Effect And Mechanism Of M6A Methyltransferase METTL14 In Inhibiting Phenotypic Transformation Of Smooth Muscle Cell

AimsAtherosclerosis(AS)constitutes the main pathological foundation of atherosclerotic cardiovascular disease(ASCVD),with the aberrant phenotypic switching of vascular smooth muscle cells(VSMCs)being a core event in AS development.RNA N6methyladenosine(m6A)modification represents the most usual RNA epigenetic modification.In recent years,the involvement of m6A modification in AS pathogenesis has progressively garnered attention;however,its regulatory role in VSMC phenotypic switching remains to be clarified.This study aims to investigate the function of m6A methyltransferase 14(METTL14)in platelet-derived growth factor BB(PDGF-BB)induced human aortic smooth muscle cells(HASMCs)phenotypic switching models,thereby providing a theoretical foundation for ASCVD prevention and treatment.Methods1.PDGF-BB was utilized to establish the HASMCs phenotypic switching model.Quantitative real-time PCR(qPCR)and western blot(WB)were employed to assess mRNA expression and protein expression levels of m6A major functional proteincoding genes in the HASMCs phenotypic switching model.2.Immunofluorescence staining technique was applied to detect the expression pattern of METTL14 in human atherosclerotic plaques.3.A gain-and loss-of-function strategy was implemented to knockdown and overexpress the METTL14 gene in HASMCs.The role of METTL14 in PDGF-BB-induced phenotypic switching of HASMCs was examined through EdU proliferation assay and transwell migration assay.4.RNA sequencing(RNA-seq)was employed to screen METTL14 target genes.The differentially up-regulated genes after overexpression of the METTL14 gene and those differentially down-regulated genes after METTL14 gene knockdown were considered intersections.QPCR and WB experiments were employed to confirm that the METTL14 gene regulates P21 gene expression in HASMCs.5.EdU proliferation assay and transwell migration assay were utilized to determine the role of the P21 gene in HASMC phenotypic switching.A phenotypic reversion assay was employed to elucidate the role of the P21 gene in suppressing HASMC phenotypic switching by the METTL14 gene.6.SRAMP software was used to analyze the mRNA sequence of the P21 gene and predict potential m6A modification sites.RNA-Binding Protein Immunoprecipitation(RIP)and qPCR experiments were employed to detect the direct binding of METTL14 protein to P21 gene mRNA in HASMCs.Results1.The PDGF-BB-induced HASMCs phenotypic switching model was successfully established.QPCR results revealed that the mRNA expression levels of fat and obesity-associated protein(FTO),alkB family protein(ALKBH5),methyltransferaseassociated protein 3(METTL3),and METTL14 were all significantly downregulated in PDGF-BB-treated HASMCs compared to the control.Meanwhile,WB results demonstrated that only METTL14 protein expression levels were significantly downregulated after PDGF-BB treatment.This suggests that the downregulation of METTL14 may play a crucial regulatory role in PDGF-BB-induced phenotypic switching of HASMCs.2.Immunofluorescence staining technique showed that METTL14 expression was significantly downregulated in human carotid plaques.This implies that the downregulation of the METTL14 gene may be involved in AS development.3.EdU proliferation assay and transwell migration assay revealed that knocking down the METTL14 gene substantially promoted the proliferation and migration of HASMCs.Conversely,overexpressing the METTL14 gene significantly inhibited the proliferation and migration of HASMCs.Moreover,overexpression of the METTL14 gene also considerably impeded PDGF-BB-induced proliferation and migration of HASMCs.4.RNA-seq results indicated that a total of 432 differentially up-regulated genes in HASMCs overexpressing the METTL14 gene,and 940 differentially down-regulated genes in HASMCs with METTL14 gene knockdown.A total of 36 genes were obtained after identifying the intersection of the two with both upregulation after overexpression of the METTL14 gene and downregulation after METTL14 gene knockdown in HASMCs.In conjunction with literature progress,the P21 gene was selected as a potential downstream target of METTL14.Subsequent qPCR and WB experiments further established that overexpression of the METTL14 gene in HASMCs significantly up-regulated the mRNA and protein expression levels of P21,while METTL14 gene knockdown significantly down-regulated P21 gene expression.Consequently,the P21 gene is the target gene of METTL14 in HASMCs.5.Knocking down the P21 gene in HASMCs significantly promoted cell proliferation and migration.Additionally,the simultaneous knockdown of the P21 gene and overexpression of the METTL14 gene considerably reversed the decreased proliferation and migration of HASMCs induced by METTL14 gene overexpression.This suggests that METTL14 inhibits HASMCs proliferation and migration by modulating the expression of the P21 gene.6.SRAMP prediction revealed multiple highly scored m6A modification sites on the P21 gene mRNA sequence.RIP-qPCR results demonstrated that P21 gene mRNA was significantly enriched by the antibody against METTL14 protein compared with IgG,indicating that METTL14 protein directly binds to P21 gene mRNA in HASMCs.Conclusions1.Immunofluorescence staining revealed that METTL14 expression levels were significantly downregulated in human atherosclerotic plaques and co-localized with smooth muscle cells,suggesting that downregulation of METTL14 in smooth muscle cells is closely associated with atherosclerosis.2.METTL14 expression levels were substantially downr egulated in the PDGF-BBinduced HASMCs phenotypic switching model.This observation implies that the downregulation of the METTL14 gene may have an important regulatory role in PDGF-BB-induced HASMCs phenotypic switching.3.Knocking down the METTL14 gene significantly promoted the proliferation and migration of HASMCs.In contrast,overexpressing the METTL14 gene markedly inhibited the proliferation and migration of HASMCs.Additionally,overexpression of the METTL14 gene also significantly impeded PDGF-BB-induced proliferation and migration of HASMCs.4.The downstream target gene of METTL14 was screened and identified,confirming that METTL14 suppressed the proliferation and migration of HASMCs by upregulating the expression level of the P21 gene.5.RIP-qPCR experiments demonstrated that METTL14 protein directly bound to P21 gene mRNA.This finding suggests that METTL14 protein-mediated m6A modification may regulate P21 gene expression in HASMCs,thereby inhibiting PDGF-BB-induced phenotypic switching of HASMCs.