MiR-30a Inhibits Cardiac Fibrosis Induced By Hypoxia Via Targeting CTGF On Cardiac Fibroblasts

Background:CTGF(connective tissue growth factor)is an important fibrosis related cytokines,which acts as efficient response element of TGF-β1(transforming growth factor β1)signal downstream and specifically mediates TGF-β1induced myocardial fibrosis.A recent study has shown that miR-30a(miRNA-30a)involved in the process of post-infarction myocardial fibrosis.miR-30a expression variation trend is contrary to the changing tendency of TGF-β1,CTGF expression level,and Ⅰ,Ⅲcollagen level.It indicated that miR-30a might through regulating the transcription and translation process of TGF-β1 and CTGF to affect the process of myocardial fibrosis.Objective:This study aimed to explore the potential correlation between the expression levels of miR-30a and TGF-β1,CTGF,I,III collagen via culturing cardiac fibroblasts(CFs)in hypoxia environment which simulated myocardial infarction model in vitro.Through this method,we aimed to confirm the anti-fibrotic effect of miRNA-30a and to determine whether miR-30a inhibits cardiac fibrosis induced by hypoxia via targeting CTGF on cardiac fibroblasts.Methods:1.Rat cardiac fibroblasts(RCF)were cultured in hypoxia environment.The mRNA level of miR-30a,TGF-β1 and CTGF were detected by realtime-quantitative PCR(RT-qPCR)analysis.The protein level of TGF-β1 CTGF and a-SMA were detected by western blot(WB)analysis.2.RCF were randomly divided into 5 groups,control group,hypoxia group,hypoxia+miR-30a mimics group,hypoxia+miR-30a mimics+AMO-miR-30a group and hypoxia+miR-30a NC group.We respectively observed the expression level of TGF-β1 and CTGF.3.Rat cardiac fibroblasts(RCF)were cultured in normal condition.RCF were transfected with miR-30a mimics,miR-30a inhibitor or miR-30a NC.Then we observe the expression of CTGF mRNA and protein level.4.Dual-luciferase reporter gene assay.We conducted the CTGF gene combination dual luciferase expression vector,CTGF-3’-UTR wild type(CTGF-3’-UTR-WT)and then co-transfected it with miR-30a to 293T cells.Moreover,we conducted combination dual luciferase expression vector with CTGF mutant gene,CTGF-3’-UTR mutant type(CTGF-3’-UTR-MU)and then co-transfected it with miR-30a to 293T cells.Negative control groups were established with miR-30a NC(scramble siRNA).Whether CTGF is a direct target of miR-30a was detected with dual luciferase reporter gene assay system.Result:1.Cultured RCF treated with hypoxia were characterized by the up-regulation of TGF-β1,CTGF,a-SMA,Ⅰ,Ⅲ collagen compared with control group(P<0.01)and down-regulation of miR-30a(P<0.01).2.The up-regulation of TGF-β1,CTGF,a-SMA,Ⅰ,Ⅲ collagen was significantly suppressed in hypoxia+miR-30a mimics group compared with hypoxia+miR-30a NC group(P<0.05).However,the suppression was abrogated by co-transfection with AMO-miR-30a compared with NC group(P>0.05).3.After the elimination of hypoxia environment impact,RCF transfected with miR-30a mimics were characterized by down-regulation of CTGF(P<0.05)and RCF transfected with miR-30a inhibitor were characterized by up-regulation of CTGF(P<0.05).4.The luciferase activity was significantly suppressed in CTGF-3’-UTR-WT+miR-30a group compared with CTGF-3’-UTR-WT+miR-30a NC group or CTGF-3’-UTR-MU+miR-30a group(P<0.01).No significant change was observed of the luciferase activity in CTGF-3’-UTR-MU+miR-30a-NC group or CTGF-3’-UTR-MU+miR-30a group compared with CTGF-3’-UTR-WT+miR-30aNC group(P>0.05).The results above indicated that CTGF is a direct target of miR-30a.Conclusion:1.Hypoxia increase the level of fibrosis relative indicators and suppress the expression of miR-30a.2.MiR-30a inhibits cardiac fibrosis induced by impacting the expression of CTGF and TGF-β1.3.CTGF is a direct target of miR-30a.miR-30a exert anti-fibrosis effect by directly targeting CTGF.