宿主肝细胞内锌指蛋白ZBTB7A调控PI3K/AKT/mTOR通路在乙型肝炎病毒复制中的作用及机制

The mechanism for effect of host genes on the regulation of HBV replication is unknown. Our study showed that the intrahepatic ZBTB7A levels have negative correlation with serum HBV DNA load. Silencing of ZBTB7A with small interfering RNA enhances HBV replication and over-expression of ZBTB7A suppresses HBV replication significantly. It's reported that ZBTB7A modulates genesis of tumors via activating PI3K/AKT/mTOR pathway.Also, it's reported that activation of PI3K/AKT/mTOR pathway may inhibit autophagy process, which facilitates HBV replication,hence decreasing viral replication.To investigate whether gene ZBTB7A modulates PI3K/AKT/mTOR pathway and the downstream autophagy, we knocked down ZBTB7A with small interfering RNA in HepG2.2.15 cells and found that expression of AKT, phospho-Akt ,phospho-mTOR down regulated. Further, we discovered that the level of autophagy-related protein LC3 increased and P62 which is a marker of autophagy degradation decreased. These results indicated that ZBTB7A can modulate PI3K/AKT/mTOR pathway and downstream autophagy to regulate HBV replication. However, how ZBTB7A modulated PI3K/AKT/mTOR pathway is still unknown. Previous reports showed that ZBTB7A could interact with protein H-Ras. Meanwhile, it's reported that H-Ras can promote PI3K activity. Based on our previous findings and reports, we hypothesize that gene ZBTB7A mediates HBV replication by regulates PI3K/AKT/mTOR pathway and downstream autophagy via interaction with H-Ras. We aim to further clarify the mechanism for effect of gene ZBTB7A on the regulation of HBV replication in hepatoma cell lines and high-pressure tail vein injection mouse model by means of knocking-down or over expression of ZBTB7A gene , immunofluorescence, co-immunoprecipitation, fluorescence co-localization and so on. This study will help to identify the mechanism of host gene ZBTB7A on regulating HBV replication and to develop new insights for antiviral therapy of chronic hepatitis B.

慢乙肝患者肝细胞调控HBV复制的机制仍未阐明。前期发现肝细胞内锌指蛋白ZBTB7A的表达水平与病毒载量负相关。体外过表达和敲除ZBTB7A基因，可分别显著下调和上调HBV复制。据报道，ZBTB7A可激活PI3K/AKT/mTOR通路调控多种肿瘤发生。另有报道激活PI3K/AKT/mTOR可抑制自噬，下调HBV复制。我们前期敲除ZBTB7A后，mTOR通路活性下降及下游自噬形成增加，提示ZBTB7A可调控PI3K/AKT/mTOR及下游自噬。目前ZBTB7A如何调控该通路尚未知。文献报道ZBTB7A可与H-Ras蛋白结合，而H-Ras蛋白可激活PI3K，故我们提出假设：ZBTB7A可通过H-Ras调控PI3K/AKT/mTOR通路及下游自噬，调控HBV复制。我们拟在体外和体内实验中，通过基因过表达与敲除、免疫荧光、免疫共沉淀、荧光共定位等方法阐明ZBTB7A调控HBV复制的机制。

慢乙肝患者肝细胞调控HBV复制的机制仍未阐明。本课题前期发现肝细胞内锌指蛋白ZBTB7A的表达水平与乙肝病毒载量负相关。为验证其对HBV复制的调控作用及机制，我们通过在Huh7细胞系内转染带GFP标签的ZBTB7A过表达质粒或表达HBV病毒的pSM2质粒，通过免疫荧光共聚焦，观察到内源性及外源性ZBTB7A蛋白均在细胞核内表达，且内源性高表达。进一步，我们在肝癌细胞系里过表达ZBTB7A可显著抑制HBV DNA和RNA水平，且降低病毒蛋白HBeAg和HBsAg分泌水平。为探究其机制，我们观察到ZBTB7A可明显抑制HBV核心启动子活性，并筛查了ZBTB7A对转录因子的影响，发现ZBTB7A能抑制核转录因子FXRα及HNF4α的RNA及蛋白表达水平，且通过免疫共沉淀方法，显示ZBTB7A蛋白可与FXRα及HNF4α蛋白直接相互作用，初步阐释其对HBV转录水平影响的机制。既往研究报道ZBTB7A可激活PI3K/Akt/mTOR通路调控多种肿瘤发生。另有研究报道激活PI3K/Akt/mTOR通路可抑制自噬，下调HBV复制。前期实验中过表达ZBTB7A后可激活Akt/mTOR通路。进一步我们检测自噬相关蛋白P62及LC3蛋白，发现过表达ZBTB7A可抑制自噬流，从而抑制病毒复制。同时，我们在慢性感染HBV小鼠模型中通过高压尾静脉注射过表达质粒，分别检测注射后1d、4d、7d、14d、21d、28d、35d、42d的HBsAg水平、HBeAg水平及HBV DNA水平。同时取注射后7d及21d的肝组织进行免疫组化检测。对照组为注射空白质粒M35，实验组为相同载体的质粒ZBTB7A。结果显示：实验组的HBsAg和HBeAg水平明显低于对照组，肝组织免疫组化结果显示实验组的HBcAg水平明显低于对照组，验证了ZBTB7A基因对HBV复制的抑制效应。病毒与宿主因素常相互作用、相互调节。由此，我们在体外和体内实验观察了感染HBV后对ZBTB7A基因表达水平的影响。体外实验显示，HBV感染后可下调细胞内ZBTB7A基因的表达水平，同时通过肝组织免疫组化显示，慢乙肝患者肝组织内ZBTB7A蛋白水平低于健康对照者，提示HBV感染后可下调ZBTB7A蛋白，可能以实现病毒逃逸。本课题发现了宿主肝细胞锌指蛋白ZBTB7A 基因对HBV复制的调控及机制，为寻找抗HBV药物的新靶位提供了新的理论依据。