妊娠期糖尿病中患者miR-138-5p通过调控缺氧诱导因子1α保护β细胞功能  

miR-138-5p protectsβcell function by regulating HIF-1αin gestational diabetes mellitus

作　　者：黄好[1] 肖芳[2] 贾虹[1] 王晓霜[1] 段亚亭 HUANG Hao;XIAO Fang;JIA Hong;WANG Xiaoshuang;DUAN Yating(Department of Gynecology and Obstetrics,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400021,China;Department of ULtrasound,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400021,China)

机构地区：[1]重庆市中医院妇产科,重庆400021 [2]重庆市中医院超声科,重庆400021

出　　处：《中国临床药理学与治疗学》2020年第5期489-497,共9页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：国家中医药管理局全国名老中医药专家传承工作室。

摘　　要：目的:探讨在妊娠期糖尿病(GDM)患者中miR-138-5p对胰岛β细胞功能的影响及其相关作用机制。方法:通过RT-qPCR对比15例GDM患者与15例的正常健康孕妇外周血中miR-138-5p的表达差异;miR-138-5p mimic及inhibitor分别转染入β细胞系INS-1细胞中,过表达或抑制其在该细胞中的表达水平,并通过RT-qPCR验证转染效率;利用MTT增殖实验、Annexin V-FITC凋亡实验及胰岛素释放实验分别检测miR-138-5p对INS-1细胞的增殖、凋亡和胰岛素释放能力的影响;通过miRNA靶基因预测软件Target Scan筛选miR-138-5p的目标靶基因,并利用双荧光素酶基因报告及Western blot实验进行验证;功能挽救实验证实miR-138-5p是否通过靶向调控其目标基因而发挥对INS-1细胞的增殖、凋亡及胰岛素释放能力影响;Western blot实验检测miR-138-5p在INS-1细胞中作用的分子信号通路。结果:与正常健康孕妇相比,GDM患者外周血中miR-138-5p的表达显著低表达;在INS-1细胞中转染miR-138-5p mimic及inhibitor后可显著促进或抑制miR-138-5p的表达;过表达miR-138-5p可明显促进INS-1细胞的增殖,抑制其发生凋亡并促进细胞对胰岛素的释放能力;而下调miR-138-5p的表达,可显著抑制INS-1细胞的增殖,促进其发生凋亡及抑制细胞的胰岛素释放能力;通过miRNA靶基因预测软件Target Scan筛选缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)为miR-138-5p的目标靶基因,双荧光素酶基因报告实验及Western blot实验提示miR-138-5p可抑制INS-1细胞中HIF-1α的表达;功能挽救实验证实miR-138-5p通过调控HIF-1α的表达,影响INS-1细胞的增殖、凋亡及胰岛素释放能力;Western blot实验明确miR-138-5p可能通过靶向调控HIF-1α的表达,影响PI3K/AKT信号通路中PI3K、AKT及其磷酸化后的p-PI3K、p-AKT蛋白而发挥对INS-1细胞的作用。结论:在GDM患者中,miR-138-5p可能通过靶向调控HIF-1α的表达,进而影响PI3K/AKT信号通路,促进β细胞的增殖、�AIM:To study the effect of miR-138-5 p on the function ofβcell in gestational diabetes mellitus(GDM)and its related mechanism.METHODS:The expression of miR-138-5 p in peripheral blood of 15 GDM pregnant women and 15 normal pregnant women were compared by RT-qPCR.miR-138-5 p mimic and inhibitor were transfected into INS-1 cells,respectively,and their expression level was over expressed or inhibited.RT-qPCR was used to verify the transfection efficiency.MTT proliferation experiment,Annexin V-FITC apoptosis experiment and insulin release experiment were used to detect the effects of miR-138-5 p on INS-1 cell proliferation,apoptosis and insulin release ability.The target gene of miR-138-5 p was screened by TargetScan,a miRNA target gene prediction software.The functional rescue experiment confirmed whether miR-138-5 p could exert its influence on INS-1 cell proliferation,apoptosis and insulin release ability by targeting its target gene.Western blot was used to detect the molecular signaling pathway of miR-138-5 p in INS-1 cells.RESULTS:The expression of miR-138-5 p in peripheral blood of GDM pregnant women was significantly lower than that of normal pregnant women.RT-qPCR showed that miR-138-5 p mimic and inhibitor could significantly promote or inhibit the expression of miR-138-5 p in INS-1 cells.The results of MTT proliferation experiment,Annexin V-FITC apoptosis experiment and insulin release experiment indicated that over expression of miR-138-5 p could significantly promote the proliferation of INS-1 cells,inhibit the apoptosis of cells and promote the insulin release ability of cells.However,down-regulating the expression of miR-138-5 p could significantly inhibit the proliferation of INS-1 cells,promote apoptosis and inhibit insulin release.HIF-1αwas selected as the target gene of miR-138-5 p by TargetScan.The double luciferase gene report and Western blot showed that miR-138-5 p could inhibit the expression of HIF-1αin INS-1 cells.The functional rescue experiment confirmed that miR-138-5 p could affect th

关 键 词：妊娠期糖尿病 miR-138-5p HIF-1Α INS-1细胞 PI3K/AKT信号通路 

分 类 号：R965.2[医药卫生—药理学] R587[医药卫生—药学]