一组新的乙肝病毒X基因的序列变异在原发性肝癌中的临床意义和功能研究

X protein is the critical effector of hepatitis B virus in the carcinogenesis and development of liver cancer and amount of previous evidences have indicated the frequent variations in sequence of X gene. However, the exact charactors, biological functions and underlying mechanisms of X gene variation in hepatocellular carcinoma remain largely unclassifed. We previously explored the whole sequence of X gene in both tumoral and nontumoral tissues from a large cohort of HCC patients and found that a group of simulteneously occurred gene sequence variations (designated as novel X genotype) could predict the better prognosis of HCC patients after hepatectomy, which lead us to hypothesize that this novel X genotype might harbor biological significances and might pretect hepatitis B virus-infected liver from carcinogenesis and tumor progression. In this project, through comprasions of HCC cell phenotypes after transfection of wild type, novel genotype of X gene and scrambel control vectors, we aimed to illustrate the functional significance of the novel X genotype in the process of hepatocellular carcinoma advanced by X protein. The gene expression profiling would be applied to investigate the downstream signalling pathways correlated with the novel X genotype and immunoprecipitation assays would be used to explore the direct effector of X protein coded by the novel X genotype. Furthermore, we would investigate the potentially clinical value of the novel X genotype as a biomarker in liver cancer. Generally, this study would firstly uncover a group of variations in X gene with benefit for hepatocellular carcinoma when compared to wild type of X gene and clarity the underlying mechanisms, leading to more attention paid on X gene variations in diagonosis and therapy of hepatocellular carcinoma.

X蛋白是乙肝病毒感染导致原发性肝癌发生和发展的关键因子，编码X蛋白的基因序列具有一定的多态性，但是肝癌病人中X基因序列变异的特征、生物学意义和作用机制尚未被系统阐述。前期考察大规模临床样本中X基因的序列特征并筛选得到与肝癌病人较好预后密切相关的一组变异（X基因新亚型），我们由此推测这组基因变异可影响X蛋白的功效，是乙肝病毒致癌过程中重要的保护性因素。本研究拟从体内、外水平考察此组变异对X蛋白调控肿瘤细胞表型过程中所具效应的影响，利用高通量表达谱检测结合免疫共沉淀等手段阐明此组X基因变异对肝癌细胞下游通路的调控作用和分子机制，再利用临床队列考察X基因变异作为肝癌诊断标志物的价值。我们将首次报道能降低X蛋白致癌作用的一组基因变异，阐明其作用机制并探讨其临床价值，对进一步揭示X基因在肝癌中发挥作用的多样性，加强肝癌诊疗过程中乙肝病毒X基因型的重视程度具有重要意义。

以往对肝癌基因组学的研究往往集中于宿主（人肝细胞）基因的突变或者结构改变，对HBV 核心效应分子HBX 蛋白的基因序列变异的生物学意义和临床价值仍缺少系统性的研究。而研究HBV 本身的序列变异，尤其是编码核心蛋白的HBX 基因序列变异，对于慢性HBV 感染者早期肝癌的筛查以及原发性肝癌病人的治疗手段选择具有同样重要的临床意义。.在本研究的前期工作中，我们通过对大队列肝癌患者癌和癌旁组织HBx基因的测序和聚类分析，对HBx基因进行了分型，发现了一型新的HBx基因型HBx-EHBH2（HBx-E2）。在肝癌组织队列中，预后分析表明携带有HBx-E2的患者预后较好，且与多个较好的临床指标相关。更重要的是，我们在外周血中也检测到了该基因型的存在，并且HBx-E2在血清队列中同样与患者较好的预后相关。在本研究中，我们发现不同地区的肝癌患者组织队列（n=171）的癌和癌旁中以及在血清队列（n=168）中，HBx-E2均与患者较好的预后相关。临床病理指标相关性分析表明HBx-E2与肝硬化缺失、肿瘤直径较小相关。按照HBx基因型分类和巴塞罗那肝癌临床（BCLC）分期在组织队列和血清队列分层进行预后分析，结果表明HBx-E2可以作为术前预测BCLC B期患者预后的分子标记物。体内外实验证明相对于其他基因型，HBx-E2失去了促进肝癌细胞和肝细胞增殖的能力。蛋白芯片和免疫共沉淀等实验发现HBx-E2失去与JAK1结合的能力，而且不能激活STAT3和STAT5。JAK1的抑制剂Upadacitinib可以抑制除HBx-E2外其他基因型的增殖能力。综上所述，HBx-E2可以作为分子标记物术前预测BCLC B期患者预后。HBx-E2失去了促进肝癌细胞和肝细胞增殖的能力，不能激活JAK1/STATs通路。JAK1是HBV相关肝癌中某些HBx基因型患者的潜在治疗靶点。