miR-660-3p靶向NFAM1对RSV诱导支气管上皮细胞凋亡及炎症因子分泌的影响  

Effect of miR-660-3p targeting NFAM1 on RSV-induced apoptosis and inflammatory factors in bronchial epithelial cells

作　　者：童珍珍 高雅丽 许娟娟 黄方 许诣 陈霞 TONG Zhenzhen;GAO Yali;XU Juanjuan;HUANG Fang;XU Yi;CHEN Xia(Department of Respiratory Medicine,Anting Hospital of Jiading District,Shanghai 201805,China;不详)

机构地区：[1]上海市嘉定区安亭医院呼吸内科,上海201805 [2]华中科技大学同济医学院附属协和医院呼吸内科 [3]湖北文理学院附属医院襄阳市中心医院儿科

出　　处：《山东医药》2024年第13期30-35,共6页Shandong Medical Journal

摘　　要：目的探讨微小RNA-660-3p(miR-660-3p)靶向具有ITAM基序1的NFAT活化蛋白(NFAM1)对呼吸道合胞病毒(RSV)诱导支气管上皮细胞凋亡和炎症因子分泌的影响。方法体外传代培养人支气管上皮细胞16HBE。取对数生长期16HBE细胞,随机分为对照组、RSV组、miR-660-3p+RSV组、miR-NC+RSV组、pcDNA-NC+RSV组、pcDNA-NFAM1+RSV组、miR-660-3p+pcDNA-NC+RSV组、miR-660-3p+pcDNA-NFAM1+RSV组,miR-NC+RSV组转染NC mimics,miR-660-3p+RSV组转染miR-660-3p mimics,pcDNA-NFAM1+RSV组转染pcDNA-NFAM1,pcDNA-NC+RSV组转染pcDNA-NC,miR-660-3p+pcDNA-NC+RSV组共转染pcDNA-NC和miR-660-3p mimics,miR-660-3p+pcDNA-NFAM1+RSV组共转染pcDNA-NFAM1和miR-660-3p mimics,对照组和RSV组不予转染;除对照组外,其余各组均予RSV感染,MOI设定为0.0001。收集各组细胞,采用RT-qPCR法检测miR-660-3p、NFAM1 mRNA表达,采用流式细胞术检测细胞凋亡率,采用Western blotting法检测Cleaved-Caspase-3、NFAM1蛋白表达;收集各组培养上清液,采用ELISA法检测TNF-α、IL-6、IL-1β。通过starBase数据库预测NFAM1与miR-660-3p的靶向结合位点,并采用双荧光素酶报告基因实验验证miR-660-3p与NFAM1的靶向调控关系。结果与对照组比较,RSV组miR-660-3p表达降低,NFAM1 mRNA与蛋白及Cleaved-Caspase-3蛋白表达升高,细胞凋亡率及培养上清液TNF-α、IL-6、IL-1β水平升高(P均<0.05)。与miR-NC+RSV组比较,miR-660-3p+RSV组miR-660-3p表达升高,Cleaved-Caspase-3蛋白表达以及细胞凋亡率和培养上清液TNF-α、IL-6、IL-1β水平降低(P均<0.05)。与pcDNA-NC+RSV组比较,pcDNA-NFAM1+RSV组NFAM1 mRNA、Cleaved-Caspase-3蛋白表达以及细胞凋亡率和培养上清液TNF-α、IL-6、IL-1β水平升高(P均<0.05)。经starBase数据库预测,NFAM1的3′非翻译区存在与miR-660-3p的结合位点。双荧光素酶报告基因实验结果显示,相较于共转染NC mimics与NFAM1-WT的16HBE细胞,共转染miR-660-3p mimics与NFAM1-WT的16HBE细胞荧光素酶活性下降(P<0.05);相较于共转Objective To investigate the effects of miR-660-3p targeting NFAM1 on the apoptosis and inflammatory factors of human bronchial epithelial cells induced by respiratory syncytial virus(RSV).Methods Human bronchial epithelial cells 16HBE were cultured in vitro.16HBE cells in the logarithmic growth phase were randomly divided into the control group,RSV group,miR-660-3p+RSV group,miR-NC+RSV group,pcDNA-NC+RSV group,pcDNA-NFAM1+RSV group,miR-660-3p+pcDNA-NC+RSV group,and miR-660-3p+pcDNA-NFAM1+RSV group,respectively.Cells in the miR-NC+RSV group were transfected with NC mimics,miR-660-3p+RSV group with miR-660-3p mimics,pcDNA-NFAM1+RSV group with pcDNA-NFAM1,pcDNA-NC+RSV group with PCDNA-NFAM1,pcDNA-NC+RSV group with PCDNA-NC,and cells in the miR-660-3p+pcDNA-NC+RSV group were co-transfected with pcDNA-NC and miR-660-3p mimics,and cells in the miR-660-3p+pcDNA-NFAM1+RSV group were co-transfected with pcDNA-NFAM1 and miR-660-3p mimics;cells in the control group and RSV group were not transfected.Then,all groups except the control group were infected with RSV and the MOI was set at 0.0001.Cells in each group were collected,and the expression levels of miR-660-3p and NFAM1 mRNA were detected by RT-qPCR,apoptosis rate was detected by flow cytometry,and protein expression levels of Cleaved Caspase-3 and NFAM1 were detected by Western blotting;the super-natant of each group was collected,and TNF-α,IL-6 and IL-1βwere detected by ELISA;the targeting binding sites of NFAM1 and miR-660-3p were predicted by starBase database,and the targeting regulatory relationship between miR-660-3p and NFAM1 was verified by double luciferase reporter gene assay.Results Compared with the control group,the expres-sion of miR-660-3p decreased,the expression of NFAM1 mRNA and protein and Cleaved Caspase-3 protein increased,the apoptosis rate and the levels of TNF-α,IL-6 and IL-1βin culture supernatance increased in the RSV group(all P<0.05).Compared with the miR-NC+RSV group,the expression of miR-660-3p increased,Cleaved Caspase-3 protein exp

关 键 词：支气管哮喘 微小RNA-660-3p 具有ITAM基序1的NFAT活化蛋白 细胞凋亡 炎症因子 

分 类 号：R562[医药卫生—呼吸系统]