Mechanism Study Of Circular RNA CircRNA＿100395 Inhibiting Myocardial Fibrosis Via Spondging MiR-144-3p In Cardiac Fibroblasts

Cardiovascular disease(CVD)has become a serious public health problem.Myocardial fibrosis(MF)is an important pathophysiological change in heart failure.Circular RNA(circRNA)is a kind of long noncoding RNA with closed loop,and a number of studies have showed that circRNAs were involved in the occurrence and development of cardiovascular diseases.This study investigated the expression of circRNA＿100395 in myocardial fibrosis of heart failure patients,and its effect on myocardial fibrosis and the mechanism involved.Aim : To investigate the regulation and mechanism of circRNA＿100395 in heart failure.Methods: 1.Masson staining and RT-q PCR were performed to detect the level of myocardial fibrosis and circRNA＿100395;2.Mouse myocardial fibroblasts(m CFs)and human atrial myofibroblasts(HAFs)were primary isolated and phenotypic identification was performed.3.AC16 cells were treated with angiotensin II(Ang-II),and circRNA＿100395expression was detected by RT-q PCR.4.Adenovirus-mediated overexpression of circRNA＿100395 was performed in myofibroblasts,Trans-well assay,EDU assay and flow cytometry were used to detect the proliferation and migration ability of myofibroblasts.5.Expression of relative fibrosis gene in myofibroblasts was detected by RT-q PCR and Western blot after overexpression of circRNA＿100395.6.Bioinformatics predicted mi RNAs interacting with circRNA＿100395,and double luciferase report assay was performed to verify their mutual binding ability.7.Relative fibrosis gene expression and Smad signaling pathway were detected by Western blot after transfection with mi R-144-3p in m Cfs;8.Western blot detection of fibrosis related genes in m CFs after co-transfection with mi R-144-3p and circRNA＿100395;9.Mutation or deletation of the binding site between circRNA＿100395 and mi R-144-3p was performed to detect the expression of fibrosis related genes by Western blot.10.Bioinformatics prediction of the coding genes interacting with mi R-144-3p,and verified the binding ability with dual luciferase report assay;11.After transfection with mi R-144-3p or overexpression of circRNA＿100395,the expression of TGIF1 was detected by RT-q PCR and Western blot.12 Silencing or overexpression of TGIF1,Western blot detection of fibrosis related genes and Smad signaling pathway.Result: 1.The degree of fibrosis in heart failure tissue was increased,and the expression of circRNA＿100395 was up-regulated;2.HAFs and m CFs isolated from primary generation had high purity and could be used for the study of fibrosis phenotypes;3.The expression of circRNA＿100395 was down-regulated in the myocardial fibrosis cell model;4.circRNA＿100395 inhibits m CFS proliferation and migration;5.CircRNA＿100395 inhibits the expression of fibrosis related genes;6.CircRNA＿100395 can bind with mi R-144-3p;7.mi R-144-3p promoted the expression of fibrosis related genes and activated the Smad signaling pathway;8.CircRNA＿100395 can inhibit the fibrosis promoting ability of mi R-144-3p;9.After binding site mutation or deletation,circRNA＿100395 decreased,even lose its ability to inhibit fibrosis;10.TGIF1 could interact with mi R-144-3p;11.Transfection of mi R-144-3p inhibited the expression of TGIF1,and overexpression of circRNA＿100395 promoted the expression of TGIF1.12.Silencing TGIF1 promoted fibrosis gene expression and activated Smad signaling pathway consistenting with the effect of mi R-144-3p transfection.Overexpression of TGIF1 could inhibit myocardial fibrosis and inhibit Smad signaling pathway,which was consistent with overexpression of circRNA＿100395.Conclusion: 1.The expression of circRNA＿100395 is up-regulated in the myocardium of heart failure patients,and circRNA＿100395 inhibits Smad3 activation and fibrosis-related gene expression in CFs;2.circRNA＿100395 spondges mi R-144-3p to enhance TGIF1 expression,contributing to inhibiting fibrosis-related gene expression in CFs.