ABSTRACT

Hepatitis B virus (HBV) exists as quasispecies (QS). However, the evolutionary characteristics of haplotypes of HBV X gene in the hepatocellular carcinoma (HCC) microenvironment remain unclear. Mutations across X gene are essential for the tumorigenicity of HBV X protein (HBx). However, the functional phenotypes of many mutant HBx remain unknown. This study aims to compare the characteristics of X gene evolution between tumour and non-tumour tissues in HCC patients and investigate the tumorigenic phenotype of HBx harbouring mutation T81P/S101P/L123S. This study included 24 HCC patients. Molecular cloning of X gene was performed to analyse characteristics of haplotypes in liver tissues. HCC cell lines stably expressing wild-type or mutant HBx and subcutaneous tumour xenograft mouse model were used to assess HBx-T81P/S101P/L123S tumorigenicity. The mean heterogeneity of HBV QS across X gene in tumour tissues was lower than that in non-tumour tissues. A location bias was observed in X gene clones with genotype C or D in tumour tissues compared to those with genotype B. Mutations in genotype-C or – D clones were mainly clustered in the dimerization region and aa110-aa140 within the transactivation region. A novel mutation combination at residues 81, 101 and 123 was identified in tumour tissues. Further, HBx-T81P/S101P/L123S promotes cell proliferation and increases genomic instability, which was mediated by MYC. This study elucidates the compartmentalized evolution patterns of HBV X gene between intra tumour and non-tumour tissues in HCC patients and provides a new mechanism underlying HBV-driven hepatocarcinogenesis, suggesting a potential viral marker for monitoring HCC.

摘要

乙型肝炎病毒 (HBV) 作为准种 (QS) 存在。然而，HBV X基因单倍型在肝细胞癌（HCC）微环境中的进化特征仍不清楚。X 基因的突变对于 HBV X 蛋白 (HBx) 的致瘤性至关重要。然而，许多突变 HBx 的功能表型仍然未知。本研究旨在比较 HCC 患者肿瘤组织和非肿瘤组织之间 X 基因进化的特征，并探讨携带突变 T81P/S101P/L123S 的 HBx 的致瘤表型。该研究包括 24 名 HCC 患者。进行X基因的分子克隆以分析肝组织中单倍型的特征。使用稳定表达野生型或突变型 HBx 的 HCC 细胞系和皮下肿瘤异种移植小鼠模型来评估 HBx-T81P/S101P/L123S 的致瘤性。肿瘤组织中跨X基因的HBV QS平均异质性低于非肿瘤组织。与基因型 B 相比，在肿瘤组织中具有基因型 C 或 D 的 X 基因克隆中观察到定位偏差。基因型 C 或 - D 克隆中的突变主要聚集在二聚化区域和反式激活区域内的 aa110-aa140。在肿瘤组织中发现了第 81、101 和 123 位残基的新突变组合。此外，HBx-T81P/S101P/L123S 促进细胞增殖并增加由 MYC 介导的基因组不稳定性。本研究阐明了 HCC 患者肿瘤内和非肿瘤组织之间 HBV X 基因的区室化进化模式，并提供了 HBV 驱动的肝癌发生的新机制，为监测 HCC 提供了潜在的病毒标志物。