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

INTRODUCTIONRNA 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.

METHODSThe 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.

RESULTSA 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.

DISCUSSIONThis 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.