Loss of DNA methylation is related to increased expression of miR-21 and miR-146b in papillary thyroid carcinoma.

BACKGROUND: DNA 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). METHODS: To 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. RESULTS: Methylation 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. CONCLUSIONS: The 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.

Genes codificadores de microRNAs regulados por metilação em carcinomas papilíferos de tireoide / MicroRNAs genes regulated by methylation in papillary thyroid carcinomas

O carcinoma papilífero (CPT) é o tipo histológico mais frequente entre os cânceres de tiroide. Geralmente são doenças indolentes, entretanto, não são conhecidas opções terapêuticas eficazes nos casos agressivos. A metilação de DNA, juntamente com a regulação por microRNAs (miRNA) são considerados mecanismos efetores do controle epigenético da expressão gênica e ainda pouco explorados em CPT. Estudos prévios de metilação global revelaram, entre outros achados, a presença de genes de miRNAs alterados por metilação. O objetivo deste estudo foi identificar e confirmar o papel de genes de miRNAs em CPT utilizando os dados prévios do grupo e os depositados no The Cancer Genome Atlas (TCGA). Para isso, o estudo foi dividido em três módulos principais. No primeiro módulo, os dados do estudo prévio foram reanalisados sendo identificadas 50 sondas diferencialmente metiladas, das quais 42 (27 genes de microRNAs) foram confirmadas pelo banco de dados do TCGA. Os dados de expressão de miRNAs provenientes do TCGA permitiu identificar 67 miRNAs diferencialmente expressos. Utilizando uma análise integrada (dados de metilação e expressão de miRNAs do TCGA), três miRNAs foram selecionados (hsa-miR-146b-5p, hsa-miR-146b-3p e hsa-miR-21-5p) como candidatos a serem regulados por metilação. Estes miRNAs apresentaram expressão aumentada e sondas diferencialmente metiladas (hipometilação). As interações entre miRNAs e mRNAs (dados do TCGA), revelaram 348 transcritos com correlação negativa e preditos como alvos dos três miRNAs (162 para o hsa-miR-146b-5p; 172 para o hsa-miR-146b-3p; 104 para o hsamiR-21-5p). No segundo módulo, os miRNAs alterados foram confirmados por pirosequenciamento e RT-qPCR. Foi observado um padrão de hipometilação significativo nos CPT em relação ao TN e LBT (lesões benignas de tireoide). A análise de RT-qPCR revelou um aumento significativo da expressão de hsa-miR-21-5p e hsa-miR-146b-5p nas amostras tumorais (N=103) em relação ao TN (N=40) e LBT (N=32). A expressão combinada dos dois miRNAs permitiu a distinção de 45/47 CPT dos 66/67 tecidos não-malignos (96% de sensibilidade e 99% de especificidade). A correlação entre os dados de metilação e expressão do gene MIR21 classificou 41/45 CPT dos 55/60 tecidos não malignos (91% de sensibilidade e 92% de especificidade). Para o gene MIR146B foi verificada a distinção entre os 45/47 CPT dos 61/61 NT/LBT (96% de sensibilidade e 100% de especificidade). Foram também selecionados e analisados por RTqPCR sete genes alvos destes miRNAs (MPPED2, STXBP5L, MRO, FHL1, FLRT1, DOK6 e MOB3B). Foram detectadas expressões significativamente mais baixas em CPT quando comparado a TN e LBT para todos os genes selecionados. Os achados de metilação e expressão dos miRNAs e mRNAs foram associados com dados clínicos-patológicos. A mutação BRAFV600E foi significativamente associada com a hipometilação e expressão aumentada de hsa-miR-21-5p e hsa-miR-146b-5p. No terceiro módulo, foi avaliado o papel da metilação na expressão dos miRNAs em três linhagens celulares de CPT (TPC1, K1 e BCPAP). Após a análise do nível de metilação basal dos MIR146B e MIR21, as células foram tratadas com 1µM e 3µM de 5-Aza-2'-deoxicitidina. A desmetilação após tratamento foi confirmada por pirosequenciamento em sequências repetitivas das regiões AluYb8 e AIR1Sat. A metilação e expressão do gene MIR146B nas três linhagens celulares revelou o MIR146B como potencialmente regulado por metilação. Este estudo deu fortes evidências que os genes MIR21 e MIR146B são regulados por metilação e atuam regulando genes alvos críticos envolvidos em CPT. Estes miRNAs tem potencial para serem utilizados na prática clínica como marcadores diagnósticos

Papillary thyroid carcinoma (PTC) is the most common histological type among thyroid cancers. Although PTC usually represents an indolent disease, no effective treatment has been described in aggressive cases. DNA methylation associated with microRNAs (miRNA) regulation is an effective epigenetic mechanism for controlling gene expression and poorly explored in CPT. Our previous studies using global DNA methylation analysis revealed, among other results, miRNAs regulated by methylation. The objective of this study was to identify and confirm the role of miRNAs genes in CPT using our previous data and those deposited in The Cancer Genome Atlas (TCGA). Therefore, this study was divided in three main modules. In the first module, data from the previous study were reanalyzed and 50 differentially methylated probes were identified, of which 42 (27 microRNA genes) were confirmed using the TCGA data. The miRNA expression data from TCGA allowed us to identify 67 differentially expressed miRNAs. An integrative analysis (methylation and expression data of miRNAs from TCGA) was performed and three miRNAs (hsa-miR-146b-5p, hsa-miR-146b-3p and hsa-miR-21-5p) were selected as candidates to be regulated by methylation. These miRNAs were overexpressed and hypomethylated. Predicted miRNAmRNA interactions (TCGA data) revealed 348 transcripts with negative correlation with three miRNAs (162 for hsa-miR-146b-5p; 172 for hsa-miR146b-3p; 104 for hsa-miR-21-5p). In the second module, the altered miRNAs were confirmed by pyrosequencing and RT-qPCR. A significant hypomethylation pattern in PTC compared to NT and TBL (thyroid benign lesions) was observed. The RT-qPCR analysis revealed a significant increase of hsa-miR-21-5p and hsa-miR-146b-5p expression in tumors (N=103) compared to NT (N=40) and TBL (N=32). Combining these two miRNAs expression values was possible to discriminate 45/47 PTC from 66/67 non-malignant tissues (96% sensitivity and 99% specificity). Similarly, the correlation between methylation and expression 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/TBL (96% sensitivity and 100% specificity) for MIR146B gene. Seven of these miRNAs target genes (MPPED2, STXBP5L, MRO, FHL1, FLRT1, DOK6 and MOB3B) were selected and analyzed by RT-qPCR. All of them were significantly down regulated in PTC compared to NT and TBL. The miRNAs and mRNAs methylation and expression findings were also correlated with clinical and pathological data. The BRAFV600E mutation was significantly associated to hypomethylation and overexpression of hsa-miR-21-5p and hsa-miR-146b5p. In the third module, the methylation role in the expression of these two miRNAs was evaluated in three PTC cell lines (TPC1, K1 and BCPAP). After evaluating the basal methylation levels of MIR146B and MIR21, the cell lines were treated with 1µM and 3µM of 5-Aza-2'-deoxycytidine. The repetitive AluYb8 and AIR1Sat regions were confirmed as demethylated using pyrosequencing. Methylation and expression data in these three cell lines revealed that MIR146B is putatively regulated by methylation. This study provides strong evidence that MIR21 and MIR146B genes are regulated by methylation, controlling crucial target genes involved in PTC. Moreover, these miRNAs have potential to be used as diagnostic markers in the clinical practice