乙肝病毒x蛋白对肝癌细胞中PUMA表达的影响

目的:探讨乙肝病毒x蛋白（hepatitis B virus x protein，HBx）对肝癌细胞中PUMA表达的影响及其机制。方法:利用质粒转染技术对p53基因不同状态的肝癌细胞转染野生型HBx基因，用RT-PCR检测转染后肝癌细胞中HBx mRNA表达和荧光定量PCR检测NFκB mRNA表达，Western blot检测HBx、p53、NFκB、PUMA蛋白表达。结果:转染后随着HBx的表达，在p53野生型肝癌细胞BEL-7402和p53突变型肝癌细胞Huh-7中NFκB mRNA和蛋白上调而p53蛋白表达下降，BEL-7402细胞PUMA蛋白表达下降而Huh-7细胞中PUMA蛋白表达略有下降。加入NFκB抑制剂PDTC后并随着PDTC浓度逐渐增加，BEL-7402细胞NFκB蛋白表达逐渐下降而p53蛋白和PUMA蛋白表达逐渐上升；但在Huh-7细胞中随着NFκB蛋白表达的抑制，p53蛋白逐渐上升而PUMA蛋白上升不明显。结论:结果提示转染HBx基因可以抑制肝癌细胞中PUMA蛋白表达，在p53野生型肝癌细胞这种抑制可能与HBx通过NFκB 的激活有关。

The effects of hepatitis B virus x protein on the expression of PUMA in hepatocellular carcinoma cells

Objective:To investigate the effects of HBx protein on the expression of PUMA in hepatocellular carcinoma (HCC) cells and its mechanism.Methods:Wild HBx plasmid was transfected into HCC cell line with different types of p53 gene.HBx mRNA expression was detected by RT-PCR.Fluorogenic quantitative PCR and Western blot were respectively administrated to the detection of nuclear factor kappa B (NFκB) mRNA and proteins of HBx，p53，NFκB and PUMA.Results:The mRNA and protein of NFκB were upregulated and the p53 protein was down-regulated in BEL-7402 with p53-wild and in Huh-7 with p53-mutation following the transfection of HBx plasmid，and the expression of PUMA protein was down-regulated.In addition，the p53 protein and PUMA protein increased all following the inactivitation of NFκB by PDTC in BEL-7402.But the HBx-mediated PUMA inhibition was not removed following the inactivitation of NFκB in Huh-7.Furthermore，it was observed that HBx induced the increase of mRNA and protein of NFκB and reduced p53 protein in BEL-7402 and Huh-7 simultaneously.But p53 protein was increased simultaneously following the inactivitation of NFκB by PDTC.Conclusion:These data show that HBx induces the down-regulation of PUMA in HCC cells，which might be related to the inhibition of p53 via NFκB signaling in p53-wild HCC cells.