The 5‐Methylcytosine RNA Modification in Hepatitis B Virus‐Negative Hepatocellular Carcinoma: Insights From Long‐Read Nanopore Sequencing

5-Methylcytosine (m5C) RNA modification contributes to tumor initiation and progression. However, its transcriptome-wide distribution patterns and biological implications in hepatitis B virus (HBV)-negative hepatocellular carcinoma (HCC) remain poorly understood. Therefore, this study employs long-read Nanopore direct RNA sequencing to systematically elucidate the mechanisms of m5C-mediated epigenetic reprogramming in HBV-negative HCC. Paired tumor and adjacent normal tissues from three HBV-negative HCC patients were collected for Nanopore sequencing. Transcriptome-wide m5C sites were profiled using the CHEUI tool, followed by a comprehensive comparison between tumor and adjacent normal tissue groups regarding the number of m5C sites, their genomic distribution characteristics, and the expression levels of m5C regulators. Finally, an integrated analysis of transcriptomic and methylation data was conducted to identify m5C-related prognostic indicators in HCC. Tumor tissues exhibited a global increase in m5C sites abundance, with differential modifications enriched on chromosomes 1-3. Genes harboring m5C modifications were significantly enriched in immune and inflammatory pathways, suggesting a potential role for this epitranscriptomic mark in remodeling the tumor immune microenvironment. Consistent upregulation of m5C regulators, including NSUN family members and ALYREF, at both gene and isoform levels, correlated with increased methylation activity. Elevated m5C coupled with decreased CES3 expression were associated with poorer overall survival. Additionally, TMEM234 showed prognostic significance despite unchanged bulk expression in public datasets. m5C modifications are globally altered in HBV-negative HCC and may contribute to post-transcriptional regulation and aberrant expression. These findings highlight the potential of m5C as both a prognostic biomarker and a therapeutic target in HBV-negative HCC.