HBx通过COX-2及其下游因子诱导小鼠发生肝癌的机制研究

Mechanism of hepatitis B virus X inducing hepatocellular carcinoma in mice through COX-2 and its downstream factors

目的利用稳定表达乙型肝炎病毒X(hepatitis B virus X,HBx)的小鼠模型探讨HBx在致癌过程中COX-2以及下游因子的动态变化过程及其作用。方法利用Western blot、RT-qPCR检测HBx-EGFP-14-19细胞中HBx的表达;通过向昆明(KM)小鼠肝门静脉注射HBx-EGFP-14-19细胞建立可长期稳定表达HBx的动物模型,分别在30、90、180和360 d处死小鼠;利用Western blot,RT-qPCR及免疫组化的方法检测HBx在肝脏中的表达,HE染色观察肝组织病理特征;Western blot,RT-qPCR检测肝组织中COX-2及其下游因子、细胞周期、凋亡相关因子mRNA和蛋白表达水平。结果成功构建了可长期稳定表达HBx的动物模型并最终发展为肝癌;HBx转入小鼠体内90 d后COX-2及其下游因子VEGF的蛋白质和mRNA表达水平被持续上调直至肿瘤发生(P<0.05);小鼠转入HBx后肝脏促凋亡因子Bad、Bax的蛋白质和mRNA表达水平降低(P<0.05),抗凋亡因子Bcl-2的mRNA和蛋白水平升高(P<0.05);细胞周期因子CyclinD1、CDK4的mRNA和蛋白质表达水平升高(P<0.05);给予HBx组小鼠COX-2抑制剂塞来昔布后可逆转HBx对上述基因的作用。结论HBx可导致肝癌发生,其机制可能与上调COX-2及其下游因子有关。

Objective To investigate the dynamic changes and the roles of COX-2 and its downstream factors in the carcinogenic process using a mouse model stably expressing hepatitis B virus X(HBx).Methods The expression of HBx in HBx-EGFP-14-19 cells was detected by Western blotting and RT-qPCR.A mouse model with stable expression of HBx was then established by injecting HBx-EGFP-14-19 cells into KM mice via hepatic portal vein.The mice were subsequently sacrificed at 30,90,180 and 360 d,respectively,and the liver tissues were collected.Western blotting,RT-qPCR and immunohistochemical assay were used to detect the expression of HBx in the liver;HE staining was adopted to observe the pathological characteristics of liver tissues.In addition,Western blotting and RT-qPCR were also employed to determine the expression changes of COX-2 and its downstream factors,cell cycle-and apoptosis-related factors in the liver tissues,at both mRNA and protein levels.Results The animal model with long-term stable expression of HBx was successfully constructed and eventually developed into liver cancer.The mRNA and protein levels of COX-2 and its downstream factor VEGF were continuously up-regulated even in 90 d after HBx was transferred into mice and the increment was continued until tumorigenesis(P<0.05).The protein and mRNA levels of liver pro-apoptotic factors Bad and Bax were decreased(P<0.05),while those of the anti-apoptotic factor Bcl-2 were increased(P<0.05),and the levels of cell cycle factors Cyclin D1 and CDK4 were increased(P<0.05).However,the effects of HBx on the above genes were reversed after administration of COX-2 inhibitor celecoxib.Conclusion HBx can cause liver cancer,which may be related to the up-regulation of COX-2 and its downstream factors.