乙肝病毒X蛋白通过CREB上调HBx结合蛋白促进肝癌细胞迁移

Hepatitis B Virus X Protein Promotes Hepatoma Cell Migration via CREB-induced Up-regulation of Hepatitis B X-interacting Protein

乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对肝癌的发生发展具有十分重要的作用.HBx具有促进肝癌迁移的作用,但其作用的分子机制不清.本研究对HBx促进肝癌细胞迁移的分子机制进行了探讨.伤口愈合和Boyden’s chamber结果表明,HBx可明显促进肝癌Hep G2细胞迁移.在稳定转染HBx的Hep G2(Hep G2-X)细胞中转染HBx结合蛋白(hepatitis B X-interacting protein,HBXIP)的RNA干扰片段,可明显抑制HBx的促迁移作用.免疫组化和实时定量PCR结果表明,HBXIP在肝癌组织中显著高表达,并且与HBx表达成正相关.荧光素酶报告基因和免疫印迹结果表明,HBx显著增强HBXIP的启动子活性和蛋白质表达水平.应用HBx的RNA干扰处理Hep G2-X细胞,HBXIP的启动子活性和蛋白质表达水平明显下降.将HBXIP启动子区的c AMP效应元件结合因子(CREB)结合位点突变后,HBx上调HBXIP的作用消失.应用CREB的RNA干扰处理肝癌细胞,在启动子水平和蛋白质水平上,HBx对HBXIP的上调作用被显著抑制.染色质免疫共沉淀结果表明,HBx能够通过CREB结合到HBXIP的启动子上,进而发挥激活HBXIP的功能.本研究结果表明,HBx促进肝癌细胞迁移的作用是通过CREB上调HBXIP实现的.这一发现对进一步揭示HBx促进肝癌细胞迁移的分子机制具有重要意义.

Hepatitis B virus X protein（ HBx） plays a crucial role in the development of hepatocellular carcinoma（ HCC）. However,the underlying mechanism by which HBx promotes the migration of hepatoma cells remains poorly understood. In the present study,we investigated the role of hepatitis B Xinteracting protein（ HBXIP） in the HBx-mediated migration of hepatoma cells. The results from wound healing assays and Boyden chamber assays showed that HBXIP contributed to HBx-elevated migration of hepatoma cells. Tissue microarrays manifested that the expression of HBXIP was up-regulated in clinicalHCC tissues. RT-q PCR analyses indicated that the expression of HBx were positively correlated with those of HBXIP in 20 clinical HCC tissues. In in vitro experiments,the over-expression of HBx resulted in the up-regulation of HBXIP,whereas HBx RNA interference reduced HBXIP expression in stably HBxtransfected Hep G2 / H7402 cell lines（ Hep G2-X / H7402-X） in a dose-dependent manner,suggesting that HBx was responsible to up-regulate HBXIP in hepatoma cells. We further demonstrated that HBx activated the promoter of HBXIP,except when the transcription factor c AMP response element binding（ CREB） site in HBXIP promoter was mutated. Moreover,CREB knockdown blocked the HBx-enhanced HBXIP up-regulation. Chromatin immunoprecipitation assay（ Ch IP） confirmed that HBx was able to bind to the promoter of HBXIP through CREB. Thus,we concluded that HBx up-regulates HBXIP through the inteaction with CREB to promote the migration of hepatoma cells.