乙型肝炎病毒X蛋白对肝癌细胞MKK3,p38MAPK表达的影响

Influences of Hepatitis B virus X protein on the expression of MKK3 and p38MAPK in hepatoma carcinoma cell

目的:观察乙型肝炎病毒X蛋白对肝癌细胞中丝裂原活化蛋白激酶(MKK3)及其下游分子p38丝裂酶原活化的蛋白激酶(p38MAPK)的表达的影响,探讨相关的信号转导机制,及其对肝癌细胞生物学行为的影响。方法:分别将质粒pCDNA3.1及pCDNA3.1-HBx重组质粒经脂质体介导转染人肝癌细胞株HepG2,G418筛选培养,并用反转录PCR和Western blot鉴定,获得表达X蛋白的稳定细胞株HepG2-HBx和阴性对照细胞株HepG2-pCDNA3.1,以HepG2细胞株作空白对照。通过RT-PCR和Western blot检测上述三种细胞株中MKK3,p38MAPK在mRNA水平和蛋白水平的表达变化,并用细胞免疫荧光法检查磷酸化p38MAPK蛋白在细胞浆及细胞核中的变化;通过MTT实验检测三种细胞株的增殖情况。采用SPSS 12软件进行统计学分析。结果:MKK3在mRNA和总蛋白水平的表达在HepG2-HBx中均高于其他两组,其他两组无明显差异;p38MAPK在mRNA和总蛋白水平三组无明显差异,而其蛋白磷酸化水平和在核蛋白中的表达在HepG2-HBx中较其他两组升高;HepG2-HBx较其他两组细胞有更强的增殖能力。结论:HBx可以通过上调肝癌细胞中MKK3的表达,促进p38MAPK磷酸化和入核,从而进一步激活下游分子发挥生物活性。p38MAPK通路在HBx促进肝癌细胞的增殖中发挥重要作用。可能是导致HBV相关性肝癌与非HBV相关性肝癌临床特点及肿瘤生物学差异的机制之一。

Objective：To observe the change of expressions of MKK3 and p38MAPK in HCC induced by hepatitis B virus X protein,and the correlative mechanisms of the effect in signal transduction,to investigate the effect of hepatitis B virus X protein to HCC.Methods：The human hepatoma carcinoma cells HepG2 were transfected with pCDNA3.1 and pCDNA-3.1-HBx recombinant plasmids by lipofectamine 2000.Then the stable cell lines expressing constantly HBx protein and the negative control cell lines were established by adding G418 to select single cell G418-resistant clones and identification with reverse transcriptase-PCR and Western blot assays,named respectively HepG2-HBx and HepG2-pCDNA3.1 cells,meanwhile with the HepG2 as blank.To detect the change on mRNA and protein level of MKK3 and p38MAPK in above three species cells by RT-PCR and Western blot assays,changes of phosphorylation-p38MAPK protein in nucleus and cytoplasm were observed with cell immunofluorescence method;To measure the proliferation,cell cycle and apoptosis of these cells by MTT assays.Data were analysed by SPSS12 system.Results：Expressing of MKK3 on mRNA and protein was higher in HepG2-HBx than in other two species cells.There was no evident change on the mRNA and total protein level of p38MAPK for all various cell models,but the phosphorylation of p38 and the quantity of it in nucleus were up-regulated in HepG2-HBx.HepG2-HBx＇s cell was stronger in proliferation than other two species cells.Conclusion：HBx can up-regulate the expression of MKK3 in hepatoma carcinoma cell and promote the phosphorylation of p38 and precipitate p38 into nucleus,further activate the downstream molecule.p38MAPK signal transductional path plays important role in the changes of proliferation hepatoma carcinoma cell induced by HBx.This may be one of the mechanisms that make an tumor biological behaviour different in clinic character between the HBV-correlated and no HBV-correlated hepatocellular carcinoma.