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RNA methylation plays a critical role in regulating all aspects of RNA function, which are implicated in the pathogenesis of various human diseases. Recent studies emphasize the role of 5-methylcytosine (m5C), an RNA modification, in key biological functions. Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as the leading chronic liver condition globally. However, the relationship between m5C methylation and MASLD remains unclear. This study aimed to investigate m5C modification in a mouse model of MASLD. In this research, using RNA transcriptome sequencing (RNA-Seq) and methylated RNA bisulfite sequencing (RNA-BS-Seq) in leptin receptor-deficient mice, we found that genes associated with hypermethylation were primarily linked to lipid metabolism. We identified 156 overlapping and differentially expressed genes (DEGs) that changed at both the mRNA expression level and the m5C modification level. Among them, 72 genes showed elevated expression and m5C modification. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that these genes were significantly associated with lipid metabolism-related signaling pathways. Our results demonstrate that m5C methylation modifications may play an important role in the development of MASLD.