miR-375启动子甲基化在MIN6细胞中存在状况的研究  

Methylation of miR-375 promoter in MIN6 cells

作　　者：高敏[1] 尹亮[2] 况雪梅 常向云[2] Gao Min;Yin Liang;Kuang Xuemei(Medical College of Shihezi University,The First Affiliated Hospital,Medical College of Shihezi University,Shihezi 832000;Dept of Endocrine and Metabolic Diseases,The First Affiliated Hospital,Medical College of Shihezi University,Shihezi 832000;Dept of Respiratory Medicine,The First Affiliated Hospital,Medical College of Shihezi University,Shihezi 832000)

机构地区：[1]石河子大学医学院,石河子832000 [2]石河子大学医学院第一附属医院内分泌代谢科,石河子832000 [3]石河子大学医学院第一附属医院呼吸内科,石河子832000

出　　处：《安徽医科大学学报》2020年第1期60-64,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81560137、81860149)

摘　　要：目的该文旨在运用MassARRAY平台分析小鼠胰岛β细胞系(MIN6)的DNA甲基化谱,并应用DNA去甲基化药物5-氮杂-2′-脱氧胞苷(5-Aza-CdR)处理MIN6细胞以探索甲基化模式的改变。方法运用MassARRAY平台检测MIN6细胞系与小鼠胚胎成纤维细胞株(NIH3T3)中miR-375基因DNA甲基化状态,并检测不同浓度5-Aza-CdR溶液处理MIN6细胞系后miR-375基因甲基化状态,评估其变化。应用噻唑蓝比色法(MTT)检测5-Aza-CdR对MIN6细胞的生长增殖的影响。使用qRT-PCR检测5-Aza-CdR 50μmol/L浓度组miR-375在MIN6细胞中和未处理的NIH3T3细胞中的表达。结果 5-Aza-CdR能抑制MIN6细胞的生长。用5-Aza-CdR处理24、48、72 h后,2μmol/L浓度组与其他各组比较,OD值均不同(P<0.05),且不同时间点之间进行比较差异有统计学意义(P<0.05)。经24、48、72 h,用不同浓度的5-Aza-CdR处理MIN6细胞后,各组mmu-miR-375的DNA均高度甲基化,50μmol/L组miR-375 DNA的平均甲基化率均低于其他组。检测出50μmol/L组5-Aza-CdR处理的MIN6细胞经24、48、72 h后,mmu-miR-375的表达高于0μmol/L组,差异有统计学意义(P<0.05)。MIN6细胞mmu-miR-375基因的DNA表达低于NIH3T3细胞,差异有统计学意义(P<0.05)。结论该研究首次运用MassARRAY平台分析mmu-miR-375启动子甲基化水平,并发现在MIN6细胞中存在mmu-miR-375 DNA广泛甲基化修饰。Objective To analyze the DNA methylation spectrum of mouse islet β cell line(MIN6) using MassARRAY platform, and to explore the changes of methylation model by treating MIN6 cells with DNA demethylation drug 5-aza-2′-deoxycytidine(5-Aza-CdR). Methods The methylation status of miR-375 gene DNA in MIN6 cell line and mouse embryonic fibroblast cell line(NIH3 T3) were detected by MassARRAY platform. The changes of miR-375 gene methylation status were evaluated after treatment with different concentrations of 5-Aza-CdR. The effect of 5-Aza-CdR on the growth and proliferation of MIN6 cells was detected by Thiazolyl blue tetrazolium bromide(MTT). The expression of miR-375 in MIN6 cells and untreated NIH3 T3 cells was detected by qRT-PCR. Results 5-Aza-CdR could inhibit the growth of MIN6 cells. After treated with 5-Aza-CdR for 24,48,72 h, the OD value of 2 μmol/L concentration group was different from that of other groups(P<0.05), and the difference was statistically significant at different time points(P<0.05). After MIN6 cells were treated with different concentrations of 5-Aza-CdR for 24,48,72 h, the DNA of mmu-miR-375 in each group was highly methylated, and the average methylation rate of miR-375 DNA in 50 μmol/L group was lower than that in other groups. The expression of mmu-miR-375 in MIN6 cells treated with 5-Aza-CdR in 50 μmol/L group was significantly higher than that in 0 μmol/L group after 24,48,72 h(P<0.05). The DNA expression of mmu-miR-375 gene in MIN6 cells was significantly lower than that in NIH3 T3 cells(P<0.05). Conclusion These results indicate that promoter hypermethylation of the mmu-miR-375 is a common event in MIN6 cells.

关 键 词：2型糖尿病 miR-375 DNA去甲基化 基质辅助激光解析电离时间飞行质谱仪 

分 类 号：R587.1[医药卫生—内分泌]