Study On RNA M~6A Modification In Coronary Heart Disease And The Role And Mechanism Of TPRG1-AS1 In Inhibiting Atherosclerotic Lesion

AimsAtherosclerotic cardiovascular disease(ASCVD),is a leading cause of disease burden and mortality worldwide.With the progress of epigenomics,numerous studies have shown that N6-methyladenosine(m6A)is closely related to atherosclerosis,and N6methyladenosine and m6A regulators are expected to be potential therapeutic targets for ASCVD such as coronary atherosclerotic heart disease(CAD).Therefore,in this study,methylated RNA immunoprecipitation sequencing(MeRIP-seq)and RNA sequencing(RNA-seq)of peripheral blood mononuclear cells(PBMCs)from patients with CAD and the controls were undertaken to delineate the m6A modification profile at the whole transcriptome level,and screen key genes and key pathways in atherosclerosis.In addition,in this study,the RNA expression profile(GSE113079)was analysed to screen m6A-related CAD biomarkers and provide potential therapeutic targets for atherosclerosis.Methods1.PBMCs from five CAD patients and five healthy controls were randomly selected from samples of previous study(GSE113079)for MeRIP-seq and RNA-seq to identify differentially methylated m6A sites(DMMSs)on mRNAs and lncRNAs between CAD patients and the controls and delineate m6A modification profiles of PBMCs from CAD patients.2.The joint analysis of MeRIP-seq and RNA-seq data were performed to identify differentially methylated genes(DMGs)that are differentially expressed between CAD patients and the controls(fold change>2,P<0.05).3.Gene set enrichment analysis(GSEA)of DMGs were performed to identify the enriched biological pathways.Hub DMGs were identified by protein-protein interaction network analysis(PPI).4.MeRIP-qPCR was performed on representative differentially methylated transcripts screened by MeRIP-seq to compare the m6A modification levels between CAD patients and controls to verify the reliability of MeRIP-seq data.5.The expression levels of m6A regulators between CAD patients and controls were further analyzed in RNA expression profile(GSE113079).6.Gene combination of m6A regulators as CAD biomarker were constructed by LASSO(least absolute shrink and selection operator)regression and Logistic regression in GSE113079,and the diagnostic efficacy was evaluated by receiver operating characteristic curve(ROC)curve in GSE66360 and GSE58294.7.The mRNAs that were correlated with the expression levels of 27 m6A regulators(r>0.5,P<0.001)in GSE113079,were intersected with differentially expressed DMGs and defined as m6A related genes.Then,CAD biomarkers were screened by LASSO regression and support vector machine recursive feature extraction(SVM-RFE)algorithm in m6A related genes.Results1.The area under curve(AUC)of total RNA m6A modification level(m6A%)in PBMCs was 0.86 with 95%confidence interval(CI)0.7467-0.9633,the best cut-off value was 0.2216(sensitivity:90.0%,specificity:73.33%).Therefore,m6A%was a potential diagnostic marker for coronary heart disease.2.This study is the first to delineate the m6A modification profile at the whole transcriptome level of PBMCs from CAD patients and the controls and systematically identify DMMSs on mRNAs and lncRNAs between CAD patients and the controls.3.The joint analysis of MeRIP-seq and RNA-seq identified 971 differentially methylated mRNAs and 31 differentially methylated lncRNAs.The direction of change in methylation levels was largely consistent with the direction of change in expression levels.4.The GSEA analysis showed that differentially expressed DMGs may be involved in the pathogenesis of CAD mediated by activation or inhibition of 12 signaling pathways.The 15 hub DMGs identified by PPI were mainly enriched in cell chemotaxis related pathways.5.MeRIP-qPCR of 12 transcripts of representative DMGs showed that the direction of change in methylation levels of 11 transcripts was consistent with that quantified by MeRIP-seq(11/12,91.7%),demonstrating the reliability of MeRIP-seq data in this study.6.The expression levels of 24 m6A regulators were significantly different between CAD patients and the controls in GSE113079.7.The AUC of gene combination of m6A regulators(ELAVL1,IGF2BP2 and FTO)was 0.98 in GSE113079.8.191 m6A related genes were obtained by the intersection of 9304 genes related to the expression of m6A regulators in GSE113079 and 971 differentially expressed DMGs,which were mainly enriched in cell adhesion related signaling pathways.9.The AUC of m6A related gene LARP4B was 0.92 in GSE113079,and the AUC in cardioembolic stroke expression profile(GSE58294)was 0.87.In atherosclerotic plaque expression profiles(GSE43292 and GSE100927),the expression level of LARP4B was significantly increased in the plaques(P<0.05).Conclusions1.This study is the first to delineate the m6A modification profile at the whole transcriptome level of PBMCs from CAD patients and the controls,and systematically identify DMMSs among mRNAs and lncRNAs.2.Differentially expressed differentially methylated genes may be involved in the pathogenesis of CAD mediated by activation or inhibition of 12 signaling pathways.Hub DMGs might be involved in cell chemotaxis-related biological processes in the pathogenesis of CAD.Differentially expressed DMGs related to m6A regulators(m6A related genes)may be involved in cell adhesion-related biological processes in the pathogenesis of CAD.3.Total RNA m6A modification levels(m6A%)in PBMCs,gene combination of m6A regulators(ELAVL1,IGF2BP2 and FTO),as well as m6A related gene LARP4B might be potential biomarkers of CAD.AimsProliferation and migration of vascular smooth muscle cells(VSMCs)are important steps in atherosclerotic plaque formation.In our previous study,the differentially expressed long non coding RNA(lncRNA)TPRG1-AS1(tumor protein p63 regulated 1,antisense 1)was identified and validated as a diagnostic biomarker of coronary atherosclerotic heart disease(CAD)(atherosclerosis,275,359-367).In our previous study,in human aortic smooth muscle cells(HASMCs),overexpression of TPRG1-AS1 significantly inhibited the migration of HASMCs,and knockdown of TPRG1-AS1 significantly promoted the migration of HASMCs.MYH9,the binding protein of TPRG1-AS1 was initially selected by in vitro RNA pull-down assay,and in HASMCs,overexpression of TPRG1-AS1 significantly downregulated MYH9 protein expression levels,and knockdown of TPRG1-AS1 did not change MYH9 mRNA expression levels.However,the binding of TPRG1-AS1 and MYH9 protein in HASMCs needs further confirmation,and the molecular mechanism by which TPRG1AS1 lead to decreased MYH9 protein levels remains to be resolved.The role of TPRG1-AS1 in neointima formation and atherosclerotic lesions induced by aberrant migration of VSMCs is still lacking to be supported by experimental evidence in vivo.This study aimed to dissect the molecular mechanism by which the interaction of TPRG1-AS 1 with MYH9 protein leads to a decrease in MYH9 protein levels and inhibits the migration of HASMCs.Using a rat carotid balloon injury model,a mice carotid wire injury model,and a mice atherosclerosis model explored the effects of TPRG1-AS1 on neointima formation and atherosclerotic lesions in vivo.Methods1.The expression level of TPRG1-AS1 in atherosclerotic plaques was verified by a series of bioinformatics analysis and qRT-PCR2.The direct binding of TPRG1-AS1 to MYH9 protein was further verified by chromatin Isolation by RNA Purification(CHIRP).3.Cycloheximide(CHX),a protein synthesis inhibitor,was applied to explore the effect of TPRG1-AS1 on MYH9 protein degradation.Using the proteasome inhibitor MG132 to treat HASMCs,the mechanism by which TPRG1-AS1 promotes MYH9 protein degradation was clarified.Treatment of HASMCs with cytochalasin D,further clarifying the molecular mechanism by which TPRG1-AS1 inhibits the migration of HASMCs by inhibiting stress fiber formation.4.To construct vascular smooth muscle cells(VSMCs)-specific TPRG1-AS1 transgenic mice and isolate their aortic smooth muscle cells,this study further verified the effect of TPRG1-AS1 on MYH9 protein expression level,F-actin stress fiber formation and migration in aortic smooth muscle cells.5.The rat carotid balloon injury model and carotid wire injury model was constructed to explore the effect of TPRG1-AS1 on neointima formation.6.VSMC specific TPRG1-AS1 transgenic apolipoprotein E(Apoe)knockout mice were given high-fat diet to evaluate the effect of TPRG1-AS1 on atherosclerosis in Apoe-/-mice.Results1.Compared with the controls,the expression of TPRG1-AS1 in human atherosclerotic plaques was significantly increased.The expression level of TPRG1AS1 in the unstable regions of plaques was further elevated compared with the stable regions of plaques.2.The CHIRP assay confirmed that TPRG1-AS1 directly bound with MYH9 protein in HASMCs.In HASMCs,TPRG1-AS1 promotes the degradation of MYH9 protein through the proteasome pathway to inhibit the formation of stress fibers,thereby inhibiting HASMCs migration.3.Compared with the controls,MYH9 protein expression levels were significantly decreased,F-actin stress fiber formation was inhibited,and cell migration capacity was significantly decreased in the aortic smooth muscle cells of VSMC specific TPRG1-AS1 transgenic mice.4.Adenovirus mediated overexpression of TPRG1-AS1 significantly inhibited rat carotid neointima formation;VSMC specific TPRG1-AS1 overexpression significantly inhibited mice carotid neointima formation.5.VSMC specific TPRG1-AS1 overexpression significantly inhibits atherosclerotic plaque formation in Apoe-/-mice.Conclusions1.Mechanistically,TPRG1-AS1 directly binds to MYH9 protein in HASMCs,and promote the degradation of MYH9 protein through the proteasomal pathway to inhibit stress fiber formation in HASMCs,thereby inhibiting HASMCs migration.2.Functionally,adenovirus mediated overexpression of TPRG1-AS1 significantly inhibited rat carotid artery neointima formation.VSMC specific overexpression of TPRG1-AS1 significantly reduced neointima formation and exerted protective effects during atherosclerotic plaque formation in Apoe-/-mice.