Implications of mC modifications in ribosomal proteins on oxidative stress, metabolic reprogramming, and immune responses in patients with mid-to-late-stage head and neck squamous cell carcinoma: Insights from nanopore sequencing.

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is a malignancy characterized by a high incidence and recurrence rate. 5-methylcytosine (mC) RNA modification is a common alteration affecting cancer progression; however, how mC operates within the tumor microenvironment of HNSCC remains to be elucidated.

Methods: We conducted Nanopore sequencing on 3 pairs of cancer and paracancerous tissues from mid- and late-stage HNSCC, obtaining 132 upregulated genes (transcriptomically upregulated, mC elevated) and 129 downregulated genes (transcriptomically downregulated, mC reduced). Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed; a differential gene interaction network (PPI) was constructed, revealing the interactions of each gene with others in the network. Co-expression analysis was performed on the genes within the PPI, unveiling their expression and regulatory relationships. Through GSVA analysis, variations in related pathways under different states were identified. Furthermore, results of mC in lncRNA were screened, followed by target gene prediction.

Results: Sequencing results from the 3 pairs of mid- and late-stage HNSCC cancer and paracancerous tissues demonstrated that , , and the lncRNAs including differentiation antagonizing nonprotein coding RNA (DANCR), DCST1 antisense RNA 1 (CCDC144NL-AS1), Growth Arrest-Specific Transcript 5 (GAS5), Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), and Small Nucleolar RNA Host Gene 3 (SNHG3), etc., under mCregulation, have close connections with surrounding genes. The differentially mCmodified genes are primarily involved in ribosomal protein synthesis, oxidative stress response, metabolic reprogramming, immunity, and other life processes; pathways like mitochondrial protein import and photodynamic therapy induced unfolded protein response are upregulated in the tumor, while pathways, including the classic P53, are suppressed. Analysis on mC-regulated long non-coding RNAs (lncRNAs) revealed tight associations with A and as well.

Conclusion: Our study identifies the key factors and signaling pathways involving mC in HNSCC. The findings suggest that ribosome-related genes might regulate ribosomal protein synthesis, oxidative stress response, metabolic reprogramming, and immune response through mC RNA modification by means like hypoxia and ferroptosis, thereby playing a pivotal role in the onset and progression of HNSCC. Hence, attention should be paid to the role of ribosomes in HNSCC. These findings may facilitate the precision and individualized treatment of patients with mid- and late-stage HNSCC in clinical settings.