乙型肝炎病毒X蛋白抑制肝癌细胞系中DKK2的表达  

Inhibition of DKK2 in hepatocellular carcinoma cell lines by hepatitis B virus X protein

作　　者：王晓艳 杜虹 王伟 权会琴 WANG Xiao-yan;DU Hong;WANG Wei;QUAN Hui-qin(Center of Infectious Diseases,Tangdu Hospital,Air Force Military Medical University,Xi an 710038,China)

机构地区：[1]空军军医大学唐都医院传染科,陕西西安710038

出　　处：《基础医学与临床》2020年第10期1348-1352,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(81602462)。

摘　　要：目的分析不同肝癌细胞系中乙型肝炎病毒X蛋白(HBx)对DKK2表达水平的影响及其可能机制。方法应用编码乙型肝炎病毒X蛋白的腺病毒(Ad-HBx)感染HepG2和SMMC7721细胞,利用免疫荧光技术和PCR技术检测X基因的表达、Western blot检测DKK2的蛋白表达水平;应用甲基化转移酶抑制剂(DAC)处理细胞,利用Western blot检测DKK2、Dnmt1、Dnmt3a和Dnmt3b的表达;通过沉默甲基化转移酶,利用Western blot检测DKK2的表达。结果Ad-HBx感染HepG2和SMMC7721细胞后,HBx mRNA相对表达倍数分别上调61.12倍和50.24倍(P<0.05),DKK2的蛋白表达水平分别下调1.61倍(P<0.05)和3.33倍(P<0.05),Dnmt1和Dnmt3a蛋白表达水平显著升高;相较于Ad-HBx感染组,DAC处理后DKK2蛋白表达水平上调2.27倍(P<0.05),Dnmt3a蛋白水平显著下调,Dnmt1和Dnmt3b蛋白水平无显著变化;沉默Dnmt1和Dnmt3a后,DKK2蛋白表达水平显著上调。结论HBx通过上调Dnmt1及Dnmt3a发挥甲基化作用来抑制DKK2的表达,进而激活Wnt/β-catenin信号通路,参与HBV相关肝癌的发生。Objective To analyze the effect of hepatitis B virus X protein on the expression of DKK2 in different hepatoma cell lines and its possible mechanism.Methods HepG2 and SMMC7721 cells were infected by Ad-HBx,and X gene expression was detected by immunofluorescence and PCR,and protein level of DKK2 was detected by Western blot.The hepatoma cell lines were incubated with methylated transferase inhibitor(DAC),the expressions of DKK2,Dnmt1,Dnmt3a and Dnmt3b were detected by Western blot.By silencing the methyltransferase,the expression of DKK2 was detected by Western blot.Results After HepG2 and SMMC7721 being infected with Ad-HBx,the relative expression of HBx mRNA was up-regulated by 61.12 times and 50.24 times respectively(P<0.05),the protein expression of DKK2 was down-regulated by 1.61 times(P<0.05)and 3.33 times(P<0.05),and the protein expression of Dnmt1 and Dnmt3a was significantly increased.Compared with the Ad-HBx infection group,DKK2 protein expression was up-regulated by 2.27 times(P<0.05),Dnmt3a protein expression was significantly down-regulated,Dnmt1 and Dnmt3b protein level was not significantly changed after DAC treatment.By silencing Dnmt1 and Dnmt3a,DKK2 protein expression was significantly up-regulated.Conclusions HBx inhibits the expression of DKK2 by up-regulation of Dnmt3a and Dnmt1,and then activates the Wnt/β-catenin signaling pathway,which is involved in the occurrence of HBV related liver cancer.

关 键 词：肝癌 乙型肝炎病毒X蛋白 DKK2 WNT/Β-CATENIN 甲基化转移酶 

分 类 号：R373[医药卫生—病原生物学]