Mir-214-3p Inhibits Cardiomyocyte Hypertrophy By Targeting Myocyte Enhancer Factor 2c

Increased morbidity and mortality of cardiovascular disease have constitute a significant economic burden around the world.Cardiac hypertrophy is the essential ingredient of the Cardiac remodeling,and which is the common complications of cardiovascular disease.The prolonged hypertrophy has potentially disastrous consequences leading a decline in ventricular function and ultimately to heart failure and death.Recent studies have demonstrated that miRNAs are involved in the pathological process of cardiac hypertrophy,and play an important role.Objective:The role of microRNA-214-3p(miR-214-3p)in cardiac hypertrophy was not well illustrated.The present study aimed to establish the mouse model and cell model of hypertrophy,to investigate the expression and potential target of miR-214-3p in angiotensin ?(Ang-?)-induced mouse cardiac hypertrophy.Methods:1.An animal model of hypertrophy was established in mice with Ang-? infusion(1.46mg/kg/2w)for 14 days,over-expression of miR-214-3p in vivo via tail vein injection of miR-214-3p.2.Neonatal mouse ventricular cardiomyocytes(NMVCs)were isolated from the hearts of 1-3-d-old newborn C57BL6 mice.A cell model of hypertrophy was established based on angiotensin-?(Ang-?,10-8M)-induced neonatal mouse ventricular cardiomyocytes(NMVCs).3.Tissue sections were stained with Wheat germ agglutinin(WGA)solution to demonstrate the size of cardiomyocytes in mouse myocardium.4.NMVCs were stained with FITC-phalloidin solution to demonstrate the size of cardiomyocytes.5.Dual luciferase reporter assay was performed to verify the interaction between miR-214-3p and the 3'UTR of MEF2C.6.The nuclear translocation of P65 in Ang-?-treated NMVCs was detected by cell immunofluorescence staining.7.The mRNA and protein expression of the associated genes in mouse hypertrophic myocardium and NMVCs were determined by RT-qPCR and Western blotting assay,respectively.Results:1.The ratio of heart weight to tibial length was increased in Ang-?infusion model group.WGA staining results revealed that the cell size of cardiomyocytes was significantly increased in the myocardium of the mouse Ang-?infusion model compared with Shan group.Result of RT-qPCR and Western blotting showed the expression of hypertrophy-related genes(ANP,ACTA1,?-MHC)both were increased at mRNA and protein levels,while the expression of miR-214-3p was markedly decreased in the hypertrophic myocardium.Cardiac hypertrophy was attenuated in Ang-?-infused mice by tail vein injection of miR-214-3p.2.The WGA staining results showed that cell size of cardiomyocyte was markedly increased in Ang-?-induced NMVCs.Our RT-qPCR and WB resulted that the mRNA and protein expression of ANP,ACTA1 and ?-MHC both were significantly increased in Ang-?-treated NMVCs.Consistently,miR-214-3p also markedly increased in Ang-?-treated NMVCs.3.Myocyte-specific enhancer factor 2C(MEF2C),which was increased in Ang-?-induced hypertrophic mouse myocardium and cardiomyocytes,was identified as a target gene of miR-214-3p.4.Functionally,miR-214-3p mimic,consistent with MEF2C siRNA,inhibited cell size increase and protein expression of ANP and ?-MHC in Ang-?-treated mouse cardiomyocytes.5.The nuclear translocation of P65 was increased in Ang-?-treated NMVCs,Our western blot results showed that Ang-? could activate the NF-?B pathway and the phosphorylation level of NF-?B P65 was significantly increased in NMVCs.6.miR-214-3p expression was enhanced after activating the NF-?B P65 pathway,while inactivating the NF-?B P65 pathway could attenuated Ang-?-stimulated miR-214-3p expression in NMVCs.Conclusion:1.we have successfully established the animal model of hypertrophy.And the expression miR-214-3p was markedly decreased in the hypertrophic myocardium.However,Cardiac hypertrophy was attenuated in Ang-?-infused mice by enforced enhancement of miR-214-3p via tail vein injection of miR-214-3p agomir,miR-214-3p had protective effects on the cardiac hypertrophy.2.A cell model of Ang-?-induced hypertrophy was established in NMVCs,Ang-? could induced hypertrophic phenotype and the expression miR-214-3p was markedly increased in Ang-?-induced NMVCs,we also found over-expression of miR-214-3p could inhibited the mRNA expression of hypertrophy-related genes.3.MEF2C is a direct target of miR-214-3p in cardiomyocytes hypertrophy,and miR-214-3p inhibits cardiomyocyte hypertrophy via suppressing MEF2C at transcriptional level in NMVCs.4.NF-?B pathway was activated in Ang-?-induced NMVCs.The up-regulation of miR-214-3p in Ang-?-induced hypertrophic cardiomyocytes results from activation of the NF-?B pathway.