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

Abstract

Hang-Tsung Liu,1,* Cheng-Shyuan Rau,2,* Yueh-Wei Liu,3 Ting-Min Hsieh,1 Chun-Ying Huang,1 Peng-Chen Chien,4 Hui-Ping Lin,4 Chia-Jung Wu,4 Pei-Chin Chuang,5 Ching-Hua Hsieh4 1Department of Trauma Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan; 2Department of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan; 3Department of General Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan; 4Department of Plastic Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, 83301, Taiwan; 5Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, 83301, Taiwan*These authors contributed equally to this workCorrespondence: Pei-Chin Chuang; Ching-Hua Hsieh, Tel [phone], Email [email protected]; [email protected]: 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: hepatocellular carcinoma, HCC, methylation, N6-methyladenosine, m6A, N1-methyladenosine, m1A, 5-methylcytidine, m5C, bulk sequencing, single-cell RNA sequencing, scRNA-seq

Keywords

• hepatocellular carcinoma (hcc)
• methylation
• n6-methyladenosine (m6a)
• n1-methyladensoine (m1a)
• 5-methylcytidine (m5c)
• bulk sequencing
• single-cell rna sequencing (scrna-seq)