Caracterização de mutações no gene MED12, dos miRNAS mapeados em 7q22 e dos candidatos a regulação do gene HMGA2 em leiomiomas uterinos

Leiomiomas uterinos (LU) são tumores benignos de origem mesenquimal frequentes sendo considerados um problema de saúde pública. A desregulação de fatores de crescimento e microRNAs, encurtamento dos telomeros, produção excessiva e desorganizada de matriz extracelular, perda de heterozigose e alterações cromossômicas recorrentes (como deleção em 7q22 e rearranjo cromossômico em 12q15) contribuem para o crescimento dos LU. Uma parcela significativa destes tumores apresenta mutação no gene MEDI2. O presente estudo teve como objetivo avaliar: a presença da mutação no MEDI2; o nível de expressão do HMGA2 e seus miRNAs preditos (let-7a, miR-26a, miR-26b); o nível de expressão do, cluster de miRNAs mapeado em 7q22 (miR-25, miR-93 e miR- 106b) e a expressão dos genes BRCAI, FANCA e seus miRNAs preditos. Foram avaliados 85 LU e 34 amostras de miométrio adjacentes obtidos de 54 pacientes submetidos à histerectomia. A análise de expressão por RT-qPCR foi realizada para os miR-let7a, miR-25, miR-93, miR-106b, miR-21, miR-26a, miR-26b, miR-197 e miR- 143, utilizando RNU44 , RNU48 e U47 como endógenos. Foi também avaliada a expressão dos genes HMGA2, BRCAI e FANCA sendo utilizados como controles endógenos RPLPO e GUSB. A mutação no MED12 foi encontrada em 50% (42/85) das amostras. Foi observado um aumento significativo (p<0,001) dos níveis de expressão do HMGA2 nos LU comparados ao miométrio adjacente, inclusive nas amostras com presença da mutação em MEDI2. Estes dados sugerem que o aumento de expressão do HMGA2 e a mutação em MEDI2 podem coexistir nestes tumores. Os miRNAs preditos para regular o HMGA2 apresentaram diminuição dos níveis de expressão: let-7a (p<O,OOl), miR-26a (p<O,OOl), miR-26b (p<O,OOl). A forte correlação negativa entre HMGA2 e seus miRNAs preditos suporta a evidência de interação entre eles. Os miRNAs mapeados...Uterine Leiomyomas (UL) are common mesenchymal benign tumors that represent a significant public health problem. The deregulation of growth factors and microRNAs (miRNAs), shortening of telomeres, excessive production of disorganized extracellular matrix, loss of heterozygosity and recurrent chromosomal aberrations (including 7q22 deletion and chromosomal rearrangements in 12q 15) have been suggested to contribute to the growth of UL. A significant number of tumors presented mutations of MED I2The aims of this study were: to investigate MED12 mutation; to evaluate the expression levels of HMGA2 and their miRNAs predicted (let-7a, miR-26a, miR-26b); the expression of miRNAs mapped at 7q22 (miR-25, miR-93, and miR- 106b) and the expression levels of BRCAI, FANC,A and their predictive miRNAs. Eight-five fresh frozen UL and 34 adjacent normal myometrium (MM) were obtained from 54 patients who had undergone a hysterectomy procedure. Quantitative real time RT-PCR was applied to evaluate the expression levels of miRs (miR-let7a, miR-25, miR-93, miR- 106b, miR-21, miR-26a, miR-26b, miR-197 and miR-143) using RNU44, RNU48 and U47 as endogenous control and to evaluate the expression of HMGA2, BRCAI and FANCA using RPLPO and GUSB as endogenous control. We detected 50% (42/85) of samples with MEDI2 mutation. A significant overexpression (p<0,001) of HMGA2 was detected in UL compared with MM, including samples with the presence of MEDI2 mutation. These data indicated that overexpression of HMGA2 may coexist with MEDI2 mutation. The miRNAs predicted to regulate HMGA2 were found significantly downregulated: let-7a (p<0,001), miR-26a (p<0,001), miR-26b (p<0,001). The negative correlation verified between HMGA2 and their predicted miRNAs support the evidence of interaction between them. The miRNAs mapped at 7q22 (miR-25, miR-93, and miR- 106b) were found as downregulated... (Complete abstract click electronic access below

PFKFB2 Promoter Hypomethylation as Recurrence Predictive Marker in Well-Differentiated Thyroid Carcinomas

Despite the low mortality rates, well-differentiated thyroid carcinomas (WDTC) frequently relapse. BRAF and TERT mutations have been extensively related to prognosis in thyroid cancer. In this study, the methylation levels of selected CpGs (5-cytosine-phosphate-guanine-3) comprising a classifier, previously reported by our group, were assessed in combination with BRAF and TERT mutations. We evaluated 121 WDTC, three poorly-differentiated/anaplastic thyroid carcinomas (PDTC/ATC), 22 benign thyroid lesions (BTL), and 13 non-neoplastic thyroid (NT) tissues. BRAF (V600E) and TERT promoter (C228T and C250T) mutations were tested by pyrosequencing and Sanger sequencing, respectively. Three CpGs mapped in PFKFB2, ATP6V0C, and CXXC5 were evaluated by bisulfite pyrosequencing. ATP6V0C hypermethylation and PFKFB2 hypomethylation were detected in poor-prognosis (PDTC/ATC and relapsed WDTC) compared with good-prognosis (no relapsed WDTC) and non-malignant cases (NT/BTL). CXXC5 was hypomethylated in both poor and good-prognosis cases. Shorter disease-free survival was observed in WDTC patients presenting lower PFKFB2 methylation levels (p = 0.004). No association was observed on comparing BRAF (60.7%) and TERT (3.4%) mutations and prognosis. Lower PFKFB2 methylation levels was an independent factor of high relapse risk (Hazard Ratio = 3.2; CI95% = 1.1&ndash;9.5). PFKFB2 promoter methylation analysis has potential applicability to better stratify WDTC patients according to the recurrence risk, independently of BRAF and TERT mutations

Characterization of BRAF mutation in patients older than 45 years with well-differentiated thyroid carcinoma

Introduction: Papillary thyroid carcinoma is the most frequent endocrine neoplasia and its incidence has tripled over the past 35 years. Although papillary thyroid carcinoma carries a good prognosis, 10%−30% of patients still develop recurrence and metastasis. Some clinical and genetic features are associated with worse prognosis. The most frequent mutation is the BRAF p.V600E, which has been associated with many clinical features of poor prognosis. However, many studies have produced controversial results without any association between BRAF mutation and clinicopathological features of poor prognosis. Objective: Since the prognostic value of BRAF mutations remains controversial, this study aims to investigate the importance of this mutation in therapeutic decisions for papillary thyroid carcinoma. Methods: Therefore, we evaluated whether the presence of BRAF mutation is associated with features of poor prognosis in 85 patients with papillary thyroid carcinoma older than 45 years treated at A.C. Camargo Cancer Center, from 1980 to 2007. BRAF mutation was evaluated by pyrosequencing. Statistical analysis was performed using SPSS. Results: The mean age of patients was 54 years (range: 45 − 77 years), 73 were women (85.8%) and 12 were men (14.2%). Among them, 39 cases (45.9%) presented extrathyroidal extension and 11 cases had recurrent disease. BRAF mutation was detected in 57 (67%) patients. No significant association was observed between BRAF mutation and gender (p  = 0.743), age (p  = 0.236), N-stage (p  = 0.423), vascular and perineural infiltration (p  = 0.085 or multifocality (p  = 1.0). Although not statistically significant, the majority of patients with recurrent disease were BRAF positive (9 out of 11) (p  = 0.325). Patients affected by BRAF mutation are associated with tumors larger than 1 cm (p  = 0.034) and with extrathyroidal extension (p  = 0.033). Conclusion: Although BRAF testing is widely available, there are no consistent data to support improvement in outcomes from incorporating it into therapeutic decision for thyroid cancer

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

Abstract 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