MirR-431 Regulates Pulmonary Surfactant Synthesis Through Targeting TGF-?/SMAD4 Pathway

Objective:1.To screen differently expressed miRNA during rat' s lung development 2.To explore whether miR-431 regulated synthesis of pulmonary surfactant through TGF-?/SMAD4 signaling pathwayMethods:1.miRNA microarray was employed to profile miRNA expressions at three different stages[embryonic(E)Day 16(E16),E19,E21]during lung development2.Functional studies were perfomed by transfecting miR-431-5p mimic and inhibitor3.Bioinformatics tools TargetScan(www.targetscan.org)and miRBase were used to predict the targets of miR-4314.Target gene of miR-431-5p was validated by dual luciferase reporter gene,qRT-PCR and WB5.CCK8 assay and flow cytometric analysis were conducted to explore the cell growth and apoptosis after transfection with miR-431-5p mimic/inhibitor/NCResults:1.miR-431 was highly downregulated at three time points in the miRNA microarray data.Microarray date indicated that miR-431 expression showed the tendency of continous downregulation at three groups(E16,E19,E21)2.The expression of SP-A,SP-B,SP-C and SP-D could be inhibited by miR-431-5p.A549 cells were transfected by miR-431-5p mimic/mimic NC/inhibitor/inhibitor NC.QRT-PCR results showed that the mRNA of SPs could be decreased by miR-431-5p mimic in A549 cells,while the SPs levels could be increased by miR-431-5p inhibitor.WB validated the same tendency of SPs in the protein level These results indicated that miR-431-5p may have a regulating role in PS metabolism by reducing the expression of SPs3.miR-431 inhibit the expression of SMAD4 by the binding to its 3'-UTRThe results of miRBase and TargetScan showed that miR-431 was highly conserved in evolution and 3'-UTR of SMAD4 binds to miR-431 with the high score,suggesting SMAD4 might be a potential target of miR-431.We then confirmed that miR-431 directly bind to the 3'-UTR of SMAD4 by luciferase reporter gene assay We further transfected A549 cells with miR-431-5p mimic and inhibitor,and found that miR-431-5p can influence the expression of SMAD4 in the level of mRNA and protein.Above knowable,miR-431-5p regulate SMAD4 expression by targeting its 3'-UTR4.miR-431-5p promoted A549 cells proliferation and reduced cells apoptosisThe results of CCK-8 assay turned out that the proliferation of A549 cells could be accelerated by miR-431-5p mimic when compared with the control group(mimic NC).In contrast,down-regulated miR-431-5p showed inverse performance Compared with the miR-431-5p mimic NC group,the apoptosis rates in the mimic group were significantly decreased(P<0.05).However,no significant differences in apoptosis rates were observed between the miR-431-5p inhibitor group and the inhibitor NC groupConclusion:miR-431 expression showed the tendency of continous downregulation during fetal lung development.miR-431-5p acts as an endogenous regulatory factor on alveolar epithelium and promotes the proliferation of A549 cells,which may mediate the down-regulation of SPs through negatively regulate TGF-?/SMAD4 pathway These results will provide a novel target for intervention and theory for neonatal lung development diseases.