Abstract

Background Nasopharyngeal carcinoma (NPC) is a widely prevalent malignant tumor with a marked tendency toward metastasis and recurrence. Ferroptosis-related genes (FRGs) are critically involved in the pathogenesis of NPC. This study aims to employ bioinformatics analysis methods to identify key genes influencing the malignant progression of NPC and to investigate the regulatory mechanisms of these genes. Methods Bulk RNA sequencing datasets (GSE53819, GSE61218, GSE64634, GSE12452, and GSE102349) and a single-cell RNA sequencing dataset (GSE150825) were downloaded from the Gene Expression Omnibus. Integrated bioinformatics analyses—including differential expression analysis, weighted gene co-expression network analysis, machine learning, and survival analysis—were conducted to identify key FRGs associated with NPC. Intracellular expression levels of adenosylhomocysteinase (AHCY), acyl-CoA synthetase long chain family member 4, glutathione peroxidase 4, macrophage stimulating 1, and Yes1 associated transcriptional regulator were detected through western blot. Cell viability was assessed using the cell counting kit-8; cell death was determined by flow cytometry; and cell migration and invasion were evaluated using wound-healing and Transwell assays. Intracellular reactive oxygen species levels were determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, and malondialdehyde and glutathione levels were detected using their respective detection kits. Results Four key FRGs—isocitrate dehydrogenase 1, AHCY, endothelial PAS domain-containing protein 1 (EPAS1), and ARHGEF26 antisense RNA 1—were identified. Survival analysis of publicly available cohorts highlighted AHCY and EPAS1 as potential biomarkers for survival in patients with NPC. We selected AHCY for further in vitro mechanistic analysis and found it to be upregulated in NPC cell lines (NPC/HK1 and c666-1) relative to the nasopharyngeal epithelial cell line NP69. Functionally, AHCY knockdow