The Regulation Of MiRNA-144-3p And MiRNA-590-3p On Cardiac Fibroblasts Function

After myocardial infarction(MI),cardiac fibroblasts(CFs)proliferate massively and differentiate into myofibroblast,causing abnormal secretion of extracellular matrix(ECM),finally leading to myocardial fibrosis(MF).Cardiac fibroblasts are the most abundant type of cells accounting for about 60% to 70% of the total number of cells in human heart.They are the main effector cells in the process of myocardial fibrosis and play a key role in the metabolism of extracellular matrix.The proliferation,differentiation and migration of cardiac fibroblasts can affect the fibrosis process and is an important part of the development of myocardial fibrosis.Numerous studies have shown that MF-associated miRNAs and genes can influence the function of CFs and subsequently affect the MF process.The healthy myocardium,infractional marginal regions and infarcted zone samples were collected respectively for miRNAs transcriptome sequencing,from which MI-related miRNAs were obtained,and miR-144-3p,miR-590-3p were screened as candidate miRNAs.In this study,the miR-144-3p,miR-590-3p and target gene biological function of cardiac fibroblasts were investigated by transwell assay,EdU fluorescence microscopy,qRT-PCR and western blot.The results were helpful for better understanding the mechanism of miRNAs regulation on myocardial fibrosis development.The main results and conclusions are as follows:1.The EdU fluorescence microscopy test results show that,miR-144-3p could promote cardiac fibroblasts proliferation,while miR-590-3p could inhibit cardiac fibroblasts proliferation.Transwell inserts were used to further detect if miRANs could regulate the migration of CFs.The results showed that miR-144-3p-transfected CFs promoted migration activity compared with the control groups,while miR-590-3p reduced migration activity compared with the control ones.Overexpression of miR-144-3p increased the expression of ?-SMA,Col1A1,Col3A1.Overexpression of miR-590-3p was shown to correlate with the reduction in ?-SMA,Col1A1,Col3A1.2.By using Targetscan,Pitar and RNAHybrid database and bioinformatics analyses,we identified PTEN as miR-144-3p target gene,ZEB1 as miR-590-3p target gene.The Dualluciferase reporter assays showed that miR-144-3p could bind to the 3'UTR of PTEN gene,and miR-590-3p could bind to the 3'UTR of ZEB1 gene;qRT-PCR and western blotting further identified that PTEN was directly and negatively regulated by miR-144-3p at both post-transcriptional and translation levels,while miR-590-3p only negatively regulated the translation of ZEB1.3.Inhibiting the expression of the target gene PTEN promoted the proliferation and migration of CFs,indicating that the function of PTEN is opposite to the miR-144-3p;and down-regulating the target gene ZEB1 inhibited the proliferation,cell migration of CFs,indicating that the function of ZEB1 also opposite to the function of miR-590-3p.To explore the regulation function of miR-144-3p and miR-590-3p in cardiac fibroblasts,Our findings may provide new insights into the mechanisms underlying cardiac fibrosis and provide novel potential therapeutic targets for cardiac fibrosis after MI.