The Molecular Mechanism Of LncRNA H19 Regulating Sympathetic Nerve Remodeling After Myocardial Infarction

BackgroundMyocardial infarction(MI)is a serious threat to human health,the incidence is on the rise,and the age of patients is gradually getting younger.Ventricular arrhythmias(VAs)and sudden cardiac death(SCD)are important causes of death and disability in MI patients.In recent years,a series of studies and our previous studies have shown that malignant VAS and SCD after MI are related to the imbalance of cardiac autonomic nerve caused by cardiac sympathetic nerve regeneration and remodeling.Therefore,exploring the specific mechanism of sympathetic nerve remodeling after MI will provide preventive measures and theoretical basis for reducing the incidence of malignant ventricular arrhythmias and sudden cardiac death,and improve the long-term prognosis.Sympathetic nerve regeneration and remodeling are closely related to inflammation.Sympathetic remodeling mainly occurs around the infarct where a large number of inflammatory cells and inflammatory cytokines are aggregated.Inflammatory cells(macrophages,fibroblasts,etc.)play a key role in the regulation of sympathetic remodeling by secreting nerve growth factor(NGF).In the process of sympathetic nerve remodeling,NGF is an important factor.Inhibiting the secretion of NGF can effectively reduce sympathetic nerve overremodeling,but its regulatory mechanism has not been fully clarified.Recent studies have shown that although long non-coding RNA(lncRNA)does not encode protein,it can interact with DNA,RNA or protein when tissue physiology and biochemistry changes,and regulate gene expression through mechanisms such as genetic imprinting and chromatin remodeling.In the cardiovascular field,lncRNA H19 has been found to be involved in the pathophysiological process of diseases such as atherosclerosis,hypertrophic cardiomyopathy and heart failure.Whether IncRNA H19 is involved in the process of sympathetic nerve regeneration and remodeling after MI is still unknown.lncRNA H19 can be used as a miRNA sponge for a variety of miRNAs,binding miRNAs through sequence complementarity,thereby controlling the number and activity of miRNAs,and regulating the expression levels of their target genes.Through bioinformatics analysis of RNAhybrid and Targetscan,we predicted that both IncRNA H19 and NGF are the target genes of miR-let-7a.Our previous study also found that overexpression of miR-let-7a inhibits the expression of NGF and improves sympathetic nerve remodeling after MI.Therefore,we propose the hypothesis that the over-expressed IncRNA H19 after MI may play a miRNA sponge effect by binding miR-let-7a,and reduce the inhibitory effect of miR-let-7a on its target gene NGF through the ceRNA mechanism,and increase the expression of NGF,And then promote the regeneration and remodeling of cardiac sympathetic nerve after MI.Intervention in the lncRNA H19-miR-let-7a-NGF signaling pathway will provide a new target for the prevention and treatment of ventricular arrhythmias after MI.ObjectiveWe aimed to explore the regulatory relationship of lncRNA H19,miR-let-7a and NGF after myocardial infarction,and whether they are involved in sympathetic nerve remodeling after myocardial infarction and the possible mechanism.MethodsExperiment 1:In order to explore the expression of lncRNA H19 and NGF 7 days after MI,14 male Sprague-Dawley(SD)rats(about 260-280g)were randomly divided into MI group and sham group,with 7 rats in either group.Seven days after MI was induced,the rats were sacrificed to determine the lncRNA H19 and NGF expression difference with qRT-PCR and Western blotting.The rat model of myocardial infarction was established by left coronary artery occlusion.In the sham group,only threaded the corresponding coronary artery without knotting.Experiment 2:In order to study the regulatory relationship of IncRNA H19,NGF and sympathetic nerve remodeling after MI,a total of 32 rats were randomly divided into 4 groups:sham+NC group,sham+siH19 group,MI+NC group and MI+siH19 group,with 8 rats in each group.H 19 siRNA or the control virus was injected into the left ventricular myocardium of the rats.Programmed electrical stimulation was performed prior to sacrifice at 7 days post MI to observe the susceptibility to VAs.Then the heart and blood were taken out.Western blotting and ELISA were used to detect the expression of NGF,the expression of lncRNA H19 was detected by qRT-PCR;the regeneration of sympathetic nerves is to observe the expression of growth-related protein 43(GAP43)and tyrosine hydroxylase(TH)around the infarct focus by immunofluorescence.Experiment 3:To explore that knockdown lncRNA H19 inhibits sympathetic nerve remodeling after MI by regulating the expression of NGF,A total of 24 rats were randomly divided into the MI+NC group,MI+siH19 group and MI+siH19+LV-NGF group for rescue test,with 8 rats in each group.The expressions of NGF,TH and GAP43 were detected with Western blotting and immunofluorescence staining to analyze the sympathetic nerve regeneration.Experiment 4:To study the regulatory mechanism of LncRNA H19,miR-let-7a and NGF,RNAhybrid and Targetscan were used to predict the targeting effects of miR-let-7a and the two respectively.qRT-PCR was performed to detect expression of miR-let-7a after silencing LncRNA H19.Dual luciferase reporter assay was performed to verify the direct effects of LncRNA H19,NGF and miR-let-7a.Results1.The expression of LncRNA H19 and NGF was up-regulated 7 days after MI;2.Knockdown of LncRNA H19 reduced sympathetic nerve remodeling after MI;compared with the sham group,the positive staining area of TH and GAP43 was significantly increased in the MI group,indicating sympathetic nerve regeneration after MI;Equal doses of LncRNA H19 knockdown lentivirus or negative control virus were injected into the left ventricular wall of the rats after MI.TH and GAP43 expression was significantly decreased in the MI+siH19 group compared with the MI+NC group,indicating that knockdown the expression of LncRNA H19 can inhibit the remodeling of cardiac sympathetic nerves after myocardial infarction;intracardiac programmed electrical stimulation shown that the rats were very likely to experience the induction of VAs following MI.However,in the rats with MI treated with the LncRNA H19 knockdown virus,the susceptibility to VAs was significantly reduced compared with that in the rats with MI injected with control virus,suggesting that knockdown of LncRNA H19 had an inhibitory effect on the incidence of VAs after MI;3.lncRNA H19 inhibits sympathetic nerve remodeling after MI by regulating NGF.Compared with the sham group,the expression levels of IncRNA H19 and NGF increased after 7 days of MI,while the expression of NGF decreased with the knockdown of lncRNA H19.Transfection of NGF overexpression virus can attenuate the inhibitory effect of lncRNA H19 silencing virus on NGF protein expression.Immunofluorescence results showed that NGF overexpression virus can partially rescue the inhibitory effect of lncRNA H19 silencing virus on TH and GAP43;the above experimental results further indicate the role of NGF in sympathetic nerve remodeling after MI,and lncRNA H19 participates in the process by regulating NGF;4.LncRNA H19 is a target of miR-let-7a.RNAhybrid analysis was performed to predict that LncRNA H19 was a target gene of miR-let-7a.Whether it is in the sham group or MI group,silencing LncRNA H19 increased the expression of miR-let-7a;The luciferase assay results showed that miR-let-7a suppressed the luciferase activity of the wild-type LncRNA H19 3’ UTR but had no significant effects on that of the mutant LncRNA H19 3’ UTR in 293T cells compared with the respective control,suggesting that LncRNA H19 is a direct target gene of miR-let-7a;5.NGF is a target of miR-let-7a.The target genes of miR-let-7a were predicted using TargetScan.Following the identification of NGF as a candidate target gene,the luciferase reporter assay was performed to confirm that NGF is directly regulated by miR-let-7a.The luciferase assay results showed that miR-let-7a suppressed the luciferase activity of the wild-type NGF 3’ UTR but not the luciferase activity of the mutant NGF 3’ UTR in 293T cells compared with the respective control,suggesting that NGF is a direct target gene of miR-let-7a.Conclusion1.The over-expressed IncRNA H19 after MI may play an important role in the remodeling of sympathetic nerves;2.lncRNA H19 binds miRNA-let-7a through the ceRNA mechanism to regulate the expression of NGF;it relieves the inhibitory effect of miR-let-7a on its target gene NGF and increases the expression of NGF3.Silencing lncRNA H19 inhibited the regeneration and remodeling of sympathetic nerves after MI.Intervention in the lncRNA H19/miR-let-7a/NGF signaling pathway will provide a new target for the prevention and treatment of ventricular arrhythmia after MI.