乙型肝炎病毒X蛋白A1762T/G1764A双突变对肝癌细胞生物钟基因表达的影响  

Effects of hepatitis B virus X protein A1762T/G1764A double mutation on expression of clock gene in liver cancer cells

作　　者：彭桦 占静 张乙凡 孙飞[2] 邱梦君 杨盛力[2] 

机构地区：[1]华中科技大学同济医学院附属梨园医院,武汉430077 [2]华中科技大学同济医学院附属协和医院 [3]贵州医科大学附属医院

出　　处：《山东医药》2017年第41期1-4,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81702885);中央高校基本科研业务费专项资金项目(0118530328);华中科技大学自主创新基金项目(2016YXM241);中国肝炎防治基金会-天晴肝病研究基金科研课题(TQGB20170005);中国博士后科学基金资助项目(2017M613001)

摘　　要：目的研究乙型肝炎病毒X蛋白(HBx)A1762T/G1764A双突变对肝癌细胞生物钟基因表达的影响。方法将Bel-7404细胞接种于6孔板中,待细胞生长至60%融合时按照Lipofectamine2000的说明书分别将1.0μg的pc DNA3.1-HBx和pc DNA3.1-m HBx(A1762T/G1764A双突变)质粒转染肝癌细胞系Bel-7404细胞。采用Realtime PCR和Western blotting法检测Bel-7404细胞中CLOCK、BMAL1、Per1、Per2、Per3、Cry1、Cry2、CKIεmRNA及蛋白表达。结果与转染pc DNA3.1-HBx相比,转染A1762T/G1764A双突变的pc DNA3.1-m HBx表达载体后Bel-7404细胞中Clock、Bmal1、Per2、Per3、Cry1、Cry2 mRNA和蛋白表达均降低(P均<0.05)。结论 HBx A1762T/G1764A双突变可引起肝癌细胞生物钟基因表达紊乱。Objective To investigate the effects of hepatitis B virus X protein A1762 T/G1764 A double mutation on the expression of clock gene in the hepatocellular carcinoma（ HCC） cells. Methods Bel-7404 cells were inoculated into into 6-hole plate,when the cells grew to 60% confluence,they were transfected with 1. 0 μg pc DNA3. 1-HBx and pc DNA3. 1-mHBx（ A1762 T/G1764 A double mutant） plasmids by using Lipofectamine 2000 according to the manufacturer＇s instructions. The mRNA and protein expression levels of the CLOCK,BMAL1,Per1,Per2,Per3,Cry1,Cry2,and CKIεgenes in the Bel-7404 cells were detected by real-time PCR and Western blotting. Results The mRNA and protein expression of Clock,Bmal1,Per2,Per3,Cry1 and Cry2 decreased in Bel-7404 cells transfected with A1762 T/G1764 A double mutant HBx vectors as compared with that of cells transfected with pc DNA3. 1-HBx vectors,and the differences were statistically significant（ all P〈0. 05）. Conclusions HBx A1762 T/G1764 A double mutation can cause the disorder of biological clock expression in HCC cells.

关 键 词：肝肿瘤 乙型肝炎病毒X蛋白 突变 生物钟 钟控基因 

分 类 号：R735.7[医药卫生—肿瘤]