Abstract: Objective The biomarkers and pathways of head and neck squamous cell carcinoma(HNSCC)are yet to be identified. The purpose of this study is to analyze differentially expressed genes associated with abnormal methylation in HNSCC and identify key genes and potential pathways. Methods The gene expression data set, GSE107591, and methylation data set, GSE33202, were obtained from the GEO database. The abnormally methylated genes and differentially expressed genes were screened using R, and the low methylation-high expression genes(Hypo-HGs)and high methylation-low expression genes(Hyper-LGs)were identified using the intersect function. Enrichr was used for functional and enrichment analysis of the two groups of genes. The protein-protein interaction(PPI)network was constructed using STRING and visualized using Cytoscape. Finally, we used survival analysis to identify key genes. We performed immunohistochemical analysis using CMap to identify small candidate molecules that may reverse HNSCC gene expression. Results A total of 28 hypomethylated, highly expressed genes were identified and GO enrichment analysis showed that they were mainly involved in the positive regulation of T cell chemotaxis, epidermal development, and cell-basal junction components. The results of WikiPathway analysis indicate that these genes are mainly involved in typical and atypical TGF-β signaling, blood clotting cascade, α6β4 signaling pathway, complementation and coagulation cascade, and aging and autophagy in cancer. Additionally, we found 24 genes with high methylation and low expression, which are mainly involved in GO biological processes including the regulation of angiogenesis and development, negative regulation of the interferon-γ response, negative regulation of the interferon-γ-mediated signaling pathway, and epithelial development. WikiPathway analysis further showed that these genes are mainly involved in the catalytic cycle of mammalian riboflavin monooxygenases(FMOs). Specifically, HIF1A and PPARG regulate glycolysis and metabolism of benzene and aflatoxin B1. Furthermore, key genes related to the prognosis of HNSCC were identified, namely SERPINE1, PLAU, MMRN1, LAMB3, LAMC2, PDPN, and CXCL13. Conclusion In this study, we have identified differentially expressed genes and pathways of abnormal methylation in HNSCC through biological analysis, thereby providing an important molecular basis for exploring the pathogenesis of HNSCC. Key genes, including SERPINE1, PLAU, MMRN1, LAMB3, LAMC2, PDPN, and CXCL13 can be used as abnormal methylation-based biomarkers and therapeutic targets for future diagnosis and treatment of HNSCC.

Key words: squamous cell carcinoma of the head and neck, methylation, gene expression, differential expression analysis