乙肝病毒X蛋白（HBx）及表观遗传学机制调控锌指蛋白ZNF382参与肝癌的发生及发展

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death in China. However, the pathogenesis of this disease remains largely unknown. Accumulated evidences have demonstrated that HBV infection is closely associated with liver carcinogenesis. In addition, like other cancers, initiation and progression of HCC involves multiple genetic and epigenetic alterations, ultimately leading to gain-of-function in oncogenes and loss-of-function in tumor suppressor genes. Zinc-finger proteins are the largest family of transcription factors with zinc finger motif, which play a critical role in cell differentiation, embryogenesis and tumorigenesis. Most of them are transcription repressors. However, the repression is commonly realized via creation of an inactive chromatin domain rather than via direct influence on preinitiation complex assembly, such histone deacetylation, histone methylation, recruitment of HP1 protein, and formation of a sufficiency stable repressed domain (heterochromatin).The previous studies show that several Zinc-finger proteins are aberrantly expressed in human cancers, contributing to carcinogenesis. However, its biological roles in HCC remains poorly understood. Notably, our preliminary data showed that ZNF382 gene was down-expressed in HCCs compared with adjacent non-tumor tissues. Moreover, this gene was frequently hypermethylated in a cohort of HCCs. Demethylation agent 5-Aza-2'-dC and histone deacetylase inhibitor SAHA significantly induced re-expression of silenced ZNF382 gene in HCC cells. Importantly, silencing ZNF382 expression in HCC cells dramatically promoted cell proliferation, colony formation, invasion and migration, and induced cell cycle entry. In contrast, ZNF382 re-expression in HCC cells significantly inhibited cell proliferation and induced apoptosis, suggesting that ZNF382 may be a novel tumor suppressor in HCC. In addition, our data showed that HBV protein x (HBx) positively regulated ZNF382 expression. The aims of this project were: 1) to explore the biological roles of ZNF382 in HCC; 2) to identify its downstream target genes using ChIP-Seq and gene expression microarray approaches; 3) to clarify mechanisms underlying ZNF382 expression modulated by HBx and epigenetic alterations.

肝癌是我国常见恶性肿瘤，其发生发展与乙肝病毒（HBV）感染有关，此外还涉及诸多遗传及表观遗传学改变。锌指蛋白具有特殊指状结构，能结合DNA等大分子，调控多种生理、病理过程，包括肿瘤。锌指蛋白ZNF382，能招募异染色质蛋白1（HP1）与DNA结合，参与靶基因抑制型转录调控，目前尚无该基因在肝癌中的相关报道。我们的前期结果显示ZNF382在肝癌组织及细胞中表达下调，且启动子区发生高频率的甲基化，DNA去甲基化试剂及组蛋白去乙酰化酶抑制剂能恢复其表达。下调肝癌细胞中该基因的表达能促进细胞增殖、克隆形成及侵袭；相反，过表达该基因能抑制细胞增殖并诱导凋亡，提示ZNF382可能是一个新的肝癌抑制因子。此外，我们还发现乙肝病毒x蛋白（HBx）能正向调控ZNF382的表达。本项目旨在进一步明确ZNF382在肝癌发生及发展中的生物学功能，鉴定其基因调控网络，阐明HBx及表观遗传学改变调控其表达的分子机制。

肝癌是我国常见恶性肿瘤，其发生发展与乙肝病毒（HBV）感染有关，此外还涉及诸多遗传及表观遗传学改变。锌指蛋白ZNF382，参与靶基因抑制型转录调控，已有报道在鼻咽癌、食管癌、胃癌、结肠癌及乳腺癌中均发现 ZNF382 由于甲基化而低表达，恢复其表达则抑制肿瘤进展，提示ZNF382的抑癌功能。在本研究中，我们检测了不同来源肝组织中ZNF382的水平，发现乙肝-肝硬化-肝癌过程中ZNF382水平在HBV感染后增高，而在癌组织中又降低。HCC组织及细胞系中HBV DNA含量及HBx水平与ZNF382水平正相关，且通过过表达和敲减HBx，从正反两方面证实HBx从转录水平及转录后水平分别上调ZNF382，且以转录后调控为主。通过定量PCR、3’UTR的双荧光素酶报告系统实验、pErk抑制剂处理等，证实miR6867能够结合ZNF382 3’UTR，从而抑制其表达，HBx通过激活Erk磷酸化而抑制miR6867前体的成熟过程，因而上调ZNF382。另外，我们发现肝癌组织普遍发生ZNF382基因启动子区甲基化等表观遗传学失活，且早期HCC患者癌组织中ZNF382的表达水平与患者预后呈正相关；通过过表达及敲减ZNF382，我们从正反两方面证实了ZNF382能够抑制HCC细胞的增殖、克隆形成、抗凋亡、裸鼠成瘤等恶性生物学行为；通过RT-qPCR、ChIP- qPCR及启动子区双荧光素酶报告等实验证实ZNF382抑制AP-1、MYC等促癌信号通路及分子活性，在肝癌的进展中发挥了关键生物学作用。综上，我们初步阐明了HBx及表观遗传学机制调控ZNF382参与肝癌发生发展的分子机制。