| BackgroundPathological cardiac hypertrophy is a cardiac pathological change secondary to hypertension,myocardial infarction,mechanical pressure overload,arrhythmia,and excessive activation of neuroendocrine hormone.It is an independent risk factor of heart failure and sudden cardiac death.Oxytocin(OT)is a cardiovascular homeostatic hormone,which can alleviate ischemia-reperfusion(I/R)injury and atherosclerosis and inhibit the development of diabetic cardiomyopathy.A recent study shows that oxytocin can also alleviate cardiac hypertrophy and fibrosis when it exerts a protective effect against myocardial ischemia and infarction.However,there is few study on the protective effect and mechanism of oxytocin on pathological cardiac hypertrophy.At present,the protective effect of oxytocin on cardiac hypertrophy and the specific molecular mechanism are still unclear.The ratio of non-coding RNA to eukaryotic genomes is approximately 98%,including long non-coding RNA,microRNA and circular RNA,which play pivotal regulatory roles in the progression of cardiac hypertrophy.LncRNA-miRNA-mRNA forms a regulatory network that transmit the intracellular hypertrophic signaling by a competing endogenous RNA mechanism.Our research group found that oxytocin protects against myocardial ischemia/reperfusion injury along with the significant down-regulation of miR-375-3p.Previous studies have reported that down-regulation of miR-375-3p can alleviate cardiac hypertrophy.Therefore,we hypothesized that the anti-hypertrophic effects of oxytocin may be related to the down-regulation of miR375-3p.At the same time,we found that lncRNA GAS5 is the upstream gene of miR375-3p and KLF4 is the target gene of miR-375-3p based on bioinformatics analysis.In this study,we observed the effects of oxytocin on cardiac hypertrophy in vivo and in vitro and investigated the molecular mechanism of anti-hypertrophic effects of oxytocin through regulating lncRNA GAS5/miR-375-3p/KLF4 axis and PI3K/AKT signaling pathway.MethodsPart Ⅰ:The experiments on anti-hypertrophic and anti-fibrosis effects of oxytocin in isoproterenol(ISO)-treated rats:Male Sprague-Dawley(SD)rats received a daily subcutaneous injection of ISO,with or without oxytocin treatment.Rats were randomly divided into six groups:Control(n=6),ISO(n=8),ISO+OT(0.03μg/kg)post(n=8),ISO+OT(3 μg/kg)post(n=8),ISO+OT(3 μg/kg)pre(n=8),and OT(3 μg/kg)(n=6);Transthoracic echocardiograms were performed on rats to evaluate changes in cardiac morphology and functions.The rat body weights and heart weights were measured to calculate heart weight/body weight ratio(HW/BW);Cardiac histopathological changes were assessed by H&E,WGA,and Masson stainings.The expressions of cardiac hypertrophy and fibrosis markers(BNP,β-MHC and α-SMA)mRNA and lncRNA GAS 5 were detected by qRT-PCR.Part Ⅱ:The experiments on anti-hypertrophic effects and mechanism of oxytocin in isoproterenol-treated cardiomyocytes:Rat primary cultured cardiomyocytes were stimulated with ISO(10μM)to induce cardiomyocyte hypertrophy.The treatment group was co-incubated with oxytocin(10 nM)for 24 h;Morphological changes of cardiomyocyte were observed by H&E staining;The expressions of lncRNA GAS5,miR-375-3p,and KLF4 mRNA were detected by qRT-PCR;The expressions of BNP,β-MHC,KLF4,PI3K,p-PI3K,AKT1,and p-AKT1 proteins were detected by Western blot.Part Ⅲ:The experiments on molecular mechanism of anti-hypertrophy effects of oxytocin via lncRNA GAS5/miR-375-3p/KLF4 axis and PI3K/AKT signaling pathway:Dual-luciferase reporter gene assays were performed to validate the interactions between lncRNA GAS5 and miR-375-3p,as well as miR-375-3p and KLF4;The cells were transfected with shRNA GAS5 to down-regulate lncRNA GAS5,and cotransfected with shRNA GAS 5 and miR-375-3p inhibitor in rescue experiment,followed by OT and ISO treatment for 24h,respectively.The cells were divided into four groups:①ISO+OT+shRNA-scramble;②ISO+OT+shRNA-GAS5;③ISO+OT+shRNA-GAS5+miR-375-3p inhibitor NC;④ISO+OT+shRNAGAS5+miR-375-3p inhibitor.The cells were transfected with miR-375-3p mimics to up-regulated miR-375-3p,and co-transfected with miR-375-3p mimics and KLF4 pcDNA in rescue experiment,followed by OT and ISO treatment for 24h,respectively.The cells were divided into four groups:①ISO+OT+miR-375-3p mimics NC;②ISO+OT+miR-375-3p mimics;③ISO+OT+miR-375-3p mimics+pcDNA-NC;④ISO+OT+miR-375-3p mimics+pcDNA-KLF4.Transfected with shRNA KLF4 to construct KLF4 low expression cells,followed by OT pretreatment and ISO stimulation.In the rescue experiment,we pretreated cardiomyocytes with LY294002(a PI3K inhibitor that prevents PI3K phosphorylation)and then treated with OT and ISO.The morphological changes of cardiomyocytes were observed by immunofluorescence staining.The expressions of lncRNA GAS5,miR-375-3p and KLF4 mRNA were detected by qRT-PCR.The expressions of BNP,β-MHC,KLF4,PI3K,p-PI3K,AKT1,and p-AKT1 proteins were detected by Western blot.ResultsPart Ⅰ:1、The results of echocardiograms suggest that LVP Wd and LVPWs were significantly increased after 14-day ISO treatment in rats.However,OT(3 μg/kg)posttreatment effectively prevented left ventricular wall from thickening(p<0.05 or p<0.01).2、WGA staining showed that cross-sectional areas of cardiomyocytes were significantly enlarged in ISO group,while OT(3 μg/kg)post-treatment reduced cardiomyocyte cross-sectional areas.Masson’s trichrome staining showed a significant increase in collagen volume fraction(CVF)in ISO-infusion group and reduced in OT(3 μg/kg)post-treatment group(p<0.05).3、The results of qRT-PCR shows that ISO markedly increased the expressions of pathological cardiac hypertrophy markers(BNP,β-MHC)and fibrosis marker(α-SMA)compared with control group,whereas OT(3μg/kg)post-treatment could significantly reduce expressions of BNP,β-MHC,and αSMA.Furthermore,compared with ISO group,oxytocin(3 μg/kg)post-treatment group significantly increased the expression of lncRNA GAS5.Part Ⅱ:1、H&E staining indicated that ISO stimulation significantly increased the surface area of cardiomyocytes,while OT treatment significantly reduced it(p<0.01).2、Compared with Control group,ISO(10μM)treatment significantly increased the expressions of BNP and β-MHC proteins(p<0.01),which were reduced by OT(10 nM)treatment(p<0.01).3、The expressions of lncRNA GAS5,KLF4 mRNA,and KLF4 protein were down-regulated in ISO group compared with control group,while up-regulated significantly after OT treatment(p<0.05 or p<0.01).Inversely,the expression of miR-375-3p was increased significantly in ISO group compared with control group and decreased after OT treatment(p<0.01).4、The ratios of phosphorylated-PI3K/PI3K(p-PI3K/PI3K)and phosphorylated AKT1/AKT1(pAKT1/AKT1)were significantly increased in ISO-induced hypertrophic cardiomyocytes and significantly decreased after OT treatment(p<0.05 or p<0.01).Part Ⅲ:1、The results of dual-luciferase reporter gene assays validated that lncRNA GAS5 could combine with miR-375-3p and KLF4 was a direct target gene of miR-375-3p.2、Knockdown of lncRNA GAS5 increased the expression of miR-3753p,decreased the KLF4 mRNA and protein expressions,and inhibited antihypertrophic effects of oxytocin.The anti-hypertrophy effect of OT was restored after co-transfection of shRNA-GAS 5 with miR-375-3p inhibitor.3、Upregulation of miR375-3p reduced the expressions of KLF4 mRNA and protein,activated PI3K/AKT signaling pathway,and blunted anti-hypertrophic effects of oxytocin.The antihypertrophy effect of OT was restored after co-transfection of miR-375-3p mimics with KLF4 pcDNA.4、Knockdown of KLF4 enhanced the activation of PI3K/AKT signaling pathway and inhibited anti-hypertrophic effects of oxytocin.LY294002 treatment rescued the anti-hypertrophy effect of OT.Conclusions1.Oxytocin significantly alleviates cardiac hypertrophy induced by ISO.2.LncRNA GAS5/miR-375-3p/KLF4 axis mediates the anti-hypertrophic effects of oxytocin.3.PI3K/AKT signaling pathway is regulated by lncRNA GAS5/miR-375-3p/KLF4 axis and implicated in anti-hypertrophic effects of oxytocin. |
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