Bioinformatics analysis and experimental validation of m³C RNA methylation regulators in hepatocellular carcinoma: expression, function, and prognostic value

Aim: Hepatocellular carcinoma (HCC) remains a major challenge due to poor prognosis. This study investigates 3-methylcytidine (m3C) RNA methylation regulators to elucidate their roles in HCC and to develop a prognostic scoring system for clinical application. Methods: We integrated data from 486 HCC patients [The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets] and 16 pairs of clinical tissue samples. The expression, mutation profiles, and prognostic significance of 6 m3C regulators were analyzed. Functional assays, including cell proliferation and migration, were performed, alongside immune infiltration analysis using single-sample gene set enrichment analysis (ssGSEA). Finally, an m3C scoring system was constructed to evaluate prognostic potential. Results: Most m3C regulators (except METTL8) were upregulated in HCC tissues. Knockdown of METTL2, METTL6, ALKBH1, or ALKBH3, as well as overexpression of METTL8, inhibited HCC cell proliferation and migration. Two distinct m3C modification modes were identified, each associated with unique clinical features. The m3C score was positively correlated with longer overall survival in high-score patients and was associated with tumor mutation burden (TMB) and expression of PD-1 and CTLA4, suggesting its potential to predict immunotherapy response. Conclusion: This study highlights the genetic variation and prognostic relevance of m3C methylation regulators in HCC and introduces a novel scoring system for prognosis prediction, providing a potential tool to guide HCC treatment strategies.