ORAL SQUAMOUS CELL CARCINOMA METHYLATION ANALYSIS IN DIFFERENT REGIONS OF TUMOR SUPPRESSOR GENES

Introduction: DNA methylation at the promoter region of tumor suppressor genes is a frequent event in oral squamous cell carcinoma (OSCC), but the methylation role at other gene regions remains unclear. Aim: We investigated the methylation profile at different regions of tumor supressor genes and correlated our findings with clinical-pathological information. Methodology: In the HumanMethylation450 Beadchip platform, we used 53 specimens from OSCC and 53 from adjacent nonneoplastic tissues. Methylation level was compared considering ∆β ≤-0.10 or ≥0.10, p<0.01, and FDR <0.01. We validated our results using TCGA data and the pyrosequencing of an independent set of patients, including 15 OSCC and 15 adjacent non-neoplastic tissues. Results: We observed hypermethylation at OSCC and adjacent non-neoplastic tissues for the following genes: BRCA1, FHIT, MGMT, RASSF1, and RUNX3 at promoter regions, 5’UTR or 1st exon, and CDH13, DAPK1, HOXA1, PDLIM4, RARB, TIMP3, TP73, and VHL at the gene body or 3’UTR. We found differentially methylated sites within OSCC and adjacent non-neoplastic tissues for BRCA1, FHIT, and HOXA1. After pyrosequencing validation, only a CpG site at HOXA1 – 3’UTR region showed a significant difference (p <0.01) between OSCC and adjacent non-neoplastic tissues, 55.5%, and 36.9%, respectively. HOXA1 – 3’UTR methylation was positively correlated with smoking burden (p=0.04), relapse (p=0.03) and higher expression (FC=10.58). Conclusion: Our in-silico analysis was effective to detect methylation targets and to compare groups. The methylation at different regions of tumor suppressor genes is a frequent event in OSCC, however, little difference is observed within tumor and adjacent non-neoplastic tissues. Methylation of HOXA1 – 3’UTR CpG site is closely associated with HOXA1 aberrant expression and OSCC recurrence.