The Role Of MiRNA-186-5p In The Bronchial Epithelial Cells Of Copd And Its Regulatory Mechanism

Objective: Early miRNA transcriptome analysis in our laboratory revealed that the expression of miR-186-5p was changed in COPD,however,the specific regulatory mechanism of miR-186-5p was unknown.Bioinformatics analysis showed that miR-186-5p could bind to lncRNA RP11-20G6.3 and HIF-1?.Therefore,this study aims to explore the biological function of miR-186-5p in COPD from the cell level,and reveal the targeted-regulating of miR-186-5p with lncRNA RP11-20G6.3 and HIF-1?,and preliminarily explore the effect of lncRNA RP11-20G6.3 on the occurrence of COPD in cell,which provides theoretical basis for its molecular mechanism.Methods:(1)LPS induction and inflammatory cell model construction.(2)miR-186-5p and lncRNA RP11-20G6.3 knockdown sequence were synthesized and transfected into cells.LPS induced cell proliferation activity was detected by CCK-8 method;cell apoptosis and cell cycle were detected by flow detection.The mRNA expressions of TNF-?,HIF-1? and IL-6 were detected by qRT-PCR,and the expression levels of TLR-4/NF-?B signaling pathway p65 and p-p65 were detected by Western blot.(3)Targeted regulation of miR-186-5p with lncRNA RP11-20G6.3 and HIF-1? were verified by double luciferase reporting experiment.(4)Observe the expression of lncRNA RP11-20G6.3 in peripheral blood of COPD patients and healthy people.Results:(1)The expression of HIF-1? was increased by LPS-induced BEAS-2B cells to simulate the COPD model,and the expression of HIF-1? was increased.IL-6 and TNF-? in the supernatant were detected by ELISA,which confirmed the successful construction of the cell inflammation model and could be used in subsequent experiments.(2)Low expression of miR-186-5p significantly decreased the proliferation of BEAS-2B.Inhibition of miR-186-5p expression can promote the apoptosis of BEAS-2B.There was no significant correlation in cell cycle.Inhibition of lncRNA RP11-20G6.3 expression can reduce cell proliferation.(3)miR-186-5p knockdown can promote the expression of TNF-?,HIF-1? and IL-6 genes related to the inflammatory pathway of NF-?B.This suggests that miR-186-5p may affect COPD through NF-?B.(4)Combined with bioinformatics analysis and luciferase reporting experiment,it was found that miR-186-5p can bind to lncRNA RP11-20G6.3 and HIF-1?,thus regulating the downstream signaling pathway and causing the occurrence and development of COPD.(5)lncRNA RP11-20G6.3 is up-regulated in serum.Conclusion:(1)Low expression of miR-186-5p can significantly reduce the proliferation ability and promote apoptosis of bronchial epithelial cells,and low expression of lncRNA RP11-20G6.3 can reduce its cell proliferation,both of which play important roles in COPD.(2)The low expression of miR-186-5p can promote the expression of HIF-1?,TNF-? and IL-6 in the NF-?B inflammatory pathway.(3)lncRNA RP11-20G6.3 targets miR-186-5p and miR-186-5p targets HIF-1?.Therefore,it is speculated that lncRNA RP11-20G6.3 may regulate HIF-1? in combination with miR-186-5p to participate in NF-?B inflammation-related signaling pathways,affect the proliferation and apoptosis of bronchial epithelial cells in COPD,and thus participate in the mechanism of COPD.(4)The expression of lncRNA RP11-20G6.3 mRNA in peripheral blood of COPD patients increased.