Uncovering the Association Between m5C Regulator-Mediated Methylation Modification Patterns and Tumour Microenvironment Infiltration Characteristics in Hepatocellular Carcinoma

Background: 5-Methylcytosine (m(5)C) plays essential roles in hepatocellular carcinoma (HCC), but the association between m(5)C regulation and immune cell infiltration in HCC has not yet been clarified. Methods: In this study, we analysed 371 patients with HCC from The Cancer Genome Atlas (TCGA) database, and the expression of 13 m(5)C regulators was investigated. Additionally, gene set variation analysis (GSVA), unsupervised clustering analysis, single-sample gene set enrichment analysis (ssGSEA), correlation analysis, and immunohistochemical (IHC) staining were performed. Results: Among the 371 patients, 41 had mutations in m(5)C regulators, the frequency of which was 11.26%. Compared with normal hepatic tissues, the expression of m(5)C regulators with copy number variations (CNVs) expansion was significantly higher than that in HCC tissues. Then, we identified three m(5)C modification patterns that had obvious tumour microenvironment (TME) cell infiltration characteristics. The prognostic analysis of the three major m(5)C modification subtypes showed that Cluster-2 had a clear survival advantage over the others. In addition, we found that DNMT1 was highly expressed in tumour tissues compared with normal tissues in a tissue microarray (TMA) and that it was positively correlated with many TME-infiltrating immune cells. High expression of the m(5)C regulator DNMT1 was related to a poor prognosis in patients with HCC. Furthermore, we developed three distinct Immu-clusters. Importantly, mRNAs related to the transcription of growth factor beta (TGF-beta)/EMT pathway were significantly up-regulated in Immu-cluster 2, indicating that this cluster is considered to be the immune rejection phenotype. Immu-cluster 3 showed elevated expression of mRNAs related to immune checkpoint genes. Conclusion: Our work revealed the association between m(5)C modification and immune regulators in the TME. These findings also suggest that DNMT1 has great potential as a prognostic biomarker and therapeutic target for HCC.