miR-21靶向PTEN调节PI3K/AKT通路对狼疮性肾炎患者肾小球系膜细胞增殖及炎性因子分泌的影响  

Effects of miR-21 Targeting PTEN to Regulate PI3K/AKT Pathway on the Proliferation of Glomerular Mesangial Cells and Secretion of Inflammatory Factors in Patients with Lupus Nephritis

作　　者：刘春雅[1] 金晶 李玉芳[1] 喻敏[1] 姜毅[1] 徐利鸳[1] LIU Chunya;JIN Jing;LI Yufang(Department of Nephrology,Quzhou People’s Hospital,Quzhou,324000)

机构地区：[1]浙江省衢州市人民医院肾内科,衢州324000 [2]温州医科大学检验医学院、生命科学学院,检验医学教育部重点实验室,浙江省医学遗传学重点实验室,温州325000

出　　处：《中国中西医结合肾病杂志》2021年第9期759-764,共6页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：衢州市科技计划项目(No.2015070);浙江省自然科学基金资助项目(No.LY13H070006)。

摘　　要：目的:探讨miR-21靶向磷酸酶和张力蛋白同源物(PTEN)调节磷酸肌醇3-激酶(PI3K)/蛋白激酶B(AKT)通路对人肾小球系膜细胞(human mesangial cells,HMCs)增殖及炎性因子分泌的影响。方法:选取在我院经肾穿刺活检诊断为LN的30例患者的肾组织标本,qRT-PCR和western blot检测狼疮性肾炎肾组织中miR-21和PTEN的表达水平;HMCs随机分为5组:NC mimics组、miR-21 mimics组、pcDNA3.1-NC组、pcDNA3.1-PTEN组和miR-21 mimics+pcDNA3.1-PTEN组;TargetScan预测miR-21与PTEN的结合位点;双荧光素酶报告基因验证miR-21与PTEN的靶向关系;细胞克隆形成实验检测miR-21靶向PTEN对HMCs增殖能力的影响;细胞流式术检测miR-21靶向PTEN对HMCs凋亡率的影响;ELISA检测miR-21靶向PTEN对HMCs炎性因子生成的影响;Western blot检测miR-21靶向PTEN调节PI3K/AKT通路相关蛋白的表达。结果:miR-21在狼疮性肾炎肾组织中高表达(P=0.043),PTEN在狼疮性肾炎肾组织中低表达(P=0.037);miR-21与PTEN存在靶向关系,且miR-21靶向下调PTEN的表达(P=0.003);与NC mimics组比较,miR-21 mimics组细胞集落数目增加(P=0.003),凋亡率下降(P=0.003),TNF-α、IL-1β和IL-6的含量增加(均P=0.000),PTEN蛋白表达水平下调(P=0.003)、PI3K和AKT蛋白表达水平上调(P=0.025,P=0.004);与pcDNA3.1-NC组比较,pcDNA3.1-PTEN组细胞集落数目减少(P=0.032),凋亡率上升(P=0.035),TNF-α、IL-1β和IL-6的含量减少(P=0.003,P=0.037,P=0.029),PTEN蛋白表达水平上调(P=0.002)、PI3K和AKT蛋白表达水平明显下调(均P=0.003);与miR-21 mimics组比较,miR-21 mimics+pcDNA3.1-PTEN组细胞集落数目减少(P=0.003),凋亡率上升(P=0.000),TNF-α、IL-1β和IL-6的含量减少(P=0.003,P=0.004,P=0.004),PTEN蛋白表达水平上调(P=0.003)、PI3K和AKT蛋白表达水平下调(P=0.031,P=0.039);与pcDNA3.1-PTEN组比较,miR-21 mimics+pcDNA3.1-PTEN组细胞集落数目增加(P=0.029),凋亡率下降(P=0.031),TNF-α、IL-1β和IL-6的含量增加(P=0.000,P=0.003,P=0.003),PTEN蛋白表达水平下Objective:To investigate the effect of miR-21 targeting phosphatase and tensin homolog(PTEN)on the regulation of phosphoinositide 3-enzyme(PI3K)/protein kinase B(AKT)pathway on human glomerular mesangial cells(HMCs)proliferation and secretion of inflammatory factors influences.Methods:Kidney tissue specimens of 30 patients diagnosed as LN by renal biopsy was selected in our hospital,qRT-PCR and western blot were used to detect the expression levels of miR-21 and PTEN in lupus nephritis kidney tissue;HMCs were randomly divided into 5 groups;NC mimics group,miR-21 mimics group,pcDNA3.1-NC group,pcDNA3.1-PTEN group and miR-21 mimics+pcDNA3.1-PTEN group;TargetScan was used to predict the binding site of miR-21 and PTEN;Dual luciferase reporter gene was used to validate the targeting relationship between miR-21 and PTEN;Cell clone formation experiment was uesd to detect the effect of miR-21 targeting PTEN on the proliferation ability of HMCs;Cell flow cytometry was uesd to detect the effect of miR-21 targeting PTEN on the apoptosis rate of HMCs;ELISA was used to detect the effect of miR-21 targeting PTEN on the production of HMCs inflammatory factors;Western blot was used to detect the miR-21 targeting PTEN regulates the expression of PI3K/AKT pathway-related proteins.Results:miR-21 was highly expressed in lupus nephritis kidney tissue(P=0.043),PTEN was low expressed in lupus nephritis(P=0.037);There was a targeting relationship between miR-21 and PTEN,and miR-21 targeted down-regulation of PTEN expression(P=0.003);Compared with the NC mimics group,the number of cell colonies in the miR-21 mimics group increased(P=0.003),the apoptosis rate decreased(P=0.003),and the levels of TNF-α,IL-1β,and IL-6 increased(all P=0.000),PTEN protein expression level was down-regulated(P=0.003),PI3K and AKT protein expression levels were up-regulated(P=0.025,P=0.004);Compared with the pcD-NA3.1-NC group,the number of colonies in the pcDNA3.1-PTEN group decreased(P=0.032),the apoptosis rate increased(P=0.035),and the levels of TNF-α,I

关 键 词：MIR-21 PTEN PI3K/AKT通路 人肾小球系膜细胞 增殖 炎性因子 

分 类 号：R593.242[医药卫生—内科学]