RAP1GAP Functions as a Tumor Suppressor Gene and Is Regulated by DNA Methylation in Differentiated Thyroid Cancer

Abstract

Inactivation of tumor suppressor genes, such as RAP1GAP, by hypermethylation of their regulatory region can give rise to thyroid tumors. The aim of this study was to investigate the expression of the RAP1GAP gene and the DNA methylation patterns of its CpG74_a, CpG74_b, and CpG24 in an Iranian population with differentiated thyroid cancer (DTC). In this study, 160 individuals who underwent thyroidectomy in the Tehran Erfan Hospital between 2018 and 2020 were selected. DNA methylation patterns of selected CpG islands (CpG74_a, CpG74_b, and CpG24) were determined using methylation-specific PCR. The mRNA expression and protein level of ­RAP1GAP were also evaluated. SW1736 and B-CPAP cells were treated with 5-aza-2′-deoxycytidine (5-Aza) to demethylate these regions. The hypermethylation rates of CpG74_a and CpG24 in DTC samples were significantly higher than in the control. The mRNA expression and protein level of ­RAP1GAP were significantly decreased in the DTC group. In the DTC group, hypermethylation in CpG74_a was correlated with decreasing RAP1GAP expression (R²: 0.34; p = 0.043). CpG74_a with a specificity of 86.4% has significant prediction power to distinguish between DTC and normal thyroid tissues. Additionally, hypermethylation of CpG74_a was significantly associated with higher tumor stages (stage III-IV: 77%; stage I-II: 23%; p = 0.012). Increasing expression of RAP1GAP after demethylation with 15 µM of 5-Aza was observed in both cell lines. These results indicate that DNA hypermethylation in CpG74_a can be considered as an epigenetic biomarker in DTC.