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

摘要/Abstract

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。

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

Abstract: 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-1α was 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 significantly reversed the effects of miR-133a-3p mimics transfection on 16-HBE cell apoptosis and inflammation. Conclusions The over-expression of miR-133a-3p inhibits the inflammatory response and apoptosis of RSV-infected bronchial epithelial cells by negatively regulating the expression of CD2AP and thereby reducing cell damage.

Key words: miR-133a-3p, CD2AP, respiratory syncytial virus, bronchial epithelial cells, apoptosis, inflammatory reaction

中图分类号:

梁敏, 李弯, 熊诗思, 边俊梅. 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[J]. 基础医学与临床, 2021, 41(10): 1463-1469.

LIANG Min, LI Wan, XIONG Shi-si, BIAN Jun-mei. miR-133a-3p targeted regulating CD2AP gene reduces RSV-infected damage of human bronchial epithelial 16-HBE cell line[J]. Basic & Clinical Medicine, 2021, 41(10): 1463-1469.

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