Oropharyngeal squamous cell carcinoma (OPSCC) is an aggressive malignancy often diagnosed at advanced stages and associated with regional lymph node metastasis. Inherited single nucleotide variants (SNVs) have been suggested to influence cancer susceptibility and tumor progression by modulating DNA repair and cellular homeostasis. Recently, our group identified an association between the AA genotype of the XPC c.2815A>C SNV and increased risk of OPSCC, as well as advanced nodal stage, but the molecular mechanisms underlying these associations remain unclear.

To evaluate whether distinct XPC c.2815A>C SNV genotypes modulate the expression of XPC and other cell cycle-related genes in the OPSCC cell line (FaDu).

FaDu cells were cultivated in complete Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% fetal bovine serum and 1% penicillin–streptomycin. FaDu cells were transfected with pcDNA3.1 expression vectors carrying either the wild-type (allele A) or variant (allele C) XPC c.2815A>C constructs, with the empty pcDNA3.1 vector used as a control. XPC expression was assessed at 24h, 48h, 72h, and 96h to confirm transfection efficiency. The 48h time point was selected for downstream analysis of the basal expression of cell cycle-related genes (CDKN2B, CCND1, CCND3, and STAT3), with ACTB used as the endogenous control. Gene expression was measured by real-time PCR, relative expression was calculated using the 2???Ct method, and statistical significance was assessed with the Student’s t-test (p = 0.05).

Transfection with both wild-type and variant XPC c.2815A>C constructs increased XPC expression compared with the empty pcDNA 3.1 vector (control; 1.00 arbitrary unit [AU]) at 24h (8.18 and 5.59 AUs, respectively). Expression decreased over time but remained higher than control at 48h (4.88 and 3.62 AUs), 72h (3.01 and 2.40 AUs), and 96h (1.85 and 2.08 AUs). At 48h under basal conditions, cells expressing the wild-type XPC c.2815A allele exhibited higher CDKN2B mRNA levels than cells expressing the variant c.2815C allele (1.66 vs. 1.00 AUs; p = 0.05). No consistent differences were observed for the other cell cycle–related genes assessed.

The present preliminary findings indicate that the XPC c.2815A>C SNV alters its basal expression as well the basal expression of CDKN2B cycle–related gene in OPSCC. Cells expressing wild-type XPC c.2815A>C A allele showed altered CDKN2B expression compared with the variant allele, indicating a potential genotype-dependent effect. Our data may provide a plausible explanation for the associations between XPC c.2815A>C SNV and risk of OPSCC and tumor behavior seen in a previous study conducted by our research group.