L02细胞乙型肝炎病毒X蛋白和SOCS-1基因水平研究

Expression of hepatitis B virus X protein and suppressor of cytokine signaling-1 in L02 cells in vitro

目的探讨乙型肝炎病毒X蛋白(HBx)影响细胞因子信号传导抑制因子-1(SOCS-1)基因水平的可能机制。方法收集22例乙型肝炎相关肝细胞癌(HCC)手术后癌组织和癌旁肝组织,采用RT-PCR法检测组织HBx、DNMT3A、DNMT3B和SOCS-1 mRNA水平。通过脂质体法构建表达HBx的L02细胞和空质粒L02细胞,采用CCK8法检测5-氮杂-2′-脱氧胞苷(5-Aza-c)对L02细胞存活率的影响,采用RT-PCR法和Western blot法分别检测细胞HBx、DNA甲基转移酶(DNMT)3A、DNMT3B和SOCS-1 mRNA水平及其蛋白表达。结果肝癌组织HBx和DNMT3A mRNA水平分别为(65.2±3.5)和(77.2±3.8),显著高于癌旁肝组织【分别为(22.5±4.0)和(42.1±2.9),P<0.05】,而SOCS-1 mRNA水平为(33.1±3.0),显著低于癌旁肝组织【(75.6±2.6),P<0.05】;与对照细胞比,表达HBx的L02细胞活力随5-Aza-c作用浓度增高而下降(P<0.05);过表达HBx的L02细胞DNMT3A mRNA水平及其蛋白表达量显著高于空载质粒组(P<0.05),而SOCS-1 mRNA水平及其蛋白表达量显著低于空载质粒组(P<0.05);在5-Aza-c干预表达HBx的L02细胞,DNMT3A mRNA水平及其蛋白表达量显著低于对照组(P<0.05),而SOCS-1 mRNA水平及其蛋白表达量显著高于对照(P<0.05)。结论HBx通过上调DNMT3A表达使SOCS-1基因表达下调,表明HBx可能通过调控DNMT3A影响抑癌基因SOCS-1表达从而促进肝癌的发生。

Objective The aim of this study was to investigate the possible mechanism of hepatitis B virus X protein(HBx)affecting cytokine signal transduction inhibitor-1(SOCS-1)gene in vitro.Methods The expressions of HBx,DNA methyltransferase(DNMT)3A/3B and SOCS-1 in cancerous and paracancerous tissues of 22 patients with HBV-related hepatocellular carcinoma(HCC)were detected by real-time PCR.The HBx expression plasmid(pcDNA-X)or an empty plasmid(pcDNA3.0)were transfected in L02 cells by liposome infection.The effect of 5-aza-2′-deoxycytidine(5-Aza-C)on the survival rate of L02 cells was detected by CCK8.The HBx,DNMT3A/3B and SOCS-1 mRNA as well their protein expression were assayed by real-time PCR and Western blot.Results The HBx and DNMT3A mRNA level in cancerous tissues were(65.2±3.5)and(77.2±3.8),much higher than[(22.5±4.0)and(42.1±2.9),respectively,P<0.05],while the expression of SOCS-1 was(33.1±3.0),significantly lower than[(75.6±2.6),P<0.05]in adjacent liver tissues;the activity of L02 cells expressing HBx decreased with the increase of 5-Aza-C concentration(P<0.05);the DNMT3A mRNA level and its protein expression in L02 cells with overexpression of HBx were significantly higher than in empty plasmid-transfected cells(P<0.05);the SOCS-1 mRNA level and its protein expression were significantly lower than in the empty plasmid-transfected cells(P<0.05);the DNMT3A mRNA level and its protein expression in L02 cells expressing HBx after 5-Aza-C intervention were significantly lower than in the control cells(P<0.05),while the SOCS-1 mRNA level and its protein expression were significantly higher than in the control(P<0.05).Conclusions The present study indicates that HBx induces epigenetic down-regulation of SOCS-1 by increasing the expression of DNMT3A,which might be reversed with DNA methyltransferase inhibitor 5-Aza-C.