MiR-625-5P Negatively Regulates LPS-induced Inflammatory Responses Through Targeting Akt2 In Bronchial Epithelial Cells

Background and objectiveBronchial asthma is a chronic inflammatory disease of the airways defined with many kinds of cells and cellular components.With the deeper study,the role of bronchial epithelial cells in the development and progression of asthma has been paid more and more attention.MicroRNAs are highly conserved endogenous non-coding small RNAs that its function primarily by targeting the 3’untranslated region(3’UTR)of mRNA.A large number of researches indicated that miRNAs play an important role in the process of immune response,airway remodeling,airway hyperresponsiveness of asthma.But we have known little about the role of miRNAs in the inflammation of bronchial epithelial cells.This experiment is aimed to investigate the effect of miR-625-5p on LPS-induced inflammation of bronchial epithelial cell(16HBE)and the potential mechanism of action.Methods 1.Constructing a Inflammatory Model of Bronchial Epithelium Cells(16HBE)which induced by LPS 1.1 After 16 HBE were stimulated with different concentrations of LPS(0,0.01,0.1,1,10,100 mg/L)for 12 h,the changes of cell viability were detected by MTT assay.1.2 we select the propriate concentration of LPS(1 mg/L)to stimulate 16 HBE for 12 h,then the expression level of IL-6 and TNF-α was measured at mRNA level by Fluorescence Quantification PCR.2.The effects of up-regulated or down-regulated mi R-625-5p on LPS-induced inflammatory reaction in 16 HBE CellsMiR-625-5p mimics/ miR-625-5p inhibitor were transfected into 16 HBE Cells for 24 hour,we apply fluorescence quantification PCR to detect the expression level of miR-625-5p.The bronchial epithelium transfected with miR-625-5p mimics was stimulated with LPS for a further 12 h,expression of inflammatory cytokines(IL-6、TNF-α)mRNA was analyse by fluorescent quantitative PCR,and secretion of inflammatory cytokines(IL-6、TNF-α)protein were detected by ELISA.3.Identifying the relationship of miR-625-5p and its potential targetsAkt2 3’UTR wild-type plasmid(pMIR-Akt2-WT)/mutant plasmid(pMIR-Akt2-MUT)and miR-625-5p were co-transfected into 16 HBE Cells for 24 h and detect luciferase activity by double fluorescent reporter assay system.4.Analyzing the probable mechanism that miR-625-5p affects 16 HBE Cells inflammatory response induced by LPS.After mi R-625-5p mimics/miR-625-5p inhibitor and relevant control have been transfected into 16 HBE cells for 24 h,then apply LPS to stimulate 16 HBE cells for 12 h,and the protein expression level of p-Akt2、Akt2、p-IκBα was detected by Western blotting.Results and conclusions 1.LPS can induce 16 HBE cells inflammatory response 1.1 Compared with control group,the vitality of cells were significantly decreased.1.2 Compared with control group,proinflammatory cytokines,such as IL-6 and TNF-α were significantly increased after LPS stimulation for 12 h.And above results indicated LPS can induce 16 HBE cell inflammation model.2.miR-625-5p negatively regulated LPS-induced inflammatory response in 16 HBE cells.2.1 miR-625-5p mimics were transfected into 16 HBE Cells for 24 h,miR-625-5p was significantly up-regulated.The bronchial epithelium transfected with miR-625-5p mimics was stimulated with LPS for a further 12 hours,the level of inflammatory cytokines mRNA and protein was significantly decreased.2.2 miR-625-5p inhibitor were transfected into 16 HBE Cells for 24 h,miR-625-5p was significantly down-regulated.The bronchial epithelium transfected with miR-625-5p inhibitor was stimulated with LPS for a further 12 hours,the level of inflammatory cytokines mRNA and protein was significantly increased.And above results indicated miR-625-5p negatively regulated LPS-induced inflammatory response in 16 HBE cells.3.miR-625-5p can work through Akt2The miR-625-5p mimic and pMIR-Akt2-WT/pMIR-Akt2-WT were transfected into cells.The results showed that mi R-625-5p could significantly decrease the luciferase activity of pMIR-Akt2-WT group,and can not inhibit the pMIR-Akt2-WT group luciferase activity.4.miR-625-5p can nagetively regulate the inflammatory reaction induced by LPS through Akt2Compared with the control group,the protein expression level of p-Akt2 and p-IκBα in LPS group was significantly hoisted.Compared with the LPS group,the protein expression level of p-Akt2 and p-IκBα was significantly declined in miR-625-5p mimic group,and miR-625-5p inhibitor group was increased.Indicating that miR-625-5p can inhibit LPS-induced phosphorylation level of Akt2 and IκBα proteins.In summary,bronchial epithelial cells were stimulated with LPS to construct an inflammatory model in vitro and treated with miR-625-5p the results revealed that miR-625-5p inhibited LPS-induced inflammatory response in 16 HBE cell.Further study confirmed its mechanism might be that miR-625-5p could regulate NF-κB signaling pathway and ultimately inhibit the release of inflammatory cytokines through targeting Akt2.