METTL3 Aggravates Intimal Hyperplasia by Facilitating the m6A-YTHDC1-dependent SGK1 Gene Transcription

Abstract

BackgroundVascular smooth muscle cell (VSMC) migration and proliferation substantially contribute to neointimal hyperplasia related to in-stent restenosis. N6-methyladenosine (m6A) catalyzed by the methyltransferase-like 3 (METTL3)-involved methyltransferase complex is the most abundant RNA epigenetic modification in eukaryotes, but the role of m6A RNA methylation in VSMC migration and proliferation as well as neointima formation remains highly controversial. MethodsPrimary human and rat VSMCs were utilized for in vitro experiments. VSMC-specific METTL3 knockout mice (Mettl3flox/floxMyh11-CreERT2) were generated to explore wire injury in carotid arteries in vivo. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was performed to screen for target genes by METTL3-catalyzed m6A RNA methylation. Methylation site mapping, MeRIP-quantitative PCR (MeRIP-qPCR), chromatin immunoprecipitation-qPCR (ChIP-qPCR) and reporter gene assays were applied to explore how METTL3 modulates its target gene expression. ResultsMETTL3 was consistently upregulated in the neointima from mice subjected to carotid wire injury and patients undergoing carotid endarterectomy. VSMC-specific METTL3 deficiency significantly attenuated neointima formation in carotid arteries following wire injury in mice. Accordingly, METTL3 ablation markedly repressed VSMC proliferation both in vitro and in vivo. Mechanistically, METTL3 directly catalyzed m6A methylation on serum/glucocorticoid regulated kinase 1 (SGK1) mRNA and subsequently facilitated its transcription, which relies on the established association between the SGK1 transcript and SGK1 promoter DNA by recruiting the m6A reader YTHDC1. Conversely, SGK1 overexpression abolished METTL3 deficiency-mediated suppression of VSMC proliferation and postinjury neointima formation. ConclusionsMETTL3-catalyzed m6A RNA methylation promoted VSMC proliferation and aggravated postinjury neointima formation by facilitating YTHDC1-dependent SGK1 gene transcription. Targeting the METTL3-YTHDC1-SGK1 axis to modulate VSMC proliferation may be a potential strategy for in-stent restenosis therapy.