YTHDF1 aggravates the progression of atherosclerosis by enhancing BRD4 mRNA stability and promoting EndMT transition in an m6A-Dependent manner

Endothelial-to-mesenchymal transition (EndMT) is a crucial process driving vascular remodeling in atherosclerosis (AS). N6-methyladenosine (m6A) RNA methylation, regulated by YTH N6-methyladenosine RNA binding protein F1 (YTHDF1), an m6A reader that modulates RNA stability and translation, influences various cellular functions, but its specific role in AS-related EndMT remains unclear. This study aimed to investigate the role of the m6A reader YTHDF1 in AS progression, focusing on its regulation of bromodomain containing 4 (BRD4), a central epigenetic regulator in the Bromodomain and Extra-Terminal (BET) family, and subsequent promotion of EndMT.

Human AS datasets (involving 6 clinical samples in GSE97210 and 104 clinical samples in GSE100927) and single-cell RNA sequencing data from Apoe-/- mice (n = 3 in total) were analyzed to assess YTHDF1 expression and correlation. YTHDF1-/-Apoe-/- mice (n = 5) were generated and fed a high-fat diet to evaluate plaque formation. In vitro, human umbilical vein endothelial cells (HUVECs) were used for gain/loss-of-function experiments. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), site-directed mutagenesis, and functional assays (Transwell, wound healing) were employed to elucidate the molecular mechanism.

YTHDF1 was significantly upregulated in human AS plaques (P < 0.001) and positively correlated with EndMT markers. ScRNA-seq analysis revealed a progressive increase in YTHDF1-expressing endothelial cells during plaque progression in Apoe^-/- mice. YTHDF1 knockout in Apoe^-/- mice markedly reduced atherosclerotic lesion area (P = 0.0023), lipid deposition (P = 0.0007), and en face plaque area (P = 0.0038), accompanied by attenuated EndMT. In HUVECs, YTHDF1 overexpression promoted EndMT, enhanced cell migration (P < 0.01), and upregulated BRD4 (1.53 ± 0.20vs.1 ± 0.19 in control, p = 0.027). Mechanistically, METTL3-mediated m6A modification at a specific site in the 5'-UTR of BRD4 mRNA was recognized by YTHDF1, which stabilized BRD4 transcripts and increased BRD4 protein levels. Conversely, BRD4 knockdown suppressed the EndMT phenotype and impaired HUVEC migration (P < 0.01).

Our findings uncover a novel pathogenic axis in AS where YTHDF1, by recognizing METTL3-mediated m6A modification on BRD4 mRNA, enhances its stability. This leads to increased BRD4 expression, which in turn drives EndMT and exacerbates disease progression. The YTHDF1-m6A-BRD4 pathway may represent a potential therapeutic target for AS which requires further preclinical validation.