Integration of miRNA, mRNA and methylation profiling reveals genes aberrantly regulated associated to RETM918T mutation in Medullary Thyroid Carcinoma [miRNA component]

Medullary thyroid carcinoma (MTC) accounts for 1-2% of thyroid malignancies. It is a disease of few genetic drivers, and the etiology specifically associated with each mutation remains unknown. Here, we investigated the role of aberrant DNA methylation in the MTC development. We performed genome-wide DNA methylation profiling assessing >27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. We observed significant differences between the methylation patterns among the samples bearing the RETM918T, RETC634 mutation and “wild-type” (WT) tumors, but not those RAS-related. In detail, the RETM918T –related tumors had a larger number of hypomethylation events when compared to RETC634 – positive and WT ones. Moreover, through the integration of methylation with mRNA and miRNA expression data of the same tumors, we identified genes whose expression is negatively correlated with the methylation status of their promoters. For PLCB2, DKK4, MMP20 as well as miR-10a, -30a and -200c, the impact of promoter methylation levels on expression of these genes in MZ-CRC-1 and TT cell lines was assessed. Finally, the aberrant methylation validation of three of the genes in an independent set of 25 MTCs by bisulfite pyrosequencing suggests their role as MTC methylation markers. For each of the MTC tumors used for profiling, 300 ng of total RNA was amplified, then labeled with Hy3 fluorescent dye and hybridized over 16 h at 56°C onto a miRNA microarray (Exiqon, v.11.0-hsa, mmu & rho) according to the manufacturer’s instructions using miRCURY LNATM microRNA Array kit (Exiqon). The slides were washed, dried, and scanned in an Agilent microarray scanner (Agilent Technologies, Palo Alto, CA, USA).