The Role And Mechanism Of MiR-543 In Myocardial Fibrosis In Diabetic Cardiomyopathy

BackgroundDiabetic cardiomyopathy(DCM)refers to the myocardial structure and function disorders that occur in diabetic patients and cannot be explained by hypertensive heart disease,coronary heart disease or other cardiac diseases.DCM is the main cause of high morbidity and mortality of heart failure(HF)in diabetic patients,but there is still a lack of effective treatment for DCM.Therefore,it is important to further study the exact mechanism of DCM occurrence and development.MicroRNAs(miRNAs)are a class of endogenous single-stranded small RNAs that are highly conserved in evolution.Their main function is to negatively regulate gene expression by binding to the 3’UTR region of the target mRNA and inducing degradation or inhibiting translation subsequently.Recently,miRNAs have emerged as potential biomarkers for the diagnosis,treatment,and prognosis of diabetes mellitus and its complications.At present,the specific role and molecular mechanism of miRNAs in myocardial fibrosis in DCM remains unclear.In this study,bioinformatics analysis of chip data was conducted to screen multiple differentially expressed miRNAs,among which miR-543 was closely associated with insulin resistance(IR)and diabetes.Due to the key role of insulin resistance in myocardial fibrosis in DCM,miR-543 may serve as a bridge among insulin resistance,myocardial fibrosis and DCM.This study examined the expression of miR-543 in cardiac tissue of DCM mice and insulinresistant cardiac fibroblasts(CFs),and investigated the effect of miR-543 on myocardial fibrosis in DCM and its possible mechanism.Objectives(1)To detect the expression of miR-543 in the myocardium of DCM mice and explore its correlation with myocardial fibrosis.(2)To study the expression of miR-543 in insulin-resistant CFs and its possible role in phenotypic transformation and collagen expression of CFs.(3)To investigate the mechanism of miR-543 in myocardial fibroblasts in diabetic cardiomyopathy.Methods(1)Differential expression analysis of chip data was performed with R software.(2)Combined application of high-fat diet and streptozocin was used to construct type 2 diabetes mouse model.(3)Cardiac function of mice was monitored by echocardiography.(4)Myocardial fibrosis was detected by HE staining,Masson staining and immunohistochemical staining.(5)RT-PCR was used to detect the expression level of miR-543.(6)Combined application of high glucose and high insulin was used to construct the cell model of insulin resistance.(7)RT-PCR and Western blot were used to detect the expression level of PTEN,α-SMA,Collagen I and Collagen III.(8)EdU kit was performed to detect cell proliferation level.(9)Transwell and scratch tests were performed to detect cell migration level.(10)Dual luciferase assay was used to verified the targeting relationship between PTEN and miR-543.Results(1)Analysis of differentially expressed miRNAsBioinformatics analysis of 8 samples of GSE17035 revealed 36 up-regulated miRNAs and 22 down-regulated miRNAs.Literature review found that miR-543 was was associated with diabetes and insulin resistance among up-regulated miRNAs.Therefore,miR-543 was selected as the research target.(2)Establishment of DCM mouse modelCompared with the control group,left ventricular end-diastolic diameter(LVEDd)and E/E’ in diabetic group were significantly increased,while left ventricular ejection fraction(LVEF)and fractional shortening(FS)were significantly decreased.The results indicate that DCM mouse model has been established successfully.(3)The degree of myocardial fibrosis increased in DCM miceCompared with the control group,collagen volume fraction(CVF)and protein expression level of Collagen I and III were significantly increased in DCM group.(4)The expression level of miR-543 was increased in DCM miceCompared with the control group,the expression level of miR-543 in myocardial tissue of DCM mice was increased.(5)The degree of myocardial fibrosis was positively correlated with the expression level of miR-543 in myocardial tissueThere was a positive correlation between collagen volume fraction and expression level of miR-543 in myocardial tissue(P<0.01).There was a positive correlation between protein expression level of Collagen I and expression level of miR-543 in myocardial tissue(P<0.01).(6)Identification of primary myocardial fibroblastsThe cells were fusiform in morphology,uniform in size,without spontaneous pulsation,and vimentin fluorescence staining was positive.(7)Establishment of myocardial fibroblast model of insulin resistanceCFs model of insulin resistance was constructed by the combination of high glucose and high insulin.Results showed that IRS-1/AKT signaling pathway was impaired and Glut4 membrane translocation decreased.These results suggested that insulin resistance model was successfully established.(8)The expression level of miR-543 was increased in insulin-resistant cardiac fibroblastsThe expression level of miR-543 in CFs was the highest when insulin resistance was stimulated for 48 h,and 48 h of insulin resistance was taken as the experimental condition of subsequent CFs model to induce insulin resistance.(9)The expression level of miR-543 was inhibited by miR-543 inhibitor significantlyCompared with the control group,the expression level of miR-543 in miR-543 inhibitor group was significantly decreased.(10)Inhibition of miR-543 expression reduced the proliferation of insulin-resistant cardiac fibroblastsCompared with the control group,the proliferation level of CFs in the insulin-resistant group was significantly increased,while the proliferation level of CFs in the insulin-resistant group was significantly decreased after miR-543 inhibitor inhibited the expression of miR-543.(11)Inhibition of miR-543 expression reduced the migration of insulin-resistant cardiac fibroblastsCompared with the control group,the migration level of CFs in the insulin-resistant group was significantly increased,while the migration level of CFs in the insulin-resistant group was significantly decreased after miR-543 inhibitor inhibited the expression of miR-543.(12)Inhibition of miR-543 expression reduced the transformation of insulin-resistant cardiac fibroblasts into myofibroblasts and collagen expressionCompared with the control group,the mRNA and protein expression level of α-SMA,Collagen I and Collagen III in CFs in insulin resistance group were significantly increased,while the expression level of above indicators were significantly decreased after miR-543 inhibitor inhibited the expression of miR-543.(13)Screening of miR-543 target genesThe intersection of TargetScan、miRDB、miRWalk、microT and PITA、miRanda、miRmap in Starbase database was used to obtain 14 predicted target genes.Among them,PTEN is closely associated with fibrosis,so PTEN may mediate the pro-fibrotic effect of miR-543.(14)The expression level of PTEN was decreased in DCM mice and insulin-resistant CFsCompared with the control group,the mRNA and protein expression level of PTEN were decreased in DCM group.Compared with the control group,the mRNA and protein expression level of PTEN in insulin-resistant CFs were significantly decreased.(15)PTEN is the target gene of miR-543Compared with the control group,miR-543 mimic decreased the luciferase activity of WTPTEN,but had no significant effect on luciferase activity of MUT-PTEN.Compared with the insulin resistance group,the mRNA and protein expression level of PTEN were increased after miR-543 inhibitor inhibited the expression of miR-543.These results indicate that PTEN is the target gene of miR-543.(16)SiPTEN significantly down-regulated PTEN expressionCompared with the control group,the three kinds of siPTEN significantly reduced the expression levels of PTEN mRNA and protein.(17)MiR-543 regulates cell proliferation by targeting PTENCompared with IR group,the proliferation level of CFs was significantly decreased after miR-543 inhibitor inhibited the expression of miR-543.Compared with IR+miR-543 inhibitor group,the proliferation level of CFs in IR+miR-543 inhibitor+siPTEN group was significantly increased.(18)MiR-543 regulates cell migration by targeting PTENCompared with IR group,the migration level of CFs was significantly decreased after miR-543 inhibitor inhibited the expression of miR-543.Compared with IRR+ miR-543 inhibitor group,the migration level of CFs in IR+miR-543 inhibitor+ siPTEN group was significantly increased.(19)MiR-543 regulates the transformation of cardiac fibroblasts into myofibroblasts and collagen expression by targeting PTENCompared with IR group,the mRNA and protein expression level of α-SMA,Collagen I and Collagen Ⅲ in CFs were significantly decreased after miR-543 inhibitor inhibited the expression of miR-543.Compared with IR+miR-543 inhibitor group,the expression level of the above indicators in CFs in IR+miR-543 inhibitor+siPTEN group was significantly increased.Conclusion(1)The level of myocardial fibrosis in diabetic cardiomyopathy mice was increased,accompanied by up-regulated expression of miR-543.(2)Inhibition of miR-543 expression can reduce the transformation of cardiac fibroblasts into myofibroblasts and collagen expression under insulin resistance.(3)MiR-543 regulates the transformation of cardiac fibroblasts into myofibroblasts and collagen expression under insulin resistance by targeting PTEN.