Study On Neuropeptide Y-induced Cardiomyocyte Hypertrophy By Regulating MiR-29a

Objective:NPY(Neuropeptide Y)is a peptide constituted by 36 amino acid,and it is widely distributed in central system and peripheral nervous system.Recently,many articles have reported the significant role of NPY in myocardial hypertrophy.Myocardial hypertrophy is a compensation with limited capacity during diseases,if the pathogenic factors are long-lasting,then it would eventually lead to decompensated heart failure.MicroRNAs are a group of endogenous small RNA constituted by 20-24 nucleotide,and have many important regulatory functions in cells.In recent reports,the complex and indispensable role of miRNA in the physiology and pathology of cardiomyocyte myocytes has always been emphasized.In this article,our research aims to identify the molecular mechanism of NPY-induced myocardial hypertrophy,to explore the role of miR-29a-3p in the process of NPY-induced myocardial hypertrophy,and to elucidate a new molecular pathway of NP Y-induced myocardial hypertrophy.Methods:In this study,the myocardial hypertrophy model induced by NPY was established in primary cardiomyocytes.After cardiomyocytes are successfully extracted,the cells were starved(with 1%FBS)for 12h,and then treated with 10nmol NPY for 24h.The cytoskeleton was stained with FITC Phalloidin(FITC Phalloidin),then we observe the cell surface change with fluorescence microscope,and the mitochondrial morphological change were observed by MitoTracker Red CMXRos.The expression of dynamin-related protein 1(Drp1)was detected by Western blot,and ANP mRNA was quantitatively analyzed by fluorescence quantitative PCR(qPCR).On this basis,we observed the corresponding change of the above indexes by regulating the(high/low)expression of miR-29a-3p,and then compared the corresponding indexes of each group through co-transfection of Inhibitor-miR-29a-3p and drp1-siRNA.Results:(1)Identification of primary cardiomyocytes from newborn SD rats After the separation and purification,the primary cardiomyocyte were cultured in vitro for 72 hours,and the cell fluctuation frequency tended to be stable.Immunofluorescence staining showed that most cells expressed cTnT,which was considered as a myocardial specific marker.These cells can be used in subsequent experiments for further treatment.That is,the cells used in this experiment were primary cardiomyocytes which were cultured in vitro for 72 hours after the isolation and purification.(2)NPY leads cardiomyocyte hypertrophy in neonatal rat primary cardiomyocytesIn our previous studies,different concentrations of NPY(0,10,20,50,100nM)were used to stimulate primary cardiomyocyte,and we found that different concentrations of NPY could stimulate the increase of ANP mRNA of myocardial embryonic gene.When the stimulus time is 24h,the concentration dependence is less than that of the 48h group.Therefore,in this study,10nM of NPY was selected as the stimulus condition for 24h.The primary cardiomyocyte treated with NPY at 10nM for 24h were stained with fluorescence,and the results confirmed the increase of myocardial cell area after NPY treatment.(3)NPY increases the expression of Drpl which regulates mitochondrial divisionCompared with the control group,the expression of Drp1 was up-regulated after treatment with NPY for 24h,which was statistically significant.After NPY treatment,the expression level of Drp1 can be inhibited when transfected with Drp1-siRNA.MitoTracker Red CMXRos was used to label the sulfhydryl in the mitochondria.The results showed that after NPY treatment,the mitochondria containing mitochondrial fragments were increased significantly.After silencing Drpl,the mitochondrial fragments were decreased.(4)NPY regulates the expression of miR-29a-3p and miR-29a-3p regulates the expression of DrplThe results of qPCR showed that the expression of miR-29a-3p decreased after treatment with 10 nmol NPY for 24h,which was statistically significant.Target scan predict that Drpl was a target of miR-29a-3p.In primary cardiomyocyte,up-regulating the expression of miR-29a-3p by transfecting miRNA mimics,the expression of Drpl was decreased.Conversely,when miR-29a-3p was down-regulated by transfecting with miR-29a-3p inhibitor,the expression of Drp1 was increased.Conclusion:(1)NPY induces high expression of Drp1 can strengthen mitochondrial fission and leads cardiomyocyte hypertrophy in neonatal rat ventricular cells.(2)miR-29a-3p inhibits Drpl.(3)miR-29a-3p relives cardiomyocyte hypertrophy by inhibiting Drp1.