Non-coding RNA CircRNA₁00395 Inhibits Myocardial Fibrosis Via Sponging MiR-144-3p

Persistent atrial fibrillation?AF?can lead to atrial electrical remodeling and fibrosis.Current research shows that's a very complex pathophysiological process involving many factors,including oxidative stress,non-coding RNA,calcium overload,atrial dilation,inflammation and activation of myofibroblasts;Myocardial fibrosis plays a key role in the production and development of AF.Myocardial fibrosis causes excessive deposition of extracellular matrix,which is an important pathological feature of AF;Emerging evidence has shown that circular RNA is involved in the development and progression of many cardiovascular diseases,and play an important role in the regulation of the disease.Aim : To investigate the effect of circ RNA₁00395 on the expression of fibrosis-related genes in human atrial fibroblasts?HAFs?and its specific mechanism.Methods: 1.Western-blot and Masson staining were performed to detect the level of myocardial fibrosis in the left atrial appendage of patients with long-standing persistent AF;2.Circ RNAs microarray was used to detect the differential expression of circ RNAs in left atrial appendage of patients with long-standing persistent AF.RT-q PCR was performed to verify the expression of circ RNA₁00395 and identify the distribution of nucleoplasm;3.HAFs were isolated and cultured in vitro and identified by ACTA2 immunofluorescence staining;4.Angiotensin II?Ang-II?induces fibrosis of HAFs,and detected the expression of fibrosis-related genes,circ RNA₁00395 and its host gene KLHL20 by RT-q PCR;5.Adenovirus-mediated overexpression of circ RNA₁00395 in HAFs.CCK-8 and Transwell assays examined the effects of circ RNA₁00395 on the proliferation and migration of HAFs,and RT-q PCR and Western-blot assays the effect of circ RNA₁00395 on fibrosis-related gene expression;6.Bioinformatics predicts micro RNAs that bind to circ RNA;7.RT-q PCR was performed to detect the expression of mi R-144-3p in HAFs overexpressed circ RNA₁00395;8.Dual luciferase reporter gene assay,RNA antisense purification technology?RAP?and RNA pull-down assay were performed to detect the binding of circ RNA₁00395 to mi R-144-3p;9.RT-q PCR and Western-blot were used to detect the expression of fibrosis-related genes and proteins in HAFs after transfection of mi R-144-3p;10.Overexpressed circ RNA₁00395 and mi R-144-3p in HAFs,Western-blot was used to detect the expression of fibrosis-related genes and the activation of smad3 signaling pathway.Result: 1.The degree of fibrosis of myocardial tissue in left atrial appendage of patients with long-standing persistent AF increased significantly;2.Expression of circ RNA₁00395 in left atrial appendages of patients with long-standing persistent AF was significantly reduced,and mainly distributed in the cytoplasm;3.The HAFs isolated from the left atrial appendage of patients with long-standing persistent AF were myofibroblasts expressing the ACTA2 phenotype,which was suitable for the study of fibrotic phenotype;4.In the Ang-II-induced fibrosis cell model of HAFs,the expression of fibrosis-related genes was up-regulated,and the expression of circ RNA₁00395 and host gene KLHL20 was significantly increased;5.Overexpression of circ RNA₁00395 significantly reduced the expression of fibrosis-related genes,proliferation and migration ability;6.Bioinformatics predicted the potential binding sites of mi R-144-3p in circ RNA₁00395.Dual luciferase reporter assay,RAP and RNA pull-down assay confirmed circ RNA₁00395 binding to mi R-144-3p;7.Overexpressed mi R-144-3p in HAFs,fibrosis-related gene expression was up-regulated and smad3 pathway was activated,while overexpression of circ RNA₁00395 eliminated profibrotic effect of mi R-144-3p.Conclusion: 1.Circ RNA₁00395 was up-regulated in the left atrial appendage of patients with long-standing persistent AF;2.Circ RNA₁00395 inhibited the expression of fibrosis-related genes in HAFs and reduce the proliferation and migration ability of HAFs;3.Circ RNA₁00395 binded to mi R-144-3p;4.Mi R-144-3p is involved in the regulation of circ RNA₁00395 inhibition of fibrosis-related gene expression in HAFs.