泡沫细胞DNA甲基化及其对脂代谢基因SREBP1表达的影响

摘要

目的 探讨泡沫细胞DNA甲基化及其对脂代谢基因胆固醇调节元件结合蛋白1(SREBP1)表达的调控作用.方法 将体外培养的单核细胞系THP-1采用乙酸酯、氧化型低密度脂蛋白等处理后建立泡沫细胞模型,采用实时荧光定量PCR 法检测 THP-1 单核细胞和泡沫细胞中DNA 甲基转移酶1(DNMT1)、DNMT3A、DNMT3B mRNA表达,结果显示在THP-1泡沫细胞中DNMT1 mRNA相对表达量低于THP-1单核细胞(P＜0.05),两种细胞DNMT3A、DNMT3B mRNA相对表达量比较差异均无统计学意义(P均＞0.05),后续研究选择DNMT1基因进行转染.将THP-1泡沫细胞随机分为三组,对照组常规培养,空载体组转染空载对照质粒,DNMT1组转染DNMT1质粒,结果显示,DNMT1组DNMT1蛋白相对表达量高于其他两组,说明转染成功.收集三组细胞,采用质谱法检测SREBP1启动子甲基化状态,采用qRT-PCR和Western blotting法检测SREBP1 mRNA和蛋白表达.结果 与对照组及空载体组比较,DNMT1组SREBP1基因启动子区CpG岛22号CG位点启动子发生高甲基化,其他28个CG位点未发生甲基化. DNMT1 组 SREBP1 mRNA 和 SREBP1 蛋白相对表达量均低于其他两组( P 均 ＜0.05 ).结论 DNMT1表达降低参与泡沫细胞的形成;上调DNMT1可抑制脂代谢基因SREBP1表达;提示DNMT表达改变通过影响脂代谢基因SREBP1的甲基化水平可能在泡沫细胞形成中发挥重要作用.%Objective To investigate the DNA methylation of foam cells and its regulation effect on the lipid metabo-lism gene sterol regulatory element-binding protein-1 ( SREBP1) expression.Methods Human monocytic cells THP-1 were stimulated with Ox-LDL to form THP-1 foam cell models.DNA methyltransferase 1 (DNMT1), DNMT3A, DNMT3B mRNA expression in THP-1 monocytes and THP-1 foam cells were detected by real-time PCR.The result showed that DN-MT1 mRNA expression level was significant lower in the THP-1 foam cells than in the THP-1 monocytes (P＜0.05), and no significant difference was found in DNMT3A and DNMT3B expression between the foam cells and monocytes (both P＞0.05).We subsequently selected the DNMT1 gene for transfection.The foam cells were randomly divided into three groups.The control group was cultured routinely.The empty vector group was transfected with the empty control plasmid , and the DNMT1 group was transfected with the DNMT1 plasmid.The results showed that the relative expression of DNMT 1 protein in the DNMT1 group was higher than that in the other two groups , indicating the transfection was successful.The methylation status of SREBP1 promoter was detected by mass spectrometry , and the expression of SREBP1 mRNA and pro-tein was detected by RT-qPCR and Western blotting.Results Compared with the control group and the empty vector group, the hypermethylation of the promoter of No.22 CG locus of CpG island in the promoter region of SREBP-1 gene was observed in the DNMT1 group, while no methylation of the other 28 CG loci was observed.The relative expression of SREBP1 mRNA and SREBP1 protein in the DNMT1 group was lower than that in other two groups (all P＜0.05).Con-clusion Abnormal alteration of DNMT1 takes part in the formation of foam cells , and the up-regulation of DNMT1 could inhibit the expression of SREBP1, which suggests that DNMT expression alteration might contribute to the hypermethylation of lipid-metabolism gene and play important role in the formation of foam cells.