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

Abstract

<b>Background:</b> 5-Methylcytosine (m⁵C) plays essential roles in hepatocellular carcinoma (HCC), but the association between m⁵C regulation and immune cell infiltration in HCC has not yet been clarified. <b>Methods:</b> In this study, we analysed 371 patients with HCC from The Cancer Genome Atlas (TCGA) database, and the expression of 13 m⁵C 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. <b>Results:</b> Among the 371 patients, 41 had mutations in m⁵C regulators, the frequency of which was 11.26%. Compared with normal hepatic tissues, the expression of m⁵C regulators with copy number variations (CNVs) expansion was significantly higher than that in HCC tissues. Then, we identified three m⁵C modification patterns that had obvious tumour microenvironment (TME) cell infiltration characteristics. The prognostic analysis of the three major m⁵C 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 m⁵C 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 β (TGF-β)/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. <b>Conclusion:</b> Our work revealed the association between m⁵C 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.