HBx抑制RXRα表达对肝细胞癌DNMT3a表达的影响及初步机制研究

Effect of HBx-mediated RXR alpha repression on DNMT3a in hepatocellular carcinoma and underlying mechanism

目的探讨维甲酸X受体α(retinoid X receptorα,RXRα)在乙肝病毒X蛋白(hepatitis B virus X protein,HBx)上调肝细胞癌DNA甲基转移酶3a(DNA methyltransferases 3a,DNMT3a)表达中的作用及机制。方法采用RT-PCR和Western blot检测12例正常肝组织、56例HBV阳性的肝细胞癌和癌旁组织中DNMT3a、RXRα的基因和蛋白表达变化;在培养的SMMC-7721细胞中,转染HBx基因和RXRα基因,观察对其DNMT3a表达的影响;在转染HBx基因的SMMC-7721细胞中,分别加入信号通路阻断剂,观察何种信号通路在HBx调节RXRα的表达中发挥作用。结果与正常肝组织相比,肝癌组织与癌旁组织中DNMT3a的基因和蛋白表达量显著升高(P<0.01),肝癌组织与癌旁组织中RXRα的基因和蛋白表达量显著降低(P<0.01);在SMMC-7721细胞中,HBx的过表达使RXRα的蛋白表达量显著降低(P<0.01),DNMT3a的蛋白表达量显著升高(P<0.01),而RXRα与HBx共转染,则可降低HBx升高的DNMT3a蛋白表达量(P<0.01);MKK/MEK抑制剂PD98059和p38 MAPK抑制剂SB203580可显著升高HBx下调的RXRα基因和蛋白表达(P<0.01)。结论 HBV产生的HBx蛋白可能通过MAPK信号通路,降低RXRα表达,引起促癌基因DNMT3a蛋白含量增加。

Objective To explore the role and mechanism of retinoic acid receptor alpha （RXRα） in the regulation of DNA methyltransferases 3a （DNMT3a） expression induced by hepatitis B virus X protein （HBx） in hepatocellular carcinoma. Methods The gene and protein expression levels of DNMT3a and RXRα were detected by RT-PCR and Western blot assay respectively in normal liver tissues of 12 healthy individual, and HBV positive hepatocellular carcinoma and paired paracancerous tissues of 56 patients. The expression of DNMT3a was measured after HBx gene and/or RXRα gene were transfected in SMMC-7721 cells. The MKK/MEK blocker PD98059 and p38 MAPK blocker SB203580 were added to observe the role of signal pathways in the regulation of RXRα in SMMC-7721 cells transfected with HBx gene. ResultsCompared with normal liver tissues, the expression of DNMT3a at mRNA and protein levels was significantly increased in hepatocellular carcinoma tissues and adjacent tissues （P〈0.01）, but the levels of RXRα were significantly decreased （P〈0.01）. Overexpression of HBx gene inhibited the level of RXRα protein and raised the level of DNMT3a protein, while RXRα co-transfected with HBx decreased the expression of DNMT3a induced by HBx （P〈0.01）. MKK/MEK inhibitor PD98059 and p38 MAPK inhibitor SB203580 both significantly increased the expression of RXRα which was down-regulated by HBx gene （P〈0.01）. Conclusion HBx encoded by HBV genome may reduce the expression of RXRα and increase the content of DNMT3a through the MAPK signaling pathway.