Mechanism Of MiR-574-5p-regulated Differentiation In Cardiac Fibroblasts

Background Acute myocardial infarction is one of the main causes of sudden human death.Although the current diagnosis and treatment technology for myocardial infarction has been improved,there is no clear treatment for myocardial fibrosis that occurs after myocardial infarction.Myocardial fibrosis leads to a decrease in myocardial compliance,which in turn leads to cardiac dysfunction,and progresses to heart failure.Therefore,studying the mechanism of myocardial fibrosis after myocardial infarction and developing new therapeutic targets are of great significance for improving the quality of patients’ life and reducing mortality.The most important pathological process of myocardial fibrosis after myocardial infarction is that cardiac fibroblasts differentiate into myofibroblasts and secrete a large amount of extracellular matrix.After cardiac fibroblasts are activated and differentiated into myofibroblasts,α-smooth muscle actin(α-SMA)is highly expressed while their proliferative and migrative abilities are also significantly enhanced.At the same time,they secrete excessive extracellular matrix such as collagen I and fibronectin.Therefore,preventing the activation and differentiation of fibroblasts and studying their molecular mechanisms are effective strategies for treating myocardial fibrosis after myocardial infarction.Method Transforming growth factor-β(TGF-β)was used to stimulate human cardiac fibroblasts to detect mi R-574-5p gene expression levels by RT-q PCR.The inhibitor and mimic of mi R-574-5p were transfected into cells by Lipofectamine transfection technology,and then the level of human cardiac fibroblasts differentiation was detected.Changes in m RNA and protein expression of fibrosis-related genes were detected by RT-qPCR technology and Western blot technology,and changes in cell proliferative and migrative abilities were observed by using EDU staining technology,wound healing assay and Transwell migration assay.In order to further verify the mechanism of mi R-574-5p regulating the differentiation of human cardiac fibroblasts,we try to find downstream target genes in mi RNA-related databases,and confirm by RT-q PCR,Western blot and dual luciferase reporter assay.Finally,it was verified by further experiments that mi R-574-5p accelerates the differentiation of human cardiac fibroblasts by regulating the downstream ARID3 A gene expression and ARID3 A is a significant key factor of cardiac fibrosis pathological progress after myocardial infarction.Result It was demonstrated that mi R-574-5p expression was significantly up-regulated upon TGF-β stimulation 24 hours.After transfection with mi R-574-5p inhibitor,the fibrosis related gene(α-sma,COL1A1,FN1)m RNA and protein levels of human cardiac fibroblasts were reduced after TGF-β stimulation.By EDU staining assay,wound healing assay and Transwell migration assay,it was verified that the proliferative ability and migrative ability of human cardiac fibroblasts in mi R-574-5p knockdown group were significantly lower than those in negative control group.However,Mi R-574-5p overexpression had completely opposite experimental results.Therefore,we confirmed that mi R-574-5p promotes the conversion of human cardiac fibroblasts into myofibroblasts.In order to study the molecular mechanism of mi R-574-5p,we studied the downstream target genes in the mi RNA-related database and found that the ARID3 A gene was included in the both three mainstream databases.Furthermore,by RT-q PCR,Western blot,and dual luciferase reporter gene assay,we confirmed that ARID3 A is a functional downstream target gene of mi R-574-5p.To further clarify the role of ARID3 A in mi R-574-5p regulated cardiac fibrosis,we designed a reverse experiment and demonstrated that depletion of ARID3 A gene can reverse the anti-fibrotic effect of mi R-574-5p inhibitor.Conclusion TGF-β can induce the differentiation of HCFs into myofibroblasts and promote the expression of mi R-574-5p.Furthermore,ARID3 A,which is the direct target gene of mi R-574-5p,was down-regulated in HCFs and the down-regulation of ARID3 A could accelerate the differentiation of HCFs.Hence,mi R-574-5p and ARID3 A could be a new therapeutic target of cardiac fibrosis after myocardial infarction.