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

Abstract

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.

Keywords

Computational biology / RNA methylation / hepatocellular carcinoma / database / early diagnosis

References

[1] Qin J,Zhou C.Differential expression pattern of CC chemokine receptor 7 guides precision treatment of hepatocellular carcinoma.Signal Transduct Target Ther2025;10:229 PMCID:PMC12277428

[2] Sung H,Siegel RL.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin2021;71:209-49

[3] Li M,Xu RH.Global trends and epidemiological shifts in gastrointestinal cancers: insights from the past four decades.Cancer Commun2025;45:774-88 PMCID:PMC12328094

[4] Donne R.The liver cancer immune microenvironment: therapeutic implications for hepatocellular carcinoma.Hepatology2023;77:1773-96 PMCID:PMC9941399

[5] Thangaraj JL,Lopez E.Disruption of TGF-β signaling pathway is required to mediate effective killing of hepatocellular carcinoma by human iPSC-derived NK cells.Cell Stem Cell2024;31:1327-1343.e5 PMCID:PMC11380586

[6] Moris D,Schiltz S.Advances in the treatment of hepatocellular carcinoma: an overview of the current and evolving therapeutic landscape for clinicians.CA Cancer J Clin2025;

[7] Sangro B,Hervás-Stubbs S.Advances in immunotherapy for hepatocellular carcinoma.Nat Rev Gastroenterol Hepatol2021;18:525-43 PMCID:PMC8042636

[8] Hu Y.Practice of precision surgery in primary liver cancer.Hepatobiliary Pancreat Dis Int2021;20:108-9

[9] Haber PK,Torres-Martin M.Molecular markers of response to anti-PD1 therapy in advanced hepatocellular carcinoma.Gastroenterology2023;164:72-88.e18 PMCID:PMC12182972

[10] Llovet JM,Kulik L.Locoregional therapies in the era of molecular and immune treatments for hepatocellular carcinoma.Nat Rev Gastroenterol Hepatol2021;18:293-313

[11] Chan YT,Wu J.Biomarkers for diagnosis and therapeutic options in hepatocellular carcinoma.Mol Cancer2024;23:189 PMCID:PMC11378508

[12] Schwabe RF.Gut microbiome in HCC - mechanisms, diagnosis and therapy.J Hepatol2020;72:230-8

[13] Arvanitakis K,Germanidis G.Tumor-associated neutrophils in hepatocellular carcinoma pathogenesis, prognosis, and therapy.Cancers2021;13:2899 PMCID:PMC8228651

[14] Suzuki T.The expanding world of tRNA modifications and their disease relevance.Nat Rev Mol Cell Biol2021;22:375-92

[15] Zhou JB,Zhou XL.Modifications of the human tRNA anticodon loop and their associations with genetic diseases.Cell Mol Life Sci2021;78:7087-105 PMCID:PMC11071707

[16] McCown PJ,Kunkler CN.Naturally occurring modified ribonucleosides.Wiley Interdiscip Rev RNA2020;11:e1595 PMCID:PMC7694415

[17] Suzuki T,Kikuchi I.Complete chemical structures of human mitochondrial tRNAs.Nat Commun2020;11:4269 PMCID:PMC7455718

[18] Huang MH,Mao XL.Molecular basis for human mitochondrial tRNA m³C modification by alternatively spliced METTL8.Nucleic Acids Res2022;50:4012-28 PMCID:PMC9023283

[19] Mao XL,Huang MH.Mutually exclusive substrate selection strategy by human m³C RNA transferases METTL2A and METTL6.Nucleic Acids Res2021;49:8309-23 PMCID:PMC8373065

[20] Cui J,Sendinc E,Gregory RI.Nucleotide resolution profiling of m³C RNA modification by HAC-seq.Nucleic Acids Res2021;49:e27 PMCID:PMC7969016

[21] Schöller E,Marchand V.Balancing of mitochondrial translation through METTL8-mediated m³C modification of mitochondrial tRNAs.Mol Cell2021;81:4810-4825.e12 PMCID:PMC11214777

[22] Ignatova VV,Ho JSY.METTL6 is a tRNA m³C methyltransferase that regulates pluripotency and tumor cell growth.Sci Adv2020;6:eaaz4551

[23] Zhang LH,Hu T.The SUMOylated METTL8 Induces R-loop and Tumorigenesis via m3C.iScience2020;23:100968 PMCID:PMC7082549

[24] Feng Q,Xue T.The role of RNA modification in hepatocellular carcinoma.Front Pharmacol2022;13:984453 PMCID:PMC9479111

[25] Ma CJ,Ye TT,Feng YQ.AlkB homologue 1 demethylates N³-methylcytidine in mRNA of mammals.ACS Chem Biol2019;14:1418-25

[26] Wang X,Li J.Multi-omics analysis of copy number variations of RNA regulatory genes in soft tissue sarcoma.Life Sci2021;265:118734

[27] Charoentong P,Angelova M.Pan-cancer immunogenomic analyses reveal genotype-immunophenotype relationships and predictors of response to checkpoint blockade.Cell Rep2017;18:248-62

[28] Finn RS,Ikeda M.IMbrave150 InvestigatorsAtezolizumab plus bevacizumab in unresectable hepatocellular carcinoma.N Engl J Med2020;382:1894-905

[29] Calderaro J,Luedde T.Artificial intelligence for the prevention and clinical management of hepatocellular carcinoma.J Hepatol2022;76:1348-61 PMCID:PMC9126418

[30] Yang C,Li Y,Lv Y.Prognosis and personalized treatment prediction in TP53-mutant hepatocellular carcinoma: an in silico strategy towards precision oncology.Brief Bioinform2021;22:bbaa164

[31] Meng Y,Liao J.Single cell transcriptional diversity and intercellular crosstalk of human liver cancer.Cell Death Dis2022;13:261 PMCID:PMC8943132

[32] Fujita M,Hasegawa T.Classification of primary liver cancer with immunosuppression mechanisms and correlation with genomic alterations.EBioMedicine2020;53:102659 PMCID:PMC7048625

[33] Kan RL,Sallam T.Crosstalk between epitranscriptomic and epigenetic mechanisms in gene regulation.Trends Genet2022;38:182-93 PMCID:PMC9093201

[34] Wang B,Zhou Z.Inhibition of 6-phosphogluconate dehydrogenase suppresses esophageal squamous cell carcinoma growth and enhances the anti-tumor effects of metformin via the AMPK/mTOR pathway.Mol Cancer2025;24:97 PMCID:PMC11938747

[35] Tian Y,Yang Y.Crosstalk between 5-methylcytosine and N⁶-methyladenosine machinery defines disease progression, therapeutic response and pharmacogenomic landscape in hepatocellular carcinoma.Mol Cancer2023;22:5 PMCID:PMC9830866

[36] Lentini JM,Faqeih E,Fu D.DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification.Nat Commun2020;11:2510 PMCID:PMC7237682

[37] Chen R,Liu L,Zhou X.Crystal structure of human METTL6, the m³C methyltransferase.Commun Biol2021;4:1361 PMCID:PMC8642396

[38] Lee SA,Kim H.METTL8 mRNA methyltransferase enhances cancer cell migration via direct binding to ARID1A.Int J Mol Sci2021;22:5432 PMCID:PMC8196784

[39] Chang Q,Yin J.Discovery and validation of bladder cancer related excreted nucleosides biomarkers by dilution approach in cell culture supernatant and urine using UHPLC-MS/MS.J Proteomics2023;270:104737

[40] Wang S,Liu J.Integrative analysis of m3C associated genes reveals METTL2A as a potential oncogene in breast cancer.J Transl Med2022;20:476 PMCID:PMC9583565