Analysis and Application of m6A-Related Factors in Oral Squamous Cell Carcinoma

Abstract

Objective: This study aimed to use N6 adenosine methylation (m6A) regulatory factors to explore the prognosis of oral squamous cell carcinoma (OSCC) patients, and investigate the regulatory mechanism of m6A regulatory factors on the prognosis of OSCC patients. Methods: A new sort-based m6A comparison pair method was proposed. The prognostic model of OSCC was established using Cox regression analysis. On this basis, the model was evaluated using the receiver operator curve (ROC), and the gene mutation pattern and prognostic prediction pattern of the high-risk and low-risk groups were described, and the key genes closely related to them were screened out. RNA sequencing (RNA-seq) technology was used, combined with protein expression in the Human Protein Atlas (HPA) database and single cell detection in the Tumor Immune Single-cell Hub (TISCH) database, to find important regulatory factors. Using human squamous cell carcinoma cell line 3 (HSC-3) cells, lentivirus overexpressing WTAP or cyclin-dependent kinase inhibitor 2A (CDKN2A) were transfected and changes in HSC-3 cell viability and apoptosis were checked for. The cluster of differentiation (CD)8+ T cells were isolated from mouse spleen, transfected with WTAP siRNA, and changes in CD8+ T cell viability and apoptosis, as well as the expression levels of CDKN2A, PD-1, and TOX were checked for. Results: Univariate Cox analysis showed that risk scores significantly correlated with prognosis in OSCC (HR 1.9, p < 0.001). The ROC curve showed that the AUC (area under curve) was 0.691, 0.720 and 0.706 in 1 year, 3 years and 5 years, respectively. In the experimental group, AUC at 1 year, 3 years and 5 years were 0.680, 0.654 and 0.691, respectively. The prediction result of 6-m6A stabilized current was significantly better than that in the control group. CDKN2A was the most common mutation site in the disease (about 20%) and significantly increased in low-risk patients (p = 0.006). CDKN2A was the most commonly lost gene, accounting for more than 30%. CDKN2A/p16INK4a was significantly associated with the prognosis of OSCC (p = 0.038). GSE139324, GSE172577 and GSE103322 confirmed the high expression of CDKN2A in T lymphocytes, glandular lymphocytes and tumor cells, respectively. Results from in vitro experiments revealed that overexpressed CDKN2A significantly inhibited HSC-3 cell viability but promoted its apoptosis (p < 0.01), while overexpressed WTAP showed an opposite effect. The viability of CD8+ T cells significantly increased, and the apoptosis rate significantly decreased after WTAP siRNA transfection (p < 0.01). Furthermore, the expression levels of PD-1 and TOX also significantly decreased, while the expression of CDKN2A increased with the downregulation of WTAP (p < 0.01). Conclusions: This study is helpful to further understand the role of 6-m6A regulator in the prognosis and development of OSCC. CDKN2A is a potential prognostic marker for OSCC.