Effect of miR-660-3p targeting NFAM1 on RSV-induced apoptosis and inflammatory factors in bronchial epithelial cells
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 0.000 1.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-1β were 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-1β in 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 expres-sion,apoptosis rate,and levels of TNF-α,IL-6,and IL-1β in the culture supernatance decreased in the miR-660-3p + RSV group(all P＜0.05).Compared with the pcDNA-NC + RSV group,NFAM1 mRNA,Cleaved Caspase-3 protein expression,apoptosis rate and TNF-α,IL-6 and IL-1β levels in culture supernatance increased in the pcDNA-NFAM1 + RSV group(all P＜0.05).The starBase database predicted that there was a binding site to miR-660-3p in the 3'non-coding region of NFAM1.The results of double luciferase reporter gene experiment showed that the luciferase activity of 16HBE cells co-transfected with NC mimics and NFAM1-WT decreased in comparison with that of 16HBE cells co-transfected with miR-660-3p mimics and NFAM1-WT(P＜0.05).Compared with 16HBE cells co-transfected with NC mimics and NFAM1-MUT,16HBE cells co-transfected with miR-660-3p mimics and NFAM1-MUT had no significant changes in luciferase activity(P＞0.05).Compared with the miR-660-3p + pcDNA-NC + RSV group,Cleaved Caspase-3 protein expression,apoptosis rate,and the levels of TNF-α,IL-6,and IL-1β in culture supernatance increased in the miR-660-3p + pcDNA-NFAM1 + RSV group(all P＜0.05).Conclusion The miR-660-3p can inhibit RSV-induced apoptosis and secretion of inflammatory factors in human bronchial epithelial cells by targeting and negatively regulating NFAM1.
国家科技期刊平台
NSTL
万方数据
维普
