ERO1α及其 DNA 甲基化在同型半胱氨酸抑制肝细胞增殖中的作用  

Role of ERO1α and its DNA methylation in homocysteine-induced inhibition of hepatocyte proliferation

作　　者：赵丽[1] 曹成建[1] 刘现梅 孔繁琪[2] 马文斌[1] 周龙霞[2] 陈久凯 张鸣号[2] 焦运[3] 杨晓玲[2] 姜怡邓[2] 

机构地区：[1]宁夏医科大学 检验学院 [2]宁夏医科大学 基础学院,宁夏银川750004 [3]宁夏医科大学 总医院,宁夏银川750004

出　　处：《中国药理学通报》2014年第12期1743-1747,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81360073,81360027);宁夏自然科学基金资助项目(No 13054,13140)

摘　　要：目的探讨内质网氧化还原酶-1α(endoplasmic reticulum oxidoreductin 1-α,ERO1α)及其DNA甲基化在同型半胱氨酸(homocysteine,Hcy)抑制肝细胞增殖中的作用。方法以0μmol·L-1Hcy为正常对照组(NC group),以100μmol·L-1Hcy为干预组(Hcy group),干预肝细胞48h后,四甲基偶氮唑盐比色法(MTT)检测肝细胞增殖活力的变化;实时定量聚合酶链反应(qRT-PCR)及Western blot分别检测ERO1αmRNA和蛋白表达水平;构建ERO1α真核表达质粒,转染肝细胞后,荧光显微镜下观察转染效率并以qRTPCR及Western blot验证ERO1α是否过表达,后用MTT法检测肝细胞增殖活力的变化;巢式降落式甲基化特异性PCR(nt MS-PCR)检测ERO1αDNA甲基化水平。结果与正常对照组相比,Hcy干预组肝细胞内ERO1αmRNA及蛋白表达水平降低,且细胞增殖活力减弱(P<0.01)。测序结果证明ERO1α重组质粒构建成功,荧光显微镜下可见绿色荧光蛋白大量表达,qRT-PCR及Western blot验证结果显示ERO1α过表达(P<0.01),而MTT法结果表明ERO1α过表达可缓解Hcy导致的肝细胞增殖抑制(P<0.01)。Hcy刺激后ERO1αDNA甲基化水平升高(P<0.05)。结论 Hcy通过下调ERO1α表达抑制肝细胞增殖,而ERO1α启动子区DNA甲基化是其重要的调控机制。Aim To explore the role of ERO1 αand its DNA methylation in homocysteine （Hcy）-induced in-hibition of hepatocytes proliferation.Methods The hepatocytes stimulated with 0 μmol·L -1 Hcy were set as the normal group （NC group）and the hepatocytes stimulated with 1 00 μmol·L -1 Hcy as the experimen-tal group （Hcy group）.Methyl thiazolyl tetrazolium （MTT）reduction assay was used to reflect the prolifer-ation of the hepatocytes;qRT-PCR and Western blot were used to detect the mRNA and protein levels of ERO1 α;the expression of green fluorescence protein was observed in hepatocytes after the recombinant plas-mid of ERO1 α was constructed,which was used to confirm if the recombinant plasmid into hepatocytes was successful,then the mRNA and protein levels of ERO1 αwere assayed and the proliferation of the hepa-tocytes was also detected;ntMSP was used to detect nbsp;the change of ERO1 αDNA methylation.Results The mRNA and protein levels of ERO1 αwere decreased in Hcy group compared with NC group,and the prolifera-tion activity of hepatocytes in Hcy group was de-creased.Sequencing result showed that the recombi-nant plasmid of ERO1 αwas constructed successfully. QRT-PCR and Western blot revealed that ERO1 αwas overexpressed. The result of MTT suggested that ERO1 αoverexpression restored hepatocyte proliferation inhibited by Hcy.Hcy caused ERO1 αDNA hyperm-ethylation.Conclusions Hcy inhibits hepatocyte pro-liferation by downregulating the expression of ERO1 α, and methylation of ERO1 αpromoter may play a role in this process.

关 键 词：同型半胱氨酸 ERO1α 肝细胞 增殖 启动子区 DNA甲基化 

分 类 号：R322.47[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]