The Effect And Mechanism Of MiR-30c On Atrial Fibrosis And Fibrogenisis Of Cardiac Fibroblasts Induced By Transforming Growth Factor-?1

ObjectiveMicroRNA-30c(miR-30c)is involved in fibrotic remodeling,such as ventricular,liver or kidney fibrosis.However,the specific role and mechanism of miR-30 c in atrial fibrosis remains unclear.This study was to investigate the role of miR-30c in atrial fibrosis and its underlying mechanisms.Methods In this study,we used abdominal aortic coarctation(AAC)model and Cardiac Fibroblasts(CFs)as the research object,making the study for three parts.Part 1: In AAC-induced atrial fibrosis model,the expression of miR-30 c and TGF?RII in the left atrial tissue was detected by real time PCR,Western Blot,respectively;Collagen distribution was estimated by masson staining.The AF inducibility was observed by rapid atrial pacing.Part 2 Primary CFs were isolated and cultured from left atrium tissues of neonatal rats.Transfection with miR-30 c mimic or miR-30 c inhibitor in TGF-?1-induced CFs,the effect of CFs proliferation,transformation,migration and collagen production was observed by cell count kit-8(CCK-8),real time PCR(RT-PCR),Western blot and Transwell assay.RNAhybrid and miRanda predicated that the3'UTR of TGF?RII contained the target site of miR-30 c.Using dual luciferase reporter assays,RT-PCR and Western blot to vertify if TGF?RII was a target gene of miR-30 c.Part 3 Reconbinated Adeno-associated virus 9(AAV9)-mediated transfer miR-30c(r AAV9-miR-30c)into Sprague-Dawley rats via inferior vena cava injection following AAC.The groups are including Sham(n=6),AAC(n=8),AAC+miR-30c(n=8)and AAC+Control(n=8).The AF inducibility was observed by rapid atrial pacing.Masson's trichrome staining to study the fibrosis of left atrial tissues.The expression of miR-30 c,TGF?RII,?-SMA,Col I and Col3?1was measured by RT-PCR or Western Blot.Results1.Compare to Sham group,the expression of miR-30 c was downregrulated,while the level of TGF?RII was higher in left atrial tissue of AAC group;Masson's Trichrome staining results showed higher content of collagen in left atrial tissues of AAC group.The ECG showed that P wave duration(PWD),PR interval was prolonged in AAC group.There was no difference between two groups.2.Compare to control group,the level of TGF?RII was significantly higher in the primary CFs induced by TGF-?1.However,the expression of miR-30 c was downregrulated in CFs induced by TGF-?1.Overexpression of miR-30 c in TGF-?1-induced CFs,marked inhibited cell proliferation,transformation,migration and collagen production.However,tranfection with miR-30 c inhibitor exhibited the pro-fibrotic effect.Dual luciferase assays showed that transfection of the miR-30 c mimic suppressed the luciferase activity driven by the TGF?RII3'UTR-WT;transfection of the miR-30 c mimic increased luciferase activity by the TGF?RII 3'UTR-mutant,compared to TGF?RII 3'UTR-WT.Overexpression of miR-30 c inhibited the expression of TGF?RII,while miR-30 c inhibitor incresed the expression of TGF?RII.This suggests that TGF?RII is a new target gene for miR-30 c.3.The levels of TGF?RII,Col I and Col3?1 were downregrulated in AAC+miR-30 c group,as well as well as PWD and PR interval,miR-30 c antentuated atrial fibrosis.Conclusions Taken together,miR-30 c inhibits Cardiac Fibroblast proliferation,myofibroblast differentiation,migration,collagen synthesis by targeting on TGF?RII,which can explain the mechanism of miR-30 c reduce left atrial fibrosis.The result indicates that miR-30 c is a potential target to modify atrial fibrosis and atrial fibrillation.