Objective: To study the effect of hepatitis B virus X protein (HBx) expression level on migration and invasion of zonula occludens protein-1 (ZO-1) in HepG2 liver cancer cells. Methods: Liver cancer cells were transfected with HBV full gene plasmid (pcDNA3.1-HBV1. 1 or pcDNA3.1-HBV1.3), empty plasmid (pcDNA3.1) and HBV-encoded protein plasmids (pHBc, pHBs, pHBp and pHBx), respectively. Western blot and RT-PCR were used to detect ZO1 protein and mRNA levels. Immunoprecipitation was used to detect transfected pHBx. Western blot was used to detect ZO1 ubiquitination levels. Transwell chambers were used to assess cell migration and invasion. Cell proliferation and lactate dehydrogenase assay was used to detect siRNA transfecting targeting ZO1. Flow cytometry was used to detect cell apoptosis and cycle. The data was compared between two and multiple groups by using an independent sample t-test and one-way analysis of variance. Results: Compared with the empty plasmid, ZO1 protein level in HepG2 cells after transiently transfected with pHBV1.1 and pHBV1.3 was decreased by 42.99% ± 6.8% and 55.0% 5 ± 4.56%, respectively, and their mRNA levels did not change significantly. ZO1 protein level in Huh7 cells was decreased by 17.46% ± 4.94% and 47.53% ± 3.38%, respectively. ZO1 protein level after transfection with pHBx was decreased by 47.02% ± 3.4%, while the ZO1 protein level after transfection with pHBc, pHBs and pHBp did not change significantly. ZO1 mRNA level was unaffected with pHBx transfection. ZO1 ubiquitin level and cell migration and invasion ability in HepG2 cells was significantly increased with transfected pHBx. HepG2 cells proliferation, apoptosis and cycle after transfection with ZO1-targeted siRNA did not change significantly, but the migration and invasion ability were significantly increased. Conclusion: HBx can increase the migration and invasion of liver cancer cells by promoting the ubiquitination and degradation of ZO1 protein level.
目的： 研究乙型肝炎病毒X蛋白（HBx）对HepG2细胞ZO1表达水平的影响及闭锁小带蛋白1（ZO-1）对肝癌细胞迁移和侵袭的作用。 方法： 分别转染HBV全基因质粒（pcDNA3.1-HBV1.1或pcDNA3.1-HBV1.3）、空载质粒（pcDNA3.1）和HBV编码蛋白质粒（pHBc、pHBs、pHBp、pHBx）至肝癌细胞，蛋白质印迹法（Western blot）和RT-PCR检测细胞中ZO1蛋白水平及mRNA水平；转染pHBx,免疫共沉淀和Western blot检测ZO1泛素化水平，Transwell小室检测细胞的迁移与侵袭。转染靶向ZO1的siRNA，乳酸脱氢酶实验检测细胞的增殖，流式细胞术检测细胞的凋亡及周期，Transwell小室检测细胞的迁移与侵袭。两组数据间比较用独立样本t检验，多组数据比较用单因素方差分析。 结果： 瞬时转染pHBV1.1和pHBV1.3后，相较于空载体对照，HepG2细胞中ZO1的蛋白水平分别下降了42.99%±6.8%和55.0%5±4.56%，其mRNA水平无显著变化；Huh7细胞中ZO1的蛋白水平分别下降了17.46%±4.94%和47.53%±3.38%。转染pHBx后，ZO1蛋白水平下降了47.02%±3.4%，而转染pHBc、pHBs和pHBp后ZO1蛋白水平比较，差异无统计学意义。转染pHBx未影响ZO1的mRNA水平。转染pHBx导致HepG2细胞中ZO1泛素化水平显著增加，细胞迁移和侵袭能力增强。转染靶向ZO1的siRNA后HepG2细胞的增殖、凋亡和周期无明显变化，而迁移和侵袭能力显著升高。 结论： HBx可通过促进ZO1蛋白的泛素化降解，增加肝癌细胞的迁移与侵袭。.
Keywords: Hepatocellular carcinoma; Invasion; Migration; Ubiquitin; Zonula occludens 1.