Deciphering the Divergent Gene Expression Landscapes of m6A/m5C/m1A Methylation Regulators in Hepatocellular Carcinoma Through Single-Cell and Bulk RNA Transcriptomic Analysis

Introduction: RNA modifications mediated by the m6A, m1A, and m5C regulatory genes are crucial for the progression of malignancy. This study aimed to explore the expression of regulator genes for m6A/m5C/m1A methylation at the single-cell level and to validate their expression in cancerous and adjacent para-cancerous liver tissues of adult patients with HCC who underwent tumor resection. Methods: The bulk sequencing from The Cancer Genome Atlas (TCGA) database and the single-cell RNA sequencing (scRNA-seq) data obtained from the Gene Expression Omnibus (GEO) database were used to identify the dysregulated m6A/m5C/m1A genes for hepatocellular carcinoma (HCC). A real-time polymerase chain reaction (real-time PCR) was used to measure the expression of dysregulated m6A/m5C/m1A genes in collected human HCC tissues and compared with adjacent para-cancerous liver tissues. Immune cell infiltration with these significantly expressed methylation-related genes was evaluated using Timer2.0. Results: A discrepancy in m6A/m5C/m1A gene expression was observed between bulk sequencing and scRNA-seq. The clustered heatmap of the scRNA-seq-identified dysregulated m6A/m5C/m1A genes in TCGA cohort revealed heterogeneous expression of these methylation regulators within the cancer, whereas their expression in the adjacent liver tissues was more homogeneous. The real-time PCR validated the significant overexpression of DNMT1, NSUN5, TRMT6, IGF2BP1, and IGFBP3, which were identified using scRNA-seq, and IGFBP2, which was identified using bulk sequencing. These dysregulated methylation genes are mainly correlated with the infiltration of natural killer cells. Discussion: This study suggests that cellular diversity inside tumors contributes to the discrepancy in the expression of methylation regulator genes between traditional bulk sequencing and scRNA-seq. This study identified five regulatory genes that will be the focus of further studies regarding the function of m6A/m5C/m1A in HCC.