| Acute myocardial infarction(AMI)can lead to myocardial fibrosis and morphological changes after myocardial injury,such as impaired left ventricular function,left ventricular remodeling,and cardiac enlargement.Although the success rate of AMI treatment has increased with the widespread implementation of emergency percutaneous coronary intervention(PCI)and thrombolytic therapy,40%had left ventricular remodeling and 14.2%had heart failure.Heart failure and arrhythmia caused by myocardial fibrosis seriously threaten human life and health.Once pathological remodeling occurs,it is difficult to reverse.However,there is still no specific treatment for the root cause of the myocardial remodeling process.Therefore,exploring the pathological mechanism of myocardial fibrosis after AMI is of great clinical significance for finding sensitive and efficient early diagnostic markers,developing targeted therapeutic drugs,and improving the prognosis of patients.Recent studies have shown that some non-coding RNAs(ncRNAs),such as micrornas(miRNAs)and lncRNAs,can act as regulatory molecules in gene regulatory networks.The regulatory role of LncRNAs in heart disease has received increasing attention in recent years.The key role of LncRNAs in controlling cardiac diseases such as cardiac hypertrophy,cardiac allograft rejection and ischemic heart failure has been preliminically elucidated.Among them,Zinc finger Antisense 1(lncRNA-ZFAS1,ZFAS1)is the antisense chain of Zinc lipoprotein-encoding gene(Znfxl).Some studies have confirmed that its function of regulating cardiovascular system and is a potential diagnostic marker of AMI.Some scholars have also found that ZFAS1 is a potential functional target for the prevention and treatment of MI,but the functional role of ZFAS1 in MI needs to be further elucidated.Wnt/β-catenin signaling pathway plays key roles in most physiological and pathological processes,including tissue and organ formation,maintenance of cellular homeostasis,post-injury repair and regeneration,and disease progression.Recent studies have shown that the occurrence and progression of fibrosis in kidney,lung,liver,heart and skin are closely associated with the abnormal activation of Wnt/β-catenin signaling pathway.Furthermore,persistent and excessive activation of Wnt signaling pathway after MI can lead to severe myocardial fibrosis and impair myocardial function.Studies have shown that inhibition of Wnt signaling prevents collagen deposition and improves cardiac function after MI.Previous studies have reported that multiple LncRNAs are involved in the activation of Wnt/β-catenin signaling pathway,and whether LncZFAS1 is involved in the regulation of Wnt/β-catenin signaling axis remains to be confirmed.Based on the above background,we propose the following scientific hypothesis:myocardial fibrosis is the key cause of cardiac dysfunction after MI.This negative myocardial remodeling is regulated by lnCzFas1-mediated Wnt/β-catenin signaling pathway.Silencing LncZFAS1 can inhibit Wnt/β-catenin signaling pathway,which subsequently inhibited the proliferation,migration,and invasion of myocardial fibroblasts,further preventing collagen deposition,and thus improving cardiac function in rats with MI.To test the above hypothesis,this study first explored the correlation between the expression level of LncZFAS1 and the degree of myocardial fibrosis and cardiac function changes after AMI,and also validated the regulatory mechanism of excessive activation of Wnt/β-catenin signaling pathway in myocardial remodeling.Secondly,lentivirus transfection technology and pathway inhibitors were used to clarify the regulation effect of LncZFAS1 on Wnt/β-catenin signaling pathway in vitro and in vivo.This study helps to provide new ideas for the prevention and treatment of MI.Part I:The correlation between LncRNA ZFAS1 and myocardial fibrosis after AMIObjective:LncRNA ZFAS1 is involved in the development of cardiovascular diseases.This part of the study aims to investigate the expression of LncRNAZFAS1 in myocardial fibrosis models in vivo and in vitro.Methods:AMI-induced rat myocardial fibrosis model was established by ligation of left anterior descending coronary artery.Rat primary CFs cells were isolated,and an in vitro myocardial fibrosis model was established by hypoxia treatment.In animal experiments,left ventricular exhalation fraction(LVEF)and left ventricular short-axis shortening rate(LVFS)were measured and recorded by echocardiography.HE staining was used to observe the pathological changes of myocardial histopathology.Masson staining was used to evaluate the accumulation of collagen,and the myocardial infarction rate was calculated.The vWF level in myocardial tissue was detected by immunohistochemistry to evaluate the changes of angiogenesis.Rt-qpcr was used to detect the expression of LncRNA ZFAS1 in rat myocardial tissue.MTT assay was used to detect cell proliferation.The invasion and migration ability of cells were determined by Transwell and Wound healing assay.The expression level of LncRNA ZFAS1 in CFs cells was determined by RT-qPCR.Results:1.Compared with the control group,the LVEF and LVFS of the AMI group were significantly decreased.2.In the AMI group,the cell arrangement in the area of myocardial ischemia and necrosis was abnormal,the tissue space was enlarged,the amount of collagen accumulation was significantly increased,the incidence of myocardial infarction was significantly increased,and the expression of vWF,a molecular marker of angiogenesis,was significantly down-regulated.3.The expression level of LncRNA ZFAS1 in myocardial tissue of AMI rats was significantly increased.4.After hypoxia treatment,the proliferation ability of CFs cells was significantly improved,and the levels of cell invasion and migration were significantly increased.5.After hypoxia treatment,the expression of LncRNA ZFAS1 was significantly up-regulated in CFs cells.Conclusion:1.Cardiac function was impaired and myocardial tissue fibrosis occurred in AMI rats.2.LncRNA ZFAS1 expression was up-regulated in animal models of AMI rat fibrosis as well as in vitro myocardial fibrosis induced by hypoxia treatment.Part II:Silencing LncRNA ZFAS1 alleviates myocardial fibrosis and inhibits myocardial fibroblast activation in AMI ratsObjective:This study aimed to verify the effect of silencing LncRNA ZFAS1 on myocardial fibrosis and CFs cell activation in AMI rats in vitro and in vivo.Methods:AMI-induced rat myocardial fibrosis model was established by ligation of left anterior descending coronary artery.Rat primary CFs cells were isolated,and an in vitro myocardial fibrosis model was established by hypoxia treatment.AMI rats were injected with lentiviral knockdown vector of LncRNA ZFAS1(shZFAS1)through tail vein,and CFs cells were transfected with lentivirus to knock down LncRNA ZFAS1(shZFAS1).LVEF and LVFS were measured and recorded by echocardiography in vitro.HE and Masson staining were used to evaluate the histopathological changes and myocardial infarction rate.The level of vWF in myocardial tissue was detected by immunohistochemistry to evaluate the changes of angiogenesis.RT-qPCR was used to detect the expression level of LncRNA ZFAS1 in rat myocardial tissue.MTT assay was used to detect cell proliferation in vitro.Transwell and Wound healing were used to determine the invasion and migration ability of cells.Rt-qpcr was used to detect the expression of LncRNA ZFAS1 in CFs cells.Results:1.Silencing LncRNA ZFAS1 significantly increased LVEF and LVFS in AMI rats.2.Silencing LncRNA ZFAS1 can significantly alleviate the symptoms of AMI rats,reduce the rate of myocardial infarction,and promote the expression of vWF.3.Silencing LncRNA ZFAS1 can significantly reverse the effect of hypoxia treatment on CFs,which can inhibit the proliferation,migration and invasion of CFs.Conclusion:1.Silencing LncRNA ZFAS1 can improve cardiac function and inhibit myocardial fibrosis in AMI rats.2.Silencing LncRNA ZFAS1 inhibited CFs cell activation.Part Ⅲ:The mechanism of LncRNA ZFAS1 involved in the regulation of myocardial fibrosis by regulating the Wnt/β-catenin signaling pathwayObjective:The abnormal activation of Wnt/β-catenin signaling pathway is involved in the pathogenesis of myocardial fibrosis after AMI,and the regulatory effect of LncRNA ZFAS1 on Wnt/β-catenin signaling axis needs to be confirmed.This part aims to investigate whether LncRNA ZFAS1 affects myocardial fibrosis and CFs cell activation in AMI rats by targeting Wnt/β-catenin signaling pathway.Methods:AMI-induced rat myocardial fibrosis model was established by ligation of left anterior descending coronary artery.Rat primary CFs cells were isolated,and an in vitro myocardial fibrosis model was established by hypoxia treatment.Cells and animals were transfected with LncRNA ZFAS1 lentiviral knockdown vector(shZFAS1),and Wnt/β-catenin pathway activator HLY78 was administered for intervention.LVEF and LVFS were measured and recorded by echocardiography in vitro.HE and Masson staining were used to evaluate the histopathological changes and myocardial infarction rate.The level of vWF in myocardial tissue was detected by immunohistochemistry to evaluate the changes of angiogenesis.The mRNA and protein expression levels of β-catenin and GSK-3β in rat myocardial tissue were detected by RT-qPCR and Western blot.MTT assay was used to detect cell proliferation in vitro.Transwell and Wound healing were used to determine the invasion and migration ability of cells.The mRNA and protein expression levels of β-catenin and GSK-3β in CFs cells were detected by RT-qPCR and Western blot.Results:1.The expression of β-catenin was significantly increased in rat AMI model and hypoxia-treated CFs cell model,while GSK-3β expression level was significantly decreased.The down-regulation of LncRNA ZFAS1 gene could significantly reverse the above trends.2.Silencing LncRNA ZFAS1 could significantly reverse the downward trend of LVEF and LVFS in AMI group rats,while the simultaneous administration of Wnt/β-catenin pathway activator HLY78 could significantly decrease LVEF and LVFS.3.Silencing LncRNA ZFAS1 can significantly alleviate the symptoms of AMI rats,reverse the pathological changes of myocardial tissue,reduce the rate of myocardial infarction,and promote the expression of vWF.Compared with the AMI+shZFAS1 group,the expression of vWF was significantly decreased after HLY78 co-treatment.4.The proliferation,migration and invasion of CFs cells were significantly promoted after hypoxia treatment.ShZFAS1 treatment could significantly reverse the effect of hypoxia on CFs and inhibit the proliferation,migration and invasion of CFs.However,the co-intervention of Wnt/β-catenin pathway activator HLY78 significantly promoted the proliferation,migration and invasion of CFs compared with shZFAS1 alone.Conclusion:1.The expression of Wnt/β-catenin signaling pathway was up-regulated in the rat model of AMI fibrosis and in the in vitro model of myocardial fibrosis induced by hypoxia.2.Silencing LncRNA ZFAS1 can inhibit Wnt/β-catenin signaling pathway.3.Silencing LncRNA ZFAS1 alleviates myocardial fibrosis and inhibits CFs cell activation after AMI by inhibiting Wnt/β-catenin signaling pathway. |
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