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

Hang-Tsung Liu; Cheng-Shyuan Rau; Yueh-Wei Liu; Ting-Min Hsieh; Chun-Ying Huang; Peng-Chen Chien; Hui-Ping Lin; Chia-Jung Wu; Pei-Chin Chuang; Ching-Hua Hsieh

doi:10.2147/JHC.S448047

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

J Hepatocell Carcinoma. 2023 Dec 28:10:2383-2395. doi: 10.2147/JHC.S448047. eCollection 2023.

Authors

Affiliations

¹ Department of Trauma Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan.
² Department of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan.
³ Department of General Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan.
⁴ Department of Plastic Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan.
⁵ Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, 83301, Taiwan.

^# Contributed equally.

Abstract

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.

Keywords: 5-methylcytidine; HCC; N1-methyladenosine; N6-methyladenosine; bulk sequencing; hepatocellular carcinoma; m1A; m5C; m6A; methylation; scRNA-seq; single-cell RNA sequencing.

Grants and funding

This research was funded by the Chang Gung Memorial Hospital (grant numbers CMRPG8N0031 & CMRPG8L1261 to Hang-Tsung Liu).