Transcriptome-wide N⁶-methyladenosine methylation profile of atherosclerosis in mice

Atherosclerosis (AS) is a critical pathological event during the progression of cardiovascular diseases. It exhibits fibrofatty lesions on the arterial wall and lacks effective treatment. N⁶-methyladenosine (m⁶A) is the most common modification of eukaryotic RNA and plays an important role in regulating the development and progression of cardiovascular diseases. However, the role of m⁶A modification in AS remains largely unknown. Therefore, in this study, we explored the transcriptome distribution of m⁶A modification in AS and its potential mechanism.

Methylation Quantification Kit was used to detect the global m⁶A levels in the aorta of AS mice. Western blot was used to analyze the protein level of methyltransferases. Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to obtain the first transcriptome range analysis of the m⁶A methylene map in the aorta of AS mice, followed by bioinformatics analysis. qRT-PCR and MeRIP-qRT-PCR were used to measure the mRNA and m⁶A levels in target genes.

The global m⁶A and protein levels of methyltransferase METTL3 were significantly increased in the aorta of AS mice. However, the protein level of demethylase ALKBH5 was significantly decreased. Through MeRIP-seq, we obtained m⁶A methylation maps in AS and control mice. In total, 26,918 m⁶A peaks associated with 13,744 genes were detected in AS group, whereas 26,157 m⁶A peaks associated with 13,283 genes were detected in the control group. Peaks mainly appeared in the coding sequence (CDS) regions close to the stop codon with the RRACH motif. Moreover, functional enrichment analysis demonstrated that m⁶A-containing genes were significantly enriched in AS-relevant pathways. Interestingly, a negative correlation between m⁶A methylation abundance and gene expression level was found through the integrated analysis of MeRIP-seq and RNA-seq data. Among the m⁶A-modified genes, a hypo-methylated but up-regulated (hypo-up) gene Fabp5 may be a potential biomarker of AS.

Our study provides transcriptome-wide m⁶A methylation for the first time to determine the association between m⁶A modification and AS progression. Our study lays a foundation for further exploring the pathogenesis of AS and provides a new direction for the treatment of AS.