Comprehensive analysis of m⁶ A methylome and transcriptome by Nanopore sequencing in clear cell renal carcinoma

N⁶ -methyladenosine (m⁶ A) is the most prevalent epigenetic modification on eukaryotic messenger RNAs. Recent studies have focused on elucidating the key role of m⁶ A modification patterns in tumor progression. However, the relationship between m⁶ A and transcriptional regulation remains elusive. Nanopore technology enables the quantification of m⁶ A levels at each genomic site. In this study, a pair of tumor tissues and adjacent normal tissues from clear cell renal cell carcinoma (ccRCC) surgical samples were collected for Nanopore direct RNA sequencing. We identified 9644 genes displaying anomalous m⁶ A modifications, with 5343 genes upregulated and 4301 genes downregulated. Among these, 5224 genes were regarded as dysregulated genes, encompassing abnormal regulation of both m⁶ A modification and RNA expression. Gene Set Enrichment Analysis revealed an enrichment of these genes in pathways related to renal system progress and fatty acid metabolic progress. Furthermore, the χ² test demonstrated a significant association between the levels of m⁶ A in dysregulated genes and their transcriptional expression levels. Additionally, we identified four obesity-associated genes (FTO, LEPR, ADIPOR2, and NPY5R) among the dysregulated genes. Further analyses using public databases revealed that these four genes were all related to the prognosis and diagnosis of ccRCC. This study introduced the novel approach of employing conjoint analysis of m⁶ A modification and RNA expression based on Nanopore sequencing to explore potential disease-related genes. Our work demonstrates the feasibility of the application of Nanopore sequencing technology in RNA epigenetic regulation research and identifies new potential therapeutic targets for ccRCC.