The Role And Mechanism Of MircroRNA-130a In Cardiac Fibrosis After Myocardial Infarction

Objective: Studies have shown that multiple micro RNAs are involved in the development of cardiac fibrosis.micro RNA-130a(miR-130a)has been reported to play an important role in a variety of fibrotic diseases.However,its role and specific mechanisms in cardiac fibrosis are unclear.To investigate the role of miR-130 a in cardiac fibrosis after myocardial infarction,we conducted this study to explore the role and specific mechanism of miR-130 a in myocardial fibrosis after myocardial infarction.Methods: Adult C57BL/6J male mice were randomly divided into control group,MI group(myocardial infarction group)and Sham group(sham-operated group).The myocardial infarction model was established by ligation operation in the left anterior descending branch of the coronary artery in mice.The sham-operated model was constructed by threading only the anterior descending branch of the coronary artery without ligation at the same location in mouse heart.The hearts of mice were removed two weeks after the infarction surgery,Masson staining was performed to observe the collagen deposition.Adult C57BL/6J mice were randomly divided into Sham group,MI group,MI+NC group(myocardial infarction model group transfected with empty virus particles)and MI+H-miR-130 a group(myocardial infarction model group transfected with adeno-associated virus 9 carrying miR-130 a plasmid).Mice in the MI+NC group were pre-injected with 100 μl of empty virus particles at a titer of 1×10^12 vg/m L by murine tail vein injection 3 weeks before the coronary ligation operation.Mice in the MI+H-miR-130 a group were pre-injected with 100 μl of adeno-associated virus 9 with miR-130 a plasmid at a titer of 1×10^12 vg/m L by murine tail vein injection 3 weeks prior to the coronary ligation operation.100 μl of sterile saline was injected with Sham group and MI group.After 2 weeks of surgical operation,cardiac function was measured by cardiac ultrasound in all four groups of mice separately,and then the mice were anesthetized and executed and hearts were obtained to measure the area of cardiac fibrosis by Masson staining.The possible target genes of miR-130 a were screened by eukaryotic transcriptome(with reference)sequencing assay,and the relationship between miR-130 a and the target genes was confirmed by dual luciferase reporter assay.Hypoxia(0.3%,24h)treated primary neonatal mouse cardiac fibroblasts,while using adenovirus as a vector,regulated the expression of miR-130 a in cardiac fibroblasts.RT-q PCR and WB were performed to detect the levels of miR-130 a,transforming growth factor β(TGF-β),transforming growth factor β receptor 1(TGFBR1),α-smooth muscle cell actin(α-SMA),collagen type 1(Col-1),p-Smad3 and t-Smad3.TGFBR1 and miR-130 a were co-transfected into cardiac fibroblasts to investigate the role of TGFBR1.Results:(1)RT-q PCR results showed that miR-130 a expression was significantly reduced in the infarcted myocardium after myocardial infarction.Masson staining results showed that collagen deposition was significantly increased in the myocardial infarct area of mice in MI group.WB and RT-q PCR results showed that α-SMA,TGF-β,TGFBR1,Col-1 expression was significantly increased in the myocardium of MI group mice.(2)The results of cardiac ultrasound showed that cardiac function was significantly impaired,and EF(ejection fraction)and FS(fractional shortening)were significantly reduced,with enlarged end-diastolic volume(EDV)and end-systolic volume(ESV)in post-MI mice.High expression of miR-130 a could reduce the impaired cardiac function and increase the EF and FS after myocardial infarction,and also reduce EDV and ESV.Masson staining revealed that the heart chambers of mice after myocardial infarction were significantly enlarged,the fibrosis scar replaced the original myocardium,and the ventricular walls were thinned.And fibrosis area after myocardial infarction was significantly reduced in the MI+H-miR-130 a group of mice compared with other groups.(3)TGFBR1 expression was significantly upregulated in hypoxia-treated cardiac fibroblasts,and its expression was inhibited by miR-130 a.Dual luciferase reporter assay showed that Tgfbr1 was a target gene of miR-130 a.RTq PCR and WB results showed that Col-1,α-SMA,TGF-β and TGFBR1 were significantly up-regulated,with elevated p-Smad/t-Smad in cardiac fibroblasts after hypoxia treatment.Under normoxia,miR-130 a inhibition upregulated Col-1,p-Smad/t-Smad,α-SMA and TGFBR1.After hypoxia treatment,overexpressed miR-130 a suppressed hypoxia-induced upregulation of Col-1,α-SMA,TGFBR1 and p-Smad3/t-Smad3.Regulation of miR-130 a expression does not change TGF-β Level.Co-transfection of TGFBR1 reversed the inhibitory effect of miR-130 a on α-SMA and Col-1.Conclusion: miR-130 a could target TGFBR1 to regulate TGF-β Signal pathway,inhibiting the conversion of cardiac fibroblasts to myofibroblasts,and alleviate cardiac fibrosis,improving cardiac function after myocardial infarction.