miR-26对TGF-β_(2)诱导的人Tenon氏囊成纤维细胞迁移、细胞外基质蛋白表达的调控作用及其机制  

Regulatory effects of miR-26 on invasion and extracellular matrix protein expression of human Tenon’s capsule fibroblasts induced by TGF-β_(2)

作　　者：李燕[1] 孟凯[2] 杜允宏 陈斐 刘文静[1] 鲍慧婧 LI Yan;MENG Kai;DU Yunhong;CHEN Fei;LIU Wenjing;BAO Huijing(Ophthalmology Department,Taian City Central Hospital Affiliated to Qingdao University,Taian 271000,China)

机构地区：[1]青岛大学附属泰安市中心医院眼科,山东泰安271000 [2]青岛大学附属泰安市中心医院肛肠外科

出　　处：《山东医药》2022年第35期34-39,共6页Shandong Medical Journal

基　　金：山东省自然科学基金资助项目(ZR2019PH065);山东省泰安市科技发展计划项目(2019NS110)。

摘　　要：目的观察miR-26对转化生长因子β_(2)(TGF-β_(2))诱导后人Tenon氏囊成纤维细胞(HTFs)的细胞迁移及细胞外基质(ECM)蛋白表达的调控作用,并探讨其可能的作用机制。方法切取青光眼手术患者的结膜囊组织,以组织贴壁法体外培养HTFs。取传至第3~5代的HTFs,随机分为空白组(不予转染)、TGF-β_(2)组(不予转染)、TGF-β_(2)+miR-26 mimics组(转染miR-26类似物)、TGF-β_(2)+mimics NC组(转染miR-26类似物阴性对照物),TGF-β_(2)+miR-26 inhibitors组(转染miR-26抑制物),TGF-β_(2)+inhibitors NC组(转染miR-26抑制物阴性对照物),转染24 h后除空白组外均加入TGF-β_(2)5μg/L刺激48 h。采用划痕实验检测细胞迁移率,Western blotting法检测Ⅰ型胶原蛋白(COLⅠ)、纤维连接蛋白(Fibronectin)、CTGF(荧光素酶报告基因实验证实miR-26和CTGF存在靶向关系)蛋白,实时荧光定量PCR法检测CTGF mRNA。将对数生长期的HTFs分为空白转染组(不予转染)、TGF-β_(2)+miR-26 inhibitors组(转染miR-26 inhibitors)、TGF-β_(2)+miR-26 inhibitors+si-NC组(转染miR-26 inhibitors+阴性对照siRNA)、TGF-β_(2)+miR-26 inhibitors+si-CTGF组(转染miR-26 inhibitors+CTGF-siRNA),转染处理24 h后均加入TGF-β_(2)5μg/L刺激48 h,比较四组细胞迁移率及COLⅠ、Fibronectin蛋白表达。结果六组细胞迁移率及COLⅠ、Fibronectin蛋白表达:空白组、TGF-β_(2)+miR-26 mimics组<TGF-β_(2)组、TGF-β_(2)+mimics NC组、TGF-β_(2)+inhibitors NC组<TGF-β_(2)+miR-26 inhibitors组(P均<0.05)。六组CTGF mRNA相对表达量:空白组<TGF-β_(2)组、TGF-β_(2)+mimics NC组、TGF-β_(2)+miR-26 mimics组、TGF-β_(2)+inhibitors NC组、TGF-β_(2)+miR-26 inhibitors组(P均<0.05)。六组CTGF蛋白相对表达量:空白组、TGF-β_(2)+miR-26 mimics组<TGF-β_(2)组、TGF-β_(2)+mimics NC组、TGF-β_(2)+inhibitors NC组<TGF-β_(2)+miR-26 inhibitors组(P均<0.05)。四组细胞迁移率及COLⅠ、Fibronectin蛋白表达:TGF-β_(2)+miR-26 inhibitors+si-CTGF组<空白转染组<TGFObjective To investigate the effects of miR-26 on the migration and the extracellular matrix(ECM)protein expression of human Tenon’s capsule fibroblasts(HTFs)induced by transforming growth factor(TGF)-β_(2).Methods The samples of conjunctival sac tissues in patients with glaucoma were taken during operation to culture HTFs in vitro by the method of tissue adhesion.The 3-5 passage cells were randomly divided into the following six groups:blank group(no transfection),TGF-β_(2)group(no transfection),TGF-β_(2)+miR-26 mimics group(miR-26 mimics were transfected),TGF-β_(2)+mimics NC group(miR-26 mimics negative controls were transfected),TGF-β_(2)+miR-26 inhibitors group(miR-26 inhibitors were transfected),and TGF-β_(2)+inhibitors NC group(miR-26 inhibitors negative controls were transfected).After 24 h of transfection,TGF-β_(2)of 5μg/L was added to all groups except blank group.Cell scratch test was applied to detect the migration of HTFs.Western blotting was used to detect the expression levels of COLⅠ,Fibronectin,and CTGF(Luciferase reporter gene assay confirmed the targeted relationship between miR-26 and CTGF)proteins.RT-PCR was applied to detect the expression of CTGF m RNA.HTFs in logarithmic growth phase was divided into the following four groups:TGF-β_(2)group(no transfection),TGF-β_(2)+miR-26 inhibitors group(miR-26 inhibitors were transfected),and TGF-β_(2)+miR-26 inhibitors+si-NC group(miR-26 inhibitors+negative control si RNA were transfected),TGF-β_(2)+miR-26 inhibitors+si-CTGF group(miR-26 inhibitors+CTGF si RNA were transfected).After 24 h of transfection,TGF-β_(2)of 5μg/L was added to all groups.The cell migration rate and protein expression levels of COL I and Fibronectin were compared among the four groups.Results Cell migration rate and expression levels of COL I and Fibronectin in the six groups were as follows:blank group,TGF-β_(2)+miR-26 mimics group2group,TGF-β_(2)+mimics NC group,TGF-β_(2)+inhibitors NC group2+miR-26 inhibitors group(all P<0.05).Relative expression levels o

关 键 词：miR-26 人Tenon氏囊成纤维细胞 细胞迁移 细胞外基质 CTGF基因 转化生长因子β_(2) 青光眼 

分 类 号：R775[医药卫生—眼科]