HBx突变体通过转录因子介导靶基因GAS2诱导肝细胞癌变机制的研究

Hepatocellular carcinoma (HCC) is a worldwide threat to public health, especially in China, where chronic hepatitis B virus (HBV) infection is found in 80–90% of all HCCs. The HBV-encoded X antigen (HBx) is a trans-regulatory protein involved in virus-induced hepatocarcinogenesis. Although the carboxyl-terminus-truncated HBx, rather than the full-length counterpart, is frequently overexpressed in human HCCs, its functional mechanisms are not fully defined. We investigated the molecular function of a naturally occurring HBx variant which has 35 amino acids deleted at the C-terminus (tHBxΔ35). Genome-wide scanning analysis and PCR validation identified growth arrest-specific 2 (GAS2) as a direct target of tHBxΔ35 at transcriptional level in human immortalized liver cells. tHBxΔ35 was found to bind the promoter region of GAS2 and attenuate its expression to promote hepatocellular proliferation and tumourigenicity. Further functional assays demonstrated that GAS2 induces p53-dependent apoptosis and senescence to counteract tHBxΔ35-mediated tumourigenesis (J Pathology,2015). As a a trans-regulatory protein, tHBxΔ35 need to bind Transcri-ption Factor(TF) to induce the hepatocarcinogenesis. In the preliminary experiment, we screen the TF according to the sequence of all the tHBx- bound target promoters including GAS2 obtained from the data of ChIP-chip in the bioinformatics analysis software, in which POUF3F2 appears to be a significant transcription factor. Therefore, in this experiment,we will explore the functional mechanisms of TF-mediate tHBxΔ35 inducing hepatocarcinogenesis. Then, we will investigate the relationship between TF(POUF3F2) and GAS2 silenced by tHBxΔ35 in the level of cell、animal and clinic. Finally, we will recover further the associations of them in the development of liver cancer,which will contribute to provide a new therapeutic method for hepatocellular carcinoma.

乙型肝炎病毒(HBV)基因编码X蛋白(HBx)是诱导肝癌(HCC)发生的重要反式调控蛋白，需要在转录因子介导下作用于直接靶基因。在HCC组织中,HBx 羧基末端频繁易丢失35aa，形成自然突变体HBx(tHBxΔ35)；但对其诱导HCC机制并不清楚。在前期实验，我们首次用染色质免疫共沉淀-基因组芯片技术(CHIP-chip)筛出tHBxΔ35直接靶基因GAS2,并论证tHBxΔ35是通过抑制其靶基因GAS2进而干预细胞周期和阻断凋亡级联反应。本课题将进一步探讨tHBxΔ35如何通过转录因子与靶基因GAS2相结合的作用机制。通过预实验将CHIP-chip数据初筛出转录因子POU3F2，它作为转录调节抑制子，可能与肿瘤发生转移有关。采用免疫共沉淀、酵母双(单)杂交、免疫印迹等技术从细胞水平、动物水平以及临床HCC组织、癌周组织上探寻三者相互作用的关系和信号传导通路，从而对HCC的防治提出新思路

原发性肝癌是我国最常见的恶性肿瘤之一。乙型肝炎病毒(HBV)基因编码X蛋白(HBx)是诱导肝癌(HCC)发生的重要反式调控蛋白，近年研究发现在HCC组织中,HBx 羧基末端频繁易丢失35aa，形成自然突变体HBx(tHBxΔ35)；但对其诱导HCC机制并不清楚。本研究通过细胞计数、MTS实验、集落形成实验、流式技术、凋亡技术等验证HBx突变体(tHBxΔ35)促癌作用，并通过调节细胞周期促进增殖；通过染色质免疫共沉淀-基因组芯片(ChIP-chip)技术 ,ChIP-Seq技术IP-MS技术反复筛选和验证tHBxΔ35可能通过组蛋白的某种修饰而抑制靶基因GAS2。然后用细胞计数、MTS实验、集落形成实验、裸鼠成瘤实验来确定靶基因GAS2抑制细胞过度生长的功能；再用流式细胞PI技术、膜联蛋白V凋亡(Annexin V)、western blot技术来验证GAS2基因通过干预细胞周期,激活 GAS2-P53-caspase凋亡级联效应而促使肝癌细胞凋亡。最后在人体临床肝癌组织/癌周组织验证出相比较其癌旁组织，突变体HBxΔ35在肝癌组织更为普遍的表达；且在肝癌组织中Gas2的丢失与突变体HBx（tHBxΔ35）显著相关。本课题首次采用ChIP-chip技术 ,ChIP-Seq技术、IP-MS技术等从细胞水平、动物水平以及临床人体HCC组织、癌周组织上探寻和验证突变体tHBxΔ35和其靶基因GAS2诱导肝癌发生的作用机制，有望为HBV相关的HCC的防治提出新思路。