Identification of immune-related targets of N6-methyladenosine regulators in hepatocellular carcinoma via RNA-seq analysis

Background N6-methyladenosine (m6A) regulators are involved in hepatocellular carcinoma (HCC) development. However, the functions of m6A regulators in HCC have not yet been elucidated. This study aimed to evaluate the potential of m6A methylation regulators as biomarkers and immunotherapeutic targets in HCC. Methods RNA sequencing data were obtained from publicly available databases including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Bioinformatics analysis was conducted, including Kaplan-Meier (KM) survival analysis, univariate and multivariate Cox regression, receiver operating characteristic (ROC) curve analysis and immune-related signatures were assessed using single-sample gene set enrichment analysis (ssGSEA) and drug sensitivity prediction was performed to identify potential small-molecule compounds. In vitro , small interfering RNA (siRNA)-mediated knockdown of selected m6A regulators was performed in three HCC cell lines (HepG2, HuH7 and MHCC-97H). Quantitative polymerase chain reaction (qPCR) and Western blot (WB) were subsequently conducted to validate gene expression changes. Additionally, WB was performed after treatment with candidate drugs to evaluate their functional impact. Results The five upregulated m6A regulators in HCC, KIAA1429, METTL3, PRRC2A, RBMX, and ZC3H3, were related to worse outcomes in HCC. ROC curves showed that m6A regulators could accurately distinguish between normal and HCC tumor tissues. Furthermore, differential m6A regulator expression was affected by immune infiltration. In advanced HCC patients, m6A regulators were important in determining clinical outcomes. Drug response analysis demonstrated m6A regulators as potential therapeutic agents that could be used as guidance for patients with HCC. PCR and WB analysis showed that m6A regulators could act on immune-related sites, interact with immune tolerance, and inhibit it. Conclusions This study indicates that KIAA1429, METTL3, PRRC2A, RBMX, and ZC3H3 may be potential immunotherapeutic targets and biomarkers in HCC.