Effects Of MicroRNA-186-5p On The Permeability Of Alveolar Epithelial Barrier In Acute Lung Injury

ObjectiveTo investigate the effect of micro RNA(miRNA,miR)-186-5p on the permeability of the alveolar epithelial barrier in Acute Lung Injury(ALI)and the regulatory mechanism,and to clarify whether miR-186-5p regulates the wnt signaling pathway and thus the permeability of the alveolar epithelial barrier.Methods1.Alveolar type II epithelial cells(A549)were cultured in vitro,and cell function assays were performed after 24 h intervention of A549 with tumor necrosis factor(TNF-α)at concentrations of 10ng/ml,40ng/ml,80ng/ml and160ng/ml,respectively: cell proliferation activity assay(CCK-8),cellular inflammatory factor secretion level(ELISA),cell barrier permeability(FITC-glucan permeability),and Western Blot、 qRT-PCR were used to detect the expression of tight junction proteins ZO-1,Ocludin and Claudin-4.2.qRT-PCR was used to detect the expression of miR-186-5p under different concentrations of TNF-α intervention.Transfection of miR-186-5p mimic,miR-186-5p inhibitor into A549,fluorescence microscopy to observe the transfection effect,qRT-PCR to detect the expression of miR-186-5p.TNF-α 40ng/ml treatment of A549 transfected with miR-186-5p mimic,miR-186-5p inhibitor After 24 h,Cell survival,secretion of inflammatory factors,cell barrier permeability and tight junction protein expression were measured.3.qRT-PCR and Western Blot were performed to detect the expression of wnt5a/β-catenin signaling pathway under different concentrations of TNF-αintervention.After applying the wnt pathway inhibitor XAV939 to interfere with TNF-α 40ng/ml treated A549 for 24 h,Cell survival,secretion of inflammatory factors,cell barrier permeability and tight junction protein expression were measured.4.Target Scan Human 8.0 database predicts the binding site of miR-186-5p to wnt5 a,and dual luciferase reporter gene assay verifies the target relationship.qRT-PCR and Western Blot were performed to detect the expression of the wnt5a/ β-catenin signaling pathway after transfected by miR-186-5p mimic / inhibitor.After transfection of miR-186-5p mimic into A549 with simultaneous application of wnt pathway activator Licl,TNF-α40ng/ml treated A549 for 24 h,Cell survival,secretion of inflammatory factors,cell barrier permeability and tight junction protein expression were measured.Results1.After intervention of A549 with different concentrations of TNF-α,cell survival decreased,secretion levels of inflammatory factors IL-1β,IL-18 and IL-6 increased,FITC-dextran permeability increased,and expression of ZO-1,Ocludin and Claudin-4 m RNA and protein decreased,with significant differences starting from TNF-α 40ng/ml(all P<0.05).2.qRT-PCR results showed that the expression of miR-186-5p decreased with increasing TNF-α concentration.After transfection of miR-186-5p mimic and miR-186-5p inhibitor to A549,respectively,the fluorescent photographs and qRT-PCR results showed that the transfection was effective.After increasing/decreasing the expression of miR-186-5p and intervening with TNF-α 40ng/ml,A549 cell survival increased/decreased,secretion levels of inflammatory factors IL-1β,IL-18,IL-6 decreased/increased,FITC-dextran permeability decreased/increased,and protein of ZO-1,Ocludin,Claudin-4expression was increased/decreased(all P<0.05).3.qRT-PCR and Western Blot result showed that the content of wnt5 a m RNA,β-catenin m RNA and tthe protein expression of wnt5 a,β-catenin,cyclin D1 of wnt5a/β-catenin signaling pathway increased under different concentrations of TNF-α intervention.After applying XAV939 intervention to TNF-α 40ng/ml treated A549 for 24 h,the survival rate of A549 cells increased,the secretion levels of inflammatory factors IL-1β,IL-18,IL-6 decreased,the permeability of FITC-dextran decreased,and the protein expression of ZO-1,Ocludin,Claudin-4 increased(all P<0.05).4.miR-186-5p was predicted to bind to wnt5 a by Target Scan Human 8.0database,and the results of dual luciferase reporter gene assay showed a target relationship between miR-186-5p and wnt5 a.The reduction / increase of wnt5 a m RNA,β-catenin m RNA content after overexpression / inhibition of miR-186-5p,and the protein expression showed a decrease / increase of wnt5 a,β-catenin,and cyclin D1.TNF-α 40ng/ml treatment of A549 cells transfected with miR-186-5p mimic and applied Licl showed that compared with MI group,the cell survival rate of MI+Licl group was decreased,increased secretion levels of inflammatory factors IL-1β,IL-18,IL-6,increased FITC-dextran permeability,and decreased protein expression of ZO-1,Ocludin,Claudin-4(all P<0.05).Conclusion1.miR-186-5p can participate in regulating the permeability of the alveolar epithelial barrier by regulating the expression of the human alveolar epithelial barrier tight junction proteins ZO-1,Ocludin,and Claudin-4.2.miR-186-5p can regulate alveolar epithelial barrier permeability by targeting wnt5 a and negatively regulating the expression of wnt5a/β-catenin signaling pathway.