RSK and REDD1 in HepG2.2.15 cells, both of which are upstream positive regulators of mTOR signaling. Interestingly, PI3K/Akt/mTOR pathway has been shown to play important role in the regulation of HBV replication and liver cell survival. Furthermore, La protein has been identified as a direct substrate of Akt, which regulates the phosphorylation of La protein. In this project, we will test our central hypothesis that H11 inhibits PI3k/Akt/mTOR pathway to exhibit anti-HBV activity. Three specific aims have been proposed: Aim 1: to identify candidate lncRNAs that mediate the anti-HBV effects of H11; Aim 2: to characterize the mechanisms by which candidate lncRNAs regulate HBV infection; Aim 3: to confirm that chemical modulation of PI3k/Akt/mTOR pathway interferes with H11-induced anti-HBV effects both in vitro and in vivo. To achieve these aims, we will use HBV genome stably transfected HepG2.2.15 cell line and HBV transgenic mouse as in vitro and in vivo model, respectively. Based on the changes of lncRNAs expression profiles in HepG2.2.15 cells upon exposure to H11, we will select several candidate lncRNAs for further functional analysis in in vitro transcription/translation coupling system, HepG2.2.15 cell line, and HBV transgenic mouse. We will apply a variety of techniques such as siRNA transfection, lentivirus transduction, PCR assay, chromatin immunoprecipitation, and RNA/protein interaction assay to investigate the role of candidate lncRNAs in the modulation of HBV infection. In particular, we will focus on PI3k/Akt/mTOR pathway. We will employ specific inhibitors or siRNAs against the components of PI3k/Akt/mTOR pathway to modulate the activation of PI3k/Akt/mTOR pathway in HepG2.2.15 cell line or HBV transgenic mouse, and examine the consequences on HBV replication. The completion of this project will help reveal the molecular mechanism of the action of H11 against HBV infection, and provide the rational for the development of novel non-nucleoside anti-HBV drugs that target La protein.

英文关键词： Hepatitis B virus;Human La protein;Novel inhibitor;PI3K/Akt/mTOR pathway;Molecular mechanism