miR-133a-3p靶向调控CD2AP基因减轻RSV感染的人支气管上皮细胞系16-HBE损伤  

miR-133a-3p targeted regulating CD2AP gene reduces RSV-infected damage of human bronchial epithelial 16-HBE cell line

作　　者：梁敏 李弯 熊诗思 边俊梅 LIANG Min;LI Wan;XIONG Shi-si;BIAN Jun-mei(Department of Pediatrics, Tongren Hospital of Wuhan University, Wuhan Third Hospital, Wuhan 430074, China)

机构地区：[1]武汉大学附属同仁医院/武汉市第三医院儿科,湖北武汉430074

出　　处：《基础医学与临床》2021年第10期1463-1469,共7页Basic and Clinical Medicine

基　　金：武汉市卫生健康科研基金资助项目(WX19C36)。

摘　　要：目的探讨miR-133a-3p对呼吸道合胞病毒(RSV)感染的支气管上皮细胞凋亡、炎性反应的影响及其对CD2相关蛋白(CD2AP)的调控作用。方法收集59例支气管哮喘儿童的支气管黏膜组织标本为AsP组,同时选取59例支气管扩张非哮喘儿童的支气管黏膜组织标本为NC组,用RT-qPCR法检测组织中miR-133a-3p、CD2AP的表达量;RSV感染支气管上皮细胞(16-HBE)建立细胞损伤模型(RSV组),分别将miR-NC、miR-133a-3p mimics、si-NC、si-CD2AP、pcDNA-NC、pcDNA-CD2AP、miR-133a-3p mimics与pcDNA-NC、miR-133a-3p mimics与pcDNA-CD2AP转染至感染RSV的16-HBE细胞;用RT-qPCR法与Western blot法分别检测细胞中miR-133a-3p、CD2AP的表达量;用ELISA法检测TNF-α、IL-6、IL-1α的水平;用流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测miR-133a-3p与CD2AP的靶向关系;Western blot法检测cleaved-caspase-3蛋白表达量。结果与NC组比较,AsP组支气管黏膜组织中miR-133a-3p的表达水平降低(P<0.05),CD2AP mRNA的表达水平升高(P<0.05);RSV感染的16-HBE细胞中miR-133a-3p的表达水平降低(P<0.05),CD2AP的表达水平及TNF-α、IL-6、IL-1α的水平升高(P<0.05),凋亡率及cleaved-caspase-3蛋白水平升高(P<0.05);RSV感染的16-HBE细胞中转染miR-133a-3p mimics或转染si-CD2AP可明显降低TNF-α、IL-6、IL-1α的水平、凋亡率及cleaved-caspase-3蛋白水平(P<0.05);双荧光素酶报告实验证实CD2AP是miR-133a-3p的靶基因;共转染miR-133a-3p mimics与pcDNA-CD2AP可明显逆转miR-133a-3p mimics对16-HBE细胞凋亡及炎性反应。结论miR-133a-3p过表达可通过负向调控CD2AP的表达而抑制RSV感染的支气管上皮细胞炎性反应及凋亡,从而减轻细胞损伤。Objective To explore the effect of miR-133a-3p on the apoptosis and inflammatory response of bronchial epithelial cells infected by respiratory syncytial virus(RSV)and its regulatory effect on CD2-associated protein(CD2AP).Methods The bronchial mucosal tissue specimens from 59 children with bronchial asthma were collected as the AsP group and 59 bronchial mucosal tissue specimens from non-asthmatic children with bronchiec-tasis were selected as the NC group.RT-qPCR method was used to detect the expression of miR-133a-3p and CD2AP in the tissues.RSV infected bronchial epithelial cells(16-HBE)were used to establish a cell injury model(RSV group).miR-NC,miR-133a-3p mimics,si-NC,si-CD2AP,pcDNA-NC,pcDNA-CD2AP,miR-133a-3p mimics and pcDNA-NC,miR-133a-3p mimics and pcDNA-CD2AP were respectively transferred into 16-HBE cells infected with RSV.RT-qPCR and Western blot were used to detect the expression of miR-133a-3p and CD2AP in the cells.ELISA was used to detect the level of TNF-α,IL-6,and IL-1α.Flow cytometry was used to detect apoptosis.The dual luciferase reporter experiment was used to detect the targeting relationship between miR-133a-3p and CD2AP.Western blot method was used to detect the protein expression of cleaved-caspase-3.Results Compared with NC group,the expression of miR-133a-3p in the bronchial mucosa tissue of the AsP group was decreased(P<0.05),and the expression of CD2AP mRNA was increased(P<0.05).The expression of miR-133a-3p in RSV-infected 16-HBE cells was decreased(P<0.05),and the expression level of CD2AP,TNF-α,IL-6 and IL-1αwas increased(P<0.05),the apoptosis rate and the protein level of cleaved-caspase-3 were increased(P<0.05).Transfection of miR-133a-3p mimics or transfection of si-CD2AP into RSV-infected 16-HBE cells significantly reduced the level of TNF-α,IL-6,IL-1α,the apoptosis rate and the protein level of cleaved-caspase-3(P<0.05).The dual luciferase report experiment confirmed that CD2AP was the target gene of miR-133a-3p.Co-transfection of miR-133a-3p mimics and pcDNA-CD2AP sig

关 键 词：miR-133a-3p CD2AP 呼吸道合胞病毒 支气管上皮细胞 凋亡 炎性反应 

分 类 号：R54[医药卫生—心血管疾病]