肝脏H19在p,p'-DDE干扰糖代谢关键基因中的作用  

Role of hepatic H19 expression in glucose metabolism disorder induced by p,p'-dichlorodiphenyldichloroethylene

作　　者：郭晓晨 高明 彭雪云 吴南翔 GUO Xiaochen;GAO Ming;PENG Xueyun;WU Nanxiang(School of Public Health,Hangzhou Medical College,Hangzhou,Zhejiang 310013,China)

机构地区：[1]杭州医学院公共卫生学院,浙江杭州310013

出　　处：《预防医学》2022年第7期659-664,共6页CHINA PREVENTIVE MEDICINE JOURNAL

基　　金：浙江省自然科学基金(LQ20H260001)。

摘　　要：目的研究肝脏长链非编码RNA H19在2,2-双(4-氯苯基)-1,1-二氯乙烯(p,p'-DDE)干扰糖代谢关键基因中的作用。方法取人胚胎肝细胞(CCC-HEL-1)分为溶剂对照(DMSO)组、0.1μmol/L p,p'-DDE组、1μmol/L p,p'-DDE组、10μmol/L p,p'-DDE组、siRNA+DMSO组、siRNA+10μmol/L p,p'-DDE组;分别采用亚硫酸氢盐法、实时荧光定量PCR法和BCA法检测各组肝细胞核因子4α(HNF4α)、叉头框转录因子O1(FoxO1)和胰岛素样生长因子2(IGF2)的启动子区域甲基化、mRNA表达和蛋白表达水平;比较各组H19 mRNA表达水平和相关基因启动子区域甲基化水平及后续转录表达的变化。结果不同剂量p,p'-DDE单独暴露后,H19表达增加,10μmol/L p,p'-DDE组H19 mRNA表达水平高于DMSO组(1.31±0.25和1.02±0.22;P<0.05)。与DMSO组比较,10μmol/L p,p'-DDE组HNF4α基因启动子甲基化水平降低[(38.59±32.77)%和(61.43±24.64)%]、mRNA表达水平升高(1.33±0.26和1.03±0.28),差异均有统计学意义(P<0.05);FoxO1和IGF2基因启动子甲基化、mRNA和蛋白表达水平无明显变化(P>0.05)。经siRNA转染H19沉默后,与10μmol/L p,p'-DDE组比较,siRNA+DMSO组HNF4α基因启动子甲基化水平升高[(71.33±22.23)%和(38.59±32.78)%]、mRNA表达水平降低(0.71±0.17和1.33±0.26),FoxO1基因启动子甲基化水平升高[(47.73±34.24)%和(25.09±25.35)%],IGF2 mRNA表达水平升高(1.39±0.25和0.80±0.20),并且IGF2蛋白表达水平高于DMSO组(1.03±0.11和0.74±0.12),差异均有统计学意义(P<0.05)。结论肝细胞H19可通过调节基因启动子甲基化修饰程度,改变HNF4α、FoxO1和IGF2的转录与表达,在p,p'-DDE暴露诱发的糖稳态失调中发挥作用。Objective To investigate the role of hepatic long-chain non-coding RNA(lncRNA)H19 in key genes associ⁃ated with glucose metabolism disorder induced by p,p′-dichlorodiphenyldichloroethylene(p,p′-DDE).Methods Human embryonic liver CCC-HEL-1 cells were divided into the DMSO group,0.1μmol/L p,p′-DDE group,1μmol/L p,p′-DDE group,10μmol/L p,p′-DDE group,small interference RNA(siRNA)+DMSO group and siRNA+10μmol/L p,p′-DDE group.The promoter region methylation,mRNA expression and protein expression of hepatocyte nuclear factor 4α(HNF4α),forkhead box transcription factor O1(FoxO1)and insulin-like growth factor(IGF2)were detected in CCC HEL-1 cells using the bisulfite method,real-time fluorescence quantitative PCR(qPCR)assay and BCA assay,respec⁃tively.The changes in H19 mRNA expression,the methylation of associated genes in the promoter region and transcrip⁃tional expression were compared in CCC-HEL-1 among groups.Results Exposure to p,p′-DDE alone at different dos⁃es resulted in an increase in H19 expression,and the H19 mRNA expression was higher in the 10μmol/L p,p′-DDE group than in the DMSO group[(1.31±0.25)vs.(1.02±0.22);P<0.05].Lower methylation of the HNF4αgene in the pro⁃moter region[(38.59±32.77)%vs.(61.43±24.64)%;P<0.05]and higher HNF4αmRNA expression[(1.33±0.26)vs.(1.03±0.28);P<0.05]were detected in the 10μmol/L p,p′-DDE group than in the DMSO group,while no significant differ⁃ences were detected between the two groups in terms of the methylation of FoxO1 and IGF2 genes in the promoter re⁃gion,FoxO1 and IGF2 mRNA and protein expression(P>0.05).Following siRNA-induced H19 knockdown,higher meth⁃ylation of the HNF4αgene in the promoter region[(71.33±22.23)%vs.(38.59±32.78)%;P<0.05],lower HNF4αmRNA expression[(0.71±0.17)vs.(1.33±0.26);P<0.05],higher methylation of FoxO1 gene in the promoter region[(47.73±34.24)%vs.(25.09±25.35)%;P<0.05]and higher IGF2 mRNA[(1.39±0.25)vs.(0.80±0.20);P<0.05]were found in the siRNA+DMSO group than in the 10μmol/L p,p′-DDE

关 键 词：H19 p p'-DDE 甲基化 糖代谢 

分 类 号：R589.1[医药卫生—内分泌]