LHBs对HNF4的调控及其在HBV转录、复制和肝细胞增殖转化中的作用机制研究

Hepatitis B virus large surface protein (LHBs) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. In our previous works, using proteomic chip assay, hepatocyte nuclear factor 4 (HNF4) was upregulated by overexpression of LHBs. HNF4, a member of liver-enriched transcription factor (LETF) family, was recognized as a key factor in process of HBV transcription and replication, and in the process of liver cell proliferation and transformation. No relationship of the two proteins were reported before. In the current case, in vivo and in vitro interactions between HNF4 and LHBs will be further confirmed by standard immunoprecipitation and GST pull-down assays. In addition, to gain a insight into the molecular action of LHBs in viral hepatitis B, we will perform a systematic proteomic analysis of LHBs-associated proteins in HepG2215 cells, which recently developed by Matthias Mann, termed quantitative immunoprecipitation combined with knockdown (QUICK), integrating RNAi, iTRAQ, immunoprecipitation and quantitative Mass technologies. Using this system coupled with bioinformatics, a protein interaction map of LHBs was generated, and the molecular regulatory mechanism from LHBs to HNF4 was revealed. These data were valuable for further to study the mechanism of viral hepatitis B, and also provide some new clues for investigating HBV and human hepatocellular carcinoma.

乙型肝炎病毒S基因及其编码的多功能蛋白LHBs是近年来发现的在HBV致病、致癌过程中起重要作用的转录激活蛋白。申请者长期关注S蛋白与宿主蛋白的相互作用，前期研究中首次发现LHBs可上调肝细胞核因子HNF4的表达水平；HBV复制细胞中，HNF4基因表达水平明显上升，调控HNF4表达的HBV编码病毒蛋白以LHBs为主。肝细胞核因子HNF4是已被证实的调节HBV转录、复制的重要蛋白，且参与肝细胞增殖转化过程。二者之间的联系未见报道。本项目拟在此基础上采用免疫共沉淀结合iTRAQ-shotgun技术分离鉴定与LHBs相互作用的蛋白分子，通过生物信息学方法，描绘与LHBs相互作用的蛋白及其下游蛋白作用网络，以探讨LHBs调控HNF4表达，进而参与HBV转录、复制和肝细胞增殖转化过程的确切机制。以期为揭示HBV复制调控的新机制、寻找以抗病毒和抗肿瘤的新靶标药物奠定基础。