Sirt1对HBV DNA转录复制的调控作用及机制研究

Hepatitis B is one of the most harmful public diseases in China because it easily develops to liver fibrosis, liver cancer and liver failure, but unfortunately current therapies for Hepatitis B are still unsatisfied. Both of HBV gene expression and replication are restricted by the level of HBV transcription which is controlled by host's type II RNA polymerase and other transcriptional factors. In previous studies, we have showed that NAD+ dependent type III deacetylase Sirt1 can regulate the replication of HBV DNA. In this project, we plan to further investigate that the influence of Sirt1 on HBV transcripts, viral replication intermediates, viral proteins in HBV replication cell models by Northern blot, Southern blot, Western blot, Radioimmunoassay, RNAi, Realtime PCR and so on. In addition, the protein targeted by Sirt1 and its pathway acting on HBV transcription will also be deeply investigated by Chromatin immunoprecipitation, immunoprecipitation, dual- fluorescence report system. Finally, the function of Sirt1 on HBV replication will be confirmed at a HBV transgenic mouse model by lentivurs technology and RNAi. Our study will firstly find Sirt1 is a new factor to regulate HBV replication and viral expression via mediating the level of HBV transcription. Our project will suggest a previously unrecognized mechanism of virus-host interaction and identifies Sirt1 as a central player in HBV life cycle, making it a potential target for future anti-HBV therapies.

慢性乙型肝炎患者易发展为肝硬化、肝癌和肝衰竭，严重危害我国人民的生命健康，迄今没有满意的治疗方法。虽然人们对HBV的生活周期有了很大的认识，但宿主因子对HBV DNA转录调控的研究尚待进一步探索和明确。我们在前期研究中首次发现，NAD+依赖的蛋白脱乙酰化酶Sirt1在HBV转录复制中起调控作用。本项目拟在前期实验结果基础上进一步研究Sirt1对HBV转录本、复制中间体及病毒蛋白的调控作用，明确Sirt1是新的HBV DNA转录相关的蛋白和信号分子；鉴定出Sirt1是HBV DNA转录过程中的必需蛋白，并阐明其调控HBV DNA转录的信号通路；最后在HBV转基因小鼠模型中验证Sirt1在HBV转录复制和病毒蛋白表达中的调控作用。本研究结果，将发现Sirt1的新功能，并可以作为抗HBV治疗的潜在作用靶点，同时，在宿主因子调控HBV DNA转录的分子机制中提出创新性见解。

慢性乙型肝炎患者易发展为肝硬化、肝癌和肝衰竭，严重危害我国人民的生命健康，迄今没有满意的治疗方法。虽然人们对HBV的生活周期有了很大的认识，但宿主因子对HBV DNA转录调控的研究尚待进一步探索和明确。我们在前期研究中首次发现，NAD+依赖的蛋白脱乙酰化酶Sirt1在HBV转录复制中起调控作用。本项目在HBV复制细胞模型HepG2.2.15细胞中证实了激活Sirt1促进HBV DNA的复制。进一步研究发现，Sirt1天然激动剂白藜芦醇（Resveratrol, RSV）激活Sirt1，促进HBV的核心启动子，增加pgRNA和核心蛋白的表达，证实Sirt1是新的促进HBV DNA转录相关蛋白和信号分子；同时找到Sirt1-PGC1a-PPARa 通路在调控HBV DNA复制中起重要作用。最后在HBV复制的小鼠模型中证实了激活Sirt1在HBV转录复制和病毒蛋白表达中的调控作用。本研究结果，证实了Sirt1的促HBV DNA复制作用，提示抑制该基因可以减轻HBV的复制，同时提出，在慢乙肝患者中，需谨慎服用Sirt1激活剂白藜芦醇，预防疾病复发，具有显著的临床意义。此外，首次提出白藜芦醇通过Sirt1-PGC1a-PPARa 通路促进HBV复制，具有较好的创新性。