BackgroundDNA methylation in miRNA genes has been reported as a mechanism that may cause dysregulation of mature miRNAs and consequently impact the gene expression. This mechanism is largely unstudied in papillary thyroid carcinomas (PTC).MethodsTo identify differentially methylated miRNA-encoding genes, we performed global methylation analysis (Illumina 450 K), integrative analysis (TCGA database), data confirmation (pyrosequencing and RT-qPCR), and functional assays.ResultsMethylation analysis revealed 27 differentially methylated miRNA genes. The integrative analyses pointed out miR-21 and miR-146b as potentially regulated by methylation (hypomethylation and increased expression). DNA methylation and expression patterns of miR-21 and miR-146b were confirmed as altered, as well as seven of 452 mRNAs targets were down-expressed. The combined methylation and expression levels of miR-21 and miR-146b showed potential to discriminate malignant from benign lesions (91–96% sensitivity and 96–97% specificity). An increased expression of miR-146b due to methylation loss was detected in the TPC1 cell line. The miRNA mimic transfection highlighted putative target mRNAs.ConclusionsThe increased expression of miR-21 and miR-146b due to loss of DNA methylation in PTC resulted in the disruption of the transcription machinery and biological pathways. These miRNAs are potential diagnostic biomarkers, and these findings provide support for future development of targeted therapies.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0579-8) contains supplementary material, which is available to authorized users.

Background: Papillary thyroid cancer (PTC) is the most common thyroid malignancy. DNA methylation associated with microRNAs (miRNA) regulation is an effective epigenetic mechanism for controlling gene expression. This mechanism is poorly explored in PTC. Patients and Methods: To investigate miRNAs genes regulated by methylation, global DNA methylation (Infinium® Human Methylation450 BeadChip, Illumina) analysis was performed in 50 matched PTC and normal tissues (NT) and compared with those deposited in The Cancer Genome Atlas (TCGA) (515 PTC and 56 NT). An integrative analysis was performed in 510 cases evaluated by whole methylation and miRNA expression analysis (TCGA). Using these findings, three miRNAs were investigated by RT-qPCR and pyrosequencing in PTC (N=103), normal thyroid tissues (NT, N=40) and benign thyroid lesions (BTL, N=32). In addition, seven target genes of these miRNAs were evaluated by RT-qPCR (44PTC, 30BTL and 28NT). Functional assays using three PTC cell lines (TPC1, K1 and BCPAP) were used to investigate the role of methylation in the miRNAs expression. Results: We found 50 differentially methylated probes of which 42 (27 microRNA genes) were confirmed in the TCGA database. The miRNA expression data from TCGA presented 67 differentially expressed miRNAs in PTC. The integrative analysis revealed three miRNAs (hsa-miR-146b-5p, hsa-miR-146b-3p and hsa-miR-21-5p) as candidates to be regulated by methylation. A significant hypomethylation pattern in PTC compared to NT and BTL (thyroid benign lesions) was found for these three miRNAs. Increased expression levels of hsa-miR-21-5p and hsa-miR-146b-5p were detected in PTC compared to NT and BTL. Combining these two miRNAs expression values, 45/47 PTC were distinguished from 66/67 non-malignant tissues (96% sensitivity and 99% specificity). Similarly, the association of methylation and expression data distinguished 41/45 PTC from 55/60 non-malignant tissues (91% sensitivity and 92% specificity) for MIR21 gene and 45/47 PTC from 61/61 NT/BTL (96% sensitivity and 100% specificity) for MIR146B gene. Seven target genes (MPPED2, STXBP5L, MRO, FHL1, FLRT1, DOK6 and MOB3B) of these miRNAs were significantly down regulated in PTC compared to NT and BTL. The BRAFV600E mutation was significantly associated to hypomethylation and overexpression of hsa-miR-21-5p and hsa-miR-146b-5p. Methylation and expression data in these three cell lines revealed that MIR146B is putatively regulated by methylation. Conclusion: We provide evidences that MIR21 and MIR146B genes are regulated by methylation having control in the expression of target genes associated with PTC. These miRNAs have a strong potential to be used as diagnostic markers in the clinical practice. Citation Format: Isabella Maria D. Ortiz, Mateus de Camargo Barros-Filho, Mariana B. dos Reis, Caroline Moraes Beltrami, Fabio Albuquerque Marchi, Hellen Kuasne, Clovis Pinto, Luiz Kowalski, Silvia Rogatto. MiRNAs genes are regulated by methylation in papillary thyroid carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4416.