新型抑癌基因ARID2对HBV感染肝细胞增殖转化及病毒复制的调控研究

ARID2 is one of newly identified tumor suppressor gene and its inactive mutation plays an important role in the development of cancer, especially in hepatocellular carcinoma (HCC). However, ARID2-involved exact molecular mechanism in HBV infection is unknown. HBx is a multifunctional regulator, which is capable of transregulation of both viral and cellular genes. Although previous data point to a critical role of HBx in HBV-related HCC development, it is unclear HBx plays a direct role in regulation of ARID2 expression. Our preliminary data indicate that 1) HBx induced markedly repression of ARID2 in HBV replication-competent cells; 2) ARID2 transcripts and protein level were significantly decreased in HBV-HCCs relative to paired non-tumorous tissues;3) HBx, not HBcAg or HBsAg meidated downregulation of ARID2 expression; 3) Exogenous expression of ARID2 in hepatoma cells leads to reduced proliferation ability and migraratory capacity; 4) EMT process was significantly blocked in Huh7-ARID2 cells compared with control cells. Based on our preliminary data, we hypothesized that HBV infection induced ARID2 repression and hence contribute to HBV-related pathogenesis. In this study, HBV replicon cells, stably HBx-transduced cells and HBV-related HCC tissue samples were used to investigate the molecular mechanism that virus-induced downregulation of ARID2 expression. By ChIP-seq, ChIP and RNAi techniques, we will further explore cell migration and invasion potential induced by ARID2 in HBV replication-competent cells through cell signaling and downstream target genes, and finally, to find the molecular mechanism that ARID2-regulated HBV replication, including cccDNA formation, and transcription of HBV-encoded protein. Our studies will provide new insights into HBV-associated pathogenesis and may be helpful in developing an effective treatment against HCC.

ARID2基因是近年来新发现的一种抑癌基因，其失活突变是原发性肝癌发生的重要致病因素之一，但具体机制不明。HBV感染能否调控ARID2基因的表达，目前无相关研究报道。我们前期研究发现，在HBV复制细胞和临床肝癌组织标本中，ARID2基因的表达明显下调；调控ARID2基因表达的病毒蛋白以乙肝病毒X蛋白（HBx）为主；ARID2基因可以抑制肝癌细胞的增殖与迁移，阻断EMT 进程。本课题拟在此基础上，从HBV复制细胞模型入手，研究HBV尤其是HBx对ARID2基因的表达调控及分子机制，并通过ChIP-Seq、ChIP、基因沉默等技术探索ARID2调控相关信号通路及下游靶基因，影响细胞增殖和迁移的机制；研究ARID2基因对HBV cccDNA形成、病毒基因转录以及病毒复制水平的调控。项目的实施将丰富我们对ARID2调控细胞增殖和病毒复制的生物学功能的认识，同时也为寻找抗病毒治疗的新靶标奠定基础。

ARID2作为染色质重塑复合物家族的一员，在肝癌及多种肿瘤中存在失活突变。但到目前为止，ARID2基因的生物学功能尚不明确，其在肿瘤发生发展特别是肝细胞肝癌中的作用仍知之甚少。HBx作为一个原癌蛋白，在HBV相关肝癌的发生和发展中起非常关键的作用，其能否调控ARID2的表达及调控的具体分子机制，无相关研究报道。本研究主要包括两方面内容，一是在体外肝癌细胞中观察抑癌基因ARID2对细胞增殖和侵袭等生物学功能的影响，并在荷瘤鼠模型中观察干预ARID2的表达对于体内成瘤的影响，探索ARID2在肿瘤发生中所起的作用及其相关分子机制；另一方面，观察HBV编码蛋白HBx是否可以调节ARID2的表达及其具体的分子机制，探讨HBx通过调控ARID2的表达，在HBV相关肝癌发生发展中的作用。实验结果表明：（1）ARID2可以抑制肿瘤细胞的增殖以及肿瘤的生长，并且可以抑制细胞周期进程，使细胞停滞于G1期，具体调控的方式是抑制激活性转录因子E2F1的表达，促进阻滞性转录因子E2F4的表达，同时抑制E2F1-pRb募集到下游靶基因如CyclinD1和CyclinE1启动子区，从而抑制细胞周期蛋白CyclinD1、CyclinE1的表达；（2）在肝癌细胞和临床肝癌组织中，HBx下调ARID2的表达，其通过调控转录因子ATOH1间接下调ARID2蛋白的表达；（3）ARID2可部分逆转HBx介导的促肝癌细胞增殖和迁移作用。本研究阐明了染色质重塑复合物ARID2的一种新的抑癌机制，并且首次证实了HBx可以通过调控转录因子ATOH1间接下调ARID2蛋白的表达，促进肝癌细胞增殖和迁移能力，进而参与肝细胞癌的发生发展过程。上述研究结果为HBx及HBV介导的肝细胞肝癌的发生发展提供了新的方向，同时也为肝细胞肝癌的预防、治疗及预后提供了新的思路。