p,p'-DDE对胰岛素瘤细胞H19差异甲基化区甲基化及胰岛素分泌的影响
Effects of p,p'-DDE on H19 DMR methylation and insulin secretion of INS-1 cells
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摘要:
背景 糖尿病是全球重要的公共卫生问题。研究表明二氯二苯基二氯乙烷（p,p'-DDE）与2型糖尿病的发病密切相关，然而相关分子机制尚不清楚。
目的 探讨p,p'-DDE对大鼠胰岛素瘤细胞（INS-1细胞）H19 差异甲基化区（DMR）甲基化及胰岛素分泌的影响。
方法 用不同浓度的p,p'-DDE（0、3.125、6.25、12.5、25、50、75 µmol·L−1）染毒INS-1细胞24 h，采用CCK-8法检测INS-1细胞活力；以0、12.5、25、50 µmol·L−1 p,p'-DDE作为后续实验浓度，染毒24 h。采用亚硫酸氢盐修饰测序法检测H19 DMR 24个CpG位点甲基化水平；采用实时荧光定量PCR检测胰岛素样生长因子2（IGF2）基因转录水平；采用Western blotting检测IGF2和1型胰岛素样生长因子受体（IGF1R）蛋白水平；采用葡萄糖刺激胰岛素分泌实验测定INS-1细胞胰岛素分泌功能（高糖和低糖浓度分别为5、25 mmol·L−1）。
结果 与对照组相比，12.5 µmol·L−1 p,p'-DDE增加INS-1细胞活力，然而，50、75 µmol·L−1 p,p'-DDE抑制细胞活力（P<0.01），故本研究选择50 µmol·L−1为最高染毒浓度。25 µmol·L−1 p,p'-DDE暴露导致H19 DMR CpG18、CpG22~CpG24位点甲基化水平下调，50 µmol·L−1 p,p'-DDE暴露导致H19 DMR CpG10~CpG24位点甲基化水平下调（P<0.05或P<0.01）。不同浓度（12.5、25、50 µmol·L−1）p,p'-DDE均可下调胰岛细胞IGF2 mRNA、蛋白表达和IGF1R蛋白表达，IGF2转录水平分别降低为对照组的67.8%、68.6%、62.5%，蛋白水平分别降低为对照组的73.3%、79.5%、80.9%，IGF1R蛋白水平分别降低为对照组的54.8%、25.6%、12.9%（P<0.01）。在高糖环境（25 mmol·L−1葡萄糖）下，不同浓度p,p'-DDE均抑制胰岛素分泌水平，分别降低为对照组的85.0%、58.6%、49.5%（P<0.01）。
结论 p,p'-DDE下调胰岛细胞H19 DMR甲基化水平，干扰IGF2/IGF1R信号通路，导致胰岛素分泌功能障碍。
Abstract:Background Diabetes is a major threat to public health across the world. Studies have shown that exposure to p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) is closely related to the occurrence of type 2 diabetes mellitus. However, the relevant molecular mechanism is not clear.
Objective To investigate the effects of p,p'-DDE on H19 differentially methylated region (DMR) methylation and insulin secretion of rat insulinoma cells (INS-1 cells).
Methods INS-1 cells were cultured with different concentrations (0, 3.125, 6.25, 12.5, 25, 50, and 75 µmol·L−1) of p,p'-DDE for 24 h, and the viability of INS-1 cells was detected by CCK-8 method. INS-1 cells were exposed to 0, 12.5, 25, and 50 µmol·L−1 p,p'-DDE for 24 h in subsequent experiments. The methylation levels of 24 CpG sites inH19 DMR were analyzed by bisulfite genomic sequencing. The expression levels of insulin-like growth factor 2 (IGF2) mRNA were detected by real-time quantitative PCR. The expression levels of IGF2 and insulin-like growth factor-1 receptor (IGF1R) proteins were detected by Western blotting. The insulin secretion function of INS-1 cells was determined by glucose-stimulatedinsulin secretion test (5 and 25 mmol·L−1 glucose, respectively).
Results Compared with the control group, the viability of INS-1 cells increased significantly after treatment with 12.5 µmol·L−1 p,p'-DDE; however, it was significantly inhibited after treatment with 50 or 75 µmol·L−1 p,p'-DDE (P<0.01); therefore, 50 µmol·L−1 was chosen as the maximum concentration of exposure for subsequent experiments. The 25 µmol·L−1 p,p'-DDE treatment decreased the methylation levels of CpG18 and CpG22-CpG24 sites in H19 DMR, and the 50 µmol·L−1 p,p'-DDE treatment decreased the methylation levels of CpG10-CpG24 sites (P<0.05 orP<0.05). Multiple concentrations (12.5, 25, and 50 µmol·L−1) of p,p'-DDE down-regulated the mRNA and protein relative expression levels of IGF2 and the protein relative expression levels of IGF1R. The transcription level of IGF2 decreased to 67.8%, 68.6%, and 62.5% of the control group, the protein level of IGF2 decreased to 73.3%, 79.5%, and 80.9% of the control group, and the protein level of IGF1R decreased to 54.8%, 25.6%, and 12.9% of the control group, respectively (P<0.01). In the high glucose context, p,p'-DDE at selected concentrations inhibited the insulin secretion levels to 85.0%, 58.6%, and 49.5% of the control group, respectively (P<0.01).
Conclusion p,p'-DDE could down-regulate methylation level of H19 DMR, interfere the IGF2/IGF1R signaling pathway, and inhibit insulin secretion of islet cells.
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