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

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.

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。

人La蛋白保护HBV RNA免受核酶降解，促进乙肝病毒复制。项目组前期发现自主筛选设计的小分子化合物H11能特异性抑制La蛋白的表达并具有良好的抗乙肝病毒活性。然而这一过程的具体机制并不清楚。本项目通过基因芯片和生物信息学分析筛选了一系列与HBV复制有关的差异表达的lncRNA和mRNA。经过抑制剂处理后的细胞结果表明，候选lncRNA的表达变化与芯片结果一致，在较低浓度下H11仍然显示出良好的抗乙肝病毒活性，对HBsAg和HBeAg也具有一定的抑制作用，结果进一步阐明了H11的抗乙肝病毒作用以及lncRNA的调控作用。随后为聚焦几个关键的lncRNA，构建lncRNA-mRNA共表达网络，发现LA16c-83F12.6和LA16c-313D11.9可能靶向MT1G和SAA4调控HBV感染。采用水动力法将pAAV/HBV1.2 HBV DNA质粒经尾静脉注入小鼠体内构建急性乙肝病毒小鼠模型，发现模型小鼠血清中La蛋白的表达显著上调。对给药模型小鼠和对照小鼠进行免疫组化研究La蛋白的定位变化，结果显示给药后肝细胞中La蛋白呈下降趋势，特别是在细胞核中，此外，肝细胞和免疫细胞之间可能存在蛋白质交换的趋势。上述结果鉴定了功能性的lncRNA抗乙肝病毒调控作用以及初步阐明H11可能通过调控关键lncRNA和La蛋白调控HBV的分子机制。未来的研究需要进一步探究潜在机制。本项目基本按照原计划执行，研究结果在国内外著名期刊发表论文7篇，在投3篇，其中SCI收录4篇，在投2篇，包括肝脏病毒学领域影响因子排名前列的杂志《J Vira Hepat》(IF 4.122)1篇和《Curr Opin Infect Dis》(IF 4.439)1 篇，其中《J Vira Hepat》的论文结果被该杂志选为期刊封面。研究结果荣获第二十七届上海市优秀发明选拔赛优秀发明铜奖，2016年第四届英国Larp Society会议优秀论文奖并获奖学金资助受邀做大会口头交流，此外还多次获得上海市药学会、中国药理学会、中国药学会、上海市医学会临床药学专科分会优秀论文奖。项目申请人也因此获得中国药学会青年医院药学奖、医院卓越医师人才培养计划、公济杰出临床工作奖、全国优秀临床药师、医院拔萃人才培养计划等荣誉。该项目培养的研究生荣获研究生国家奖学金、校优秀毕业生、优秀三好学生、校一等奖学金等多项荣誉。