甲基双加氧酶2在高糖刺激肾小球系膜细胞转化生长因子β1表达中的作用

Effect of tet methylcytosine dioxygenase 2 on the regulation of transforming growth factor-β1 expression in mesangial cells induced by high glucose

目的探讨甲基双加氧酶（TET）2在高糖诱导。肾小球系膜细胞转化生长因子β1（TGF-β1）表达中的作用。方法将人肾小球系膜细胞分为正常对照组（5．5mmol／L葡萄糖）、高糖（30．0mmol／L葡萄糖）组，分别观察12—72h。用小分子化学物质SCl抑制系膜细胞TET2基因表达，分为高糖组（30．0mmol／L葡萄糖＋DMEM）、二甲亚砜（DMSO）组（30．0mmol／L葡萄糖＋终浓度0．1％DMSO）、SCl组（30．0mmol／L葡萄糖＋终浓度3μmol／LSCl）。采用实时荧光定量PCR、Western印迹法分别检测TGF-β1、TET1—3、α平滑肌肌动蛋白（α-SMA）的mRNA及蛋白水平。亚硫酸盐修饰后测序法（BSP）检测TGF-β1基因启动子及第一外显子区CpG岛的甲基化。噻唑蓝比色法（MTT）检测系膜细胞增殖情况。结果与正常对照组比较，高糖诱导系膜细胞TET2mRNA和蛋白表达升高，且存在时间依赖效应（均P〈0．05），但TET1和TET3的表达在两组间差异无统计学意义（均P〉0．05）。同时发现高糖组TGF-β1基因第一外显子区4个CG位点出现持续去甲基化，24—72h甲基化率均低于对照组（均P〈0．01），而启动子区甲基化水平无明显变化。与高糖组比较，TET2抑制剂SCl组高糖诱导的TGF-β1第一外显子区CpG岛去甲基化降低（即甲基化率升高），TGF-β1和α—SMAmRNA和蛋白表达均降低，系膜细胞增殖下调（均P〈0．05）；DMSO组与高糖组上述各项差异均无统计学意义。结论TET2可能通过催化TGF-β1基因第一外显子区的DNA去甲基化参与高糖诱导系膜细胞TGF-β1表达及细胞表型转化。

Objective To investigate the role of tet methylcytosine dioxygenase 2 （TET2） in the regulation of transforming growth factor-β1 （TGF-β1） expression in human glomerular mesangial cells induced by high glucose. Methods Cultured human glomerular mesangial cells were divided into normal control group （5.5 mmol/L glucose） and high glucose group （30.0 mmol/L glucose） which was cultured for 12 h to 72 h. The gene expression of TET2 in mesangial cells were inhibited by small molecule chemical called SC1, and which were divided into high glucose group （30.0 mmol/L glucose＋ DMEM）, DMSO group （30.0 mmol/L glucose＋0.1%DMSO） and SC1 group （30.0 mmol/L glucose＋3 μmol/L SC1）. The mRNA and protein expression of TGF-β1, TET1 to 3 and α-smooth muscle actin （α- SMA） was detected by quantitative real-time PCR and Western blotting. Methylation of CpG islands in the regulation region of TGF- β1 was detected by bisulfite sequencing PCR （BSP）. The activity of mesangial cell proliferation was assessed by colofimetry of thiazolyl blue （MTT）. Results Compared with normal control group, the mRNA and protein expression of TET2 in mesangial cells induced by high glucose was increased significantly in a time- dependent manner （all P 〈 0.05）, but the expression of TET1 and TET3 was not affected. Meanwhile methylation rate of 4 CG sites from 24 h to 72 h were decreased in the first exon of TGF-β1 （P 〈 0.01）, but not in the promoter. Compared with high glucose group, when the expression of TET2 was inhibited by SC1, the methylation rate of TGF-β1 was recovered evidently （P 〈 0.05）, the mRNA and protein expression of TGF-β1 and α-SMA was suppressed, and the proliferation of mesangial cells was decreased （all P 〈 0.05）. Conclusions Demethylation of the CpG island mediated by TET2 may play an important role in the expression of TGF-β1 and mesangial cell phenotype transformation induced by high glucose.