HBV核心抗原通过直接激活核受体CAR促进肝细胞自噬参与HBV相关肝癌发生的新型分子机制

HBV core antigen (HBc) is the key protein of hepatitis B virus replication. Our team has taken the lead in presenting the important role of HBc in the carcinogenesis of HBV related hepatocellular carcinoma, but its detailed mechanism is not clear. We previously screened out the nuclear receptor CAR that directly interacted with HBc protein using Protein chip and proteomics methods. The functional experiment showed that HBc activated CAR and made it play a role as transcription factor. Antisense blocking CAR significantly reversed HBc-induced hepatocytes autophagy and proliferation. However, the regulation of autophagy and related proteins by CAR has not been reported. Therefore, we proposed the hypothesis: HBc can directly activate CAR to regulate the expression of the key proteins of autophagy at the transcriptional level, and promote the autophagy and proliferation of hepatocytes, therefore, participate in the occurrence of HBV related hepatocellular carcinoma(HCC). Using clinical samples, cells, transgenic and gene knockout mouse models, we will revealed that HBc interacts with CAR and CAR activation site by HBc using gene transfection and functional assays and ChIP-seq et al. Additionally, we will discover and verify the key protein targeting autophagy and its promoter binding sites regulating by CAR, as well as key autophagic pathway. This study will elucidate the new molecular mechanism of HBc in the pathogenesis of HBV-related HCC and provide a new target for the treatment of HBV-related HCC.

课题组曾率先提出HBV核心抗原HBc在肝癌发生中的促癌作用，但其详尽机制尚未明确。为进行深入机制探讨，申请者前期利用蛋白质芯片筛选出与HBc直接相互作用的核受体CAR，并发现HBc可使CAR激活入核，发挥其转录因子的转录调节功能；CAR基因沉默后，能明显逆转HBc引起的细胞的自噬、增殖。然而，CAR对自噬及相关基因的调控机制未见报道。基于以上结果，本课题提出假说：HBc可使CAR激活入核，转录调控自噬关键蛋白的表达，从而促进肝细胞自噬和增殖，最终导致HBV相关肝癌的发生。本课题拟在前期蛋白质组学筛查的基础上，利用临床标本、细胞、转基因/敲基因鼠模型及基因转染、功能实验、ChIP-seq测序等技术，揭示HBc与CAR相互作用及激活位点；明确CAR激活后，其靶向调控的自噬关键蛋白及启动子结合位点、自噬关键通路等。该研究将阐明HBc在HBV相关肝癌发生中的新型分子机制，为肝癌治疗提供新靶标。

我国近九成肝癌由慢性乙型肝炎转归而来。因此，探讨乙型肝炎病毒导致的肝癌的发生发展的分子病理机制，是解决肝癌早期诊断和治疗的关键。课题组在前期研究中深入探讨了HBV各基因片段的表达对外源性诱导剂TRAIL诱导肝细胞凋亡的影响及分子机制。研究结果表明，HBV全基因组及HBx蛋白、截短型中蛋白MHBs(t)可明显提高肝癌细胞对TRAIL诱导凋亡的敏感性，从而参与了HBV感染后的急性损伤 。然而，在研究HBV核心抗原（HBc）时，课题组意外发现：HBV 核心抗原（HBc）能够抵抗TRAIL诱导的肝细胞凋亡。据此，课题组首次提出假说： HBV 核心抗原(HBc)表达可能致使HBV感染慢性化，从而促使HBV相关肝癌的发生课题组曾率先提出HBV核心蛋白HBc在肝癌发生中的促癌作用，但其详尽机制尚未明确。为进行深入机制的探讨，申请者前期利用蛋白质芯片筛选出与HBc直接相互作用的核受体CAR，并发现HBc可使CAR激活入核，发挥其转录因子的转录调节功能；CAR基因沉默后，能明显逆转HBc引起的细胞的自噬、增殖。然而，CAR对自噬及相关基因的调控机制未见报道。基于以上结果，本课题提出假说：HBc可使CAR活化入核，转录调控自噬关键蛋白的表达，从而促进肝细胞自噬和增殖，最终导致HBV相关肝癌的发生。本课题拟在蛋白质组学筛查的基础上，利用基因转染、功能实验及ChIP-seq测序等技术,揭示HBc与CAR相互作用及激活位点；明确CAR激活后，其靶向调控的自噬关键蛋白及启动子结合位点、自噬关键通路等。按照上述研究计划，明确了HBc通过直接激活CAR促进肝细胞的自噬、增殖的生物学效应，同时确定HBc与CAR相互作用的位点为CAR的ZNF结构域。明确了CAR的下游靶基因ATG5，其通过靶向调节自噬蛋白ATG5的表达，最终促进肝细胞自噬促进肝癌细胞自噬的新型分子机制。该研究将阐明HBc在HBV相关肝癌发生中的新型分子机制，为肝癌治疗提供新靶标。