miR-27b-3p调控RUNX1对高糖诱导的肾小球系膜细胞增殖及炎症反应的影响  

Effects of miR-27b-3p on hyperglycemia-induced proliferation and inflammatory response of glomerular mesangial cells by regulating RUNX1

作　　者：韩晓静 布海霞 王焕 徐可 何国斌 HAN Xiaojing;BU Haixia;WANG Huan;XU Ke;HE Guobin(Department of Nephrology,Xinxiang Central Hospital,the Fourth Clinical College of Xinxiang Medical University,Xinxiang 453000,Henan,China)

机构地区：[1]新乡市中心医院新乡医学院第四临床学院肾内科,新乡453000

出　　处：《医学研究与战创伤救治》2025年第3期231-237,共7页Journal of Medical Research & Combat Trauma Care

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20220988)。

摘　　要：目的探究miR-27b-3p对高糖诱导的肾小球系膜细胞增殖和炎症反应的作用及潜在机制。方法体外培养人肾小球系膜细胞,分为葡萄糖组(含5.6 mmol/L D-葡萄糖的培养基孵育48 h)、高糖组(含25 mmol/L D-葡萄糖的培养基孵育48 h)、miR-NC组(转染miR-NC)、miR-27b-3p组(转染miR-27b-3p mimics)、miR-27b-3p+pc-NC组(共转染miR-27b-3p mimics和pcD NA3.1-NC)、miR-27b-3p+pc-RUNX1组(共转染miR-27b-3p mimics和pcD NA3.1-RUNX1)。采用qR T-PCR检测miR-27b-3p和RUNX1mR NA表达;CCK-8法检测细胞活力;流式细胞术检测细胞凋亡;ELISA检测上清液炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-1β水平;Western blot检测细胞中RUNX1、凋亡[剪切的多聚ADP核糖多聚酶(CleavedPARP)、剪切的半光天冬氨酸蛋白酶3(Cleaved-caspase 3)]和纤维化[纤维连接蛋白(FN)、Ⅳ型胶原蛋白(CollagenⅣ)、转化生长因子β_(1)(TGF-β_(1))]相关蛋白表达;双荧光素酶报告基因、Pulldown分析系膜细胞细胞中miR-27b-3p和RUNX1的调控作用。结果与葡萄糖组相比,高糖组细胞中miR-27b-3p表达水平、细胞凋亡率、Cleaved-PARP和Cleaved-caspase 3蛋白水平降低,细胞活力、细胞培养上清液TNF-α、IL-6和IL-1β水平、RUNX1 mR NA和蛋白表达、FN、CollagenⅣ、TGF-β_(1)蛋白水平升高(P<0.05);与miR-NC组相比,miR-27b-3p组miR-27b-3p表达水平、细胞凋亡率、Cleaved-PARP、Cleaved-caspase 3蛋白水平升高,细胞活力、细胞培养上清液TNF-α、IL-6和IL-1β水平、RUNX1mR NA和蛋白表达、FN、CollagenⅣ、TGF-β_(1)蛋白水平降低(P<0.05)。上调RUNX1表达部分削弱了miR-27b-3p过表达对高糖诱导的系膜细胞增殖和炎症反应的抑制作用。双荧光素酶报告基因、Pull-down实验证实RUNX1是miR-27b-3p的靶标。结论过表达miR-27b-3p可能通过靶向下调RUNX1表达,抑制高糖诱导的系膜细胞增殖、炎症和纤维化。Objective To explore the effects of miR-27b-3p on hyperglycemia-induced proliferation and inflammation of glo-merular mesangial cells and its potential mechanism.Methods Human glomerular mesangial cells were cultured in vitro and divided into glucose group(culture medium containing 5.6 mmol/L glucose for 48 h),high glucose group(culture medium containing 25 mmol/L glucose for 48 h),miR-NC group(transfected with miR-NC)and miR-27b-3p group(transfected with miR-27b-3p mimics),miR-27b-3p+pc-NC group(co-transfected miR-27b-3p mimics and pcDNA3.1-NC),miR-27b-3p+pc-RUNX1 group(co-transfected miR-27b-3p mimics and pcDNA3.1-RUNX1).The qRT-PCR was used to detect mRNA expressions of miR-27b-3p and RUNX1.CCK-8 method was used to detect Cell viability.Flow cytometry was used to detect apoptosis.ELISA was used to detect the levels of inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1βin the supernatant.Western blot was applied to detect RUNX1 of cells,apoptosis(Cleaved-PARP,Cleaved-caspase 3)and fibrosis(FN,CollagenⅣin cells,TGF-β_(1) related protein expression.Dual luciferase reporter gene and Pull down were applied to analyze the regulatory effects of miR-27b-3p and RUNX1 in mesangial cells cells.Results Compared with the glucose group,the expression level of miR-27b-3p,apoptosis rate,Cleaved-PARP and Cleaved-caspase 3 protein levels were decreased in the high glucose group.Cell viability,levels of TNF-α,IL-6 and IL-1βin cell culture su-pernatant,expression of RUNX1 mRNA and protein,FN,CollagenⅣand TGF-β_(1) protein levels were increased(P<0.05).Com-pared with the miR-NC group,the expression level of miR-27b-3p,cell apoptosis rate,Cleaved-PARP,Cleaved-caspase 3 protein lev-els were increased in the miR-27b-3p group.Cell viability,levels of TNF-α,IL-6 and IL-1βin cell culture supernatant,expression of RUNX1 mRNA and protein,FN,CollagenⅣand TGF-β_(1) protein levels were decreased(P<0.05).Upregulation of RUNX1 partially weakened the inhibitory effect of miR-27b-3p overexpression on h

关 键 词：糖尿病肾病 肾小球系膜细胞 高糖 增殖 miR-27b-3p runt相关转录因子1 

分 类 号：R587.2[医药卫生—内分泌]