RESUMO
BACKGROUND: The inactivation of the tumor-suppressor homeodomain-only protein X (HOPX) usually involves promoter methylation in several cancer types. This study aimed to investigate the HOPX-ß mRNA expression and promoter methylation and their clinical relevance in differentiated thyroid cancer (DTC). PATIENTS AND METHODS: Clinicopathological data and paraffin-embedded thyroid tumor tissues from 21 patients with DTC and 6 with benign tumors (T) and their non-tumor parenchyma (NT) were investigated. Tumor cell lines (FTC238, FTC236 and WRO) were treated with demethylating agent. HOPX-ß mRNA expression was assessed by qRT-PCR and methylation status by Q-MSP. Thyroid cancer data from Cancer Genome Atlas (TCGA) was also collected. RESULTS: HOPX-ß mRNA re-expression in two cell lines treated with demethylating agent was observed concomitantly with reduced promoter methylation. Reduced mRNA expression in T group compared to their NT was observed, and reduced protein expression in T compared to NT was observed in three cases. Low mRNA expression with high methylation status was detected in 6/14 DTC samples. High methylation status was associated with older age at diagnosis, recurrent or progressive disease and with the presence of new neoplasm event post initial therapy while hyper-methylation correlated with worse overall survival, worse disease-free status and older age. CONCLUSION: A moderate coupling of downregulation of HOPX-ß mRNA expression in DTC followed by high HOPX-ß promoter methylation was observed however; high HOPX promoter methylation status was associated with the worse prognosis of DTC patients.
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We previously reported that ABI3 expression was decreased in thyroid cancer tissues and that ectopic expression of ABI3 in a follicular thyroid carcinoma cell line delayed cell cycle progression and inhibited cell proliferation, invasion, migration and tumor formation in athymic mice. These data indicated that ABI3 is a tumor suppressor gene; however the mechanism through which ABI3 is silenced in thyroid carcinomas is unknown. We here show that treatment of four follicular thyroid carcinoma cell lines with 5-aza-dC induced demethylation of a specific region of the ABI3 promoter and restored ABI3 expression. In contrast, 5-aza-dC treatment did not restore ABI3 expression in a non-thyroid cell line, suggesting a tissue-specific regulation. We additionally show that 8 CpG sites located within the ABI3 promoter are hypermethylated in most thyroid carcinoma samples and the degree of methylation correlated with ABI3 expression. Narrowing the region to specific CpG sites, the CpG4-6 sites showed the largest difference between benign and malignant lesions. In silico analysis revealed that these CpG sites flank a canonical binding site for NKX2-1, a thyroid specific transcriptional factor. Analysis of thyroid samples shows a correlation between NKX2-1 and ABI3 expression. In vitro assays demonstrate that NKX2-1 was required for ABI3 expression. Luciferase assay further confirmed the promoter activity of this region, which was increased when the cells were co-transfected with NKX2-1. Our study shows that the transcriptional silencing of ABI3 in cancer cells occurs via methylation and uncovered a previously unrecognized role for NKX2-1 in the regulation of ABI3.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias da Glândula Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ilhas de CpG , Inativação Gênica , Humanos , Regiões Promotoras Genéticas , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Roux-en-Y gastric bypass (RYGB) modifies the anatomical structure of the upper intestine tract, reduces gastric acid secretion, and may impair LT4 absorption. The aim of this study was to evaluate the LT4 absorption in morbidly obese patients before and after RYGB. METHODS: Thirty morbidly obese patients were divided in two groups: The NS group included 15 patients before RYGB surgery (BMI = 43.1 ± 4 kg/m(2)), and the S group included 15 patients after surgery (BMI = 37.3 ± 4 kg/m(2)). Two baseline samples were collected, and 600 µg of oral LT4 tablets were administered. Blood samples were collected at 30, 60, 120, 180, 240, 300, and 1440 min. Serum-free T4 (FT4), total T4 (TT4), and TSH were measured at each time point. The increase in TT4, FT4, and TSH (ΔTT4, ΔFT4, and ΔTSH) was calculated, subtracting from the baseline mean value. RESULTS: The pharmacokinetics parameters regarding LT4 absorption, maximum ΔTT4, and area under the curve(AUC) of both ΔTT4 and ΔFT4 were significantly higher in the S group compared with the NS group (p < 0.05). It was observed, however, that there was a significant delay in the absorption of LT4 in the S group. Basal serum TSH and leptin levels were higher in the NS group (p = 0.016 and 0.026, respectively), whereas basal serum TT4, FT4, ΔTSH, and the AUC of ΔTSH were similar between groups. CONCLUSIONS: In this study, we have demonstrated that Roux-en-Y bypass surgery does not diminish LT4 absorption. A small but significant delayed absorption of LT4, however, was observed in patients after surgery.
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Derivação Gástrica , Hipotireoidismo/tratamento farmacológico , Absorção Intestinal/efeitos dos fármacos , Obesidade Mórbida/metabolismo , Obesidade Mórbida/cirurgia , Tiroxina/farmacocinética , Adolescente , Adulto , Estudos de Coortes , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Humanos , Hipotireoidismo/complicações , Hipotireoidismo/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/tratamento farmacológico , Obesidade Mórbida/etiologia , Cuidados Pós-Operatórios , Cuidados Pré-Operatórios , Estudos Prospectivos , Tiroxina/metabolismo , Redução de Peso , Adulto JovemRESUMO
O iodo é transportado para a célula tireoideana através do simportador sódio-iodo (NIS). Estudo anterior de nosso grupo mostrou que a expressão do RNAm de NIS estava reduzida nos tecidos tumorais (T) tireoideanos quando comparada à dos tecidos não tumorais (NT) adjacentes. A metilação de ilhas CpG localizadas em promotores é um dos mecanismos epigenéticos que regula a expressão gênica. Já foi demonstrado que a metilação aberrante desempenha papel importante na tumorigênese humana, porém, estudos qualitativos não conseguiram definir um padrão de metilação do promotor de NIS no câncer diferenciado de tireóide. Assim, o presente estudo visou (1) investigar o padrão de metilação do promotor do gene NIS em amostras T (benignos e malignos) e NT adjacentes, e correlacionar o grau de metilação com os valores de RNAm, e (2) identificar novas sequencias regulatórias, alvos de metilação do DNA, que pudessem também modular a expressão de NIS. Foram estudados 30 pares de amostras de tecido tireoideano T e NT adjacente, sendo 10 benignos e 20 malignos. Também foi avaliado um par de amostras de paciente com nódulo maligno com alta captação de iodo (hipercaptante). A metilação da ilha1 CpG foi avaliada por PCR Metilação Específica (MSP) semiquantitativa e a metilação da ilha2 CpG foi analisada por bissulfito seqüenciamento; ambas com DNA bissulfito convertido. Novas ferramentas de bioinformática foram utilizadas na análise in silico para identificar ilhas CpG funcionais na região 5' upstream da seqüência promotora de NIS. Plasmídios foram construídos contendo o fragmento de DNA da Ilha2 CpG na frente de gene reporter (luciferase) e usados em transfecções de células HEK293 para avaliar a atividade regulatória desta ilha...
Iodide is transported from the blood into thyroid cell through the sodium iodide symporter (NIS). We have previously detected reduced NIS mRNA expression in thyroid tumoral tissue (T) when compared with the non tumoral tissue (NT). Methylation of DNAs CpG islands, very common in promoters, is an epigenetic alteration that regulates gene expression. Aberrant gene methylation plays an important role in human tumorigenesis. Previous qualitative studies have failed to define a NIS promoter methylation pattern in thyroid cancer. This study aimed (1) to investigate the methylation pattern of the NIS gene promoter in thyroid tumors (benign and malignant), and to correlate the methylation status with cellular levels of NIS mRNA, in T and adjacent NT samples; (2) to identify new regulatory DNA sequences that could modulate NIS gene expression by methylation of the DNA. Thirty pairs of thyroid samples (tumoral and non-tumoral), 10 benign and 20 malignant tumors were included. A pair of samples of a malignant nodule with high iodide uptake was also included. Methylation of CpG Island1 was evaluated by semiquantitative Methylation Specific PCR (MSP) and methylation of CpG island2 was analyzed by bisulfite sequencing; both using bisulfite converted DNA. New bioinformatic tools were used in in silico analysis to identify functional CpG islands in the 5 upstream region of the NIS promoter. Plasmids were constructed containing the CpG island2 DNA fragment in front of a reporter gene (lucyferase) and used in transfections assays of HEK293 cells to assess the regulatory activity of this island. Tumor thyroid cell cultures with low NIS expression were treated with the demethylating agent 5Aza and NIS mRNA expression was quantified by real time PCR. Methylation of CpG island1 was detected in all the samples (NT and T) of benign and malignant group, no differences were observed in the methylation degree between the groups...
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Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Metilação de DNA , Iodo , Simportadores , Neoplasias da Glândula TireoideRESUMO
OBJECTIVE: To extend the molecular analysis of the IVS30+1G>T intronic thyroglobulin (TG) mutation, and to report the eleven year follow-up of the affected patients. METHOSD: Two siblings with severe congenital hypothyroidism with fetal and neonatal goiter, harboring the IVS30+1G>T mutation were included. Nodular and non-nodular thyroid tissue specimens were collected. Specific thyroid genes expression was evaluated by real-timePCR and by immunohistochemistry. RESULTS: In non-nodular tissue specific thyroid genes mRNA were reduced when compared to normal thyroid sample. In the nodule, TPO and NIS expression was very low. Microscopic examinations showed very large follicular-lumina and swollen vesicles of endoplasmatic-reticulum. Strong cytoplasmatic and low follicular-lumen TG immunostaining were detected. Intracellular NIS, membrane TPO and TSHR immunostaining had higher positivity in non-nodular sample. Both patients had a long-term adequate developmental outcome, besides one patient have been lately-treated. CONCLUSIONS: IVS30+1G>T mutation not only lead to very enlarge endoplasmatic-reticulum, but also to alterations of specific thyroid genes expression. The clinical evolution of patients harboring these mutations strengthen the concept of the influence of environment, like iodine nutrition, to determine the final phenotypic appearance.
OBJETIVO: Aprofundar a análise molecular da mutação intrônica IVS30+1G>T do gene tireoglobulina (TG) e relatar a clínica de pacientes portadores da mutação, acompanhados por 11 anos. MÉTODOS: Foram estudados dois irmãos com hipotireoidismo congênito grave com bócio fetal e bócio neonatal, portadores da mutação IVS30+1G>T. Foram coletadas amostras de tecido nodular e não-nodular. Avaliou-se a expressão de genes específicos da tireóide por PCR em tempo real e imunohistoquímica. RESULTADOS: A expressão de genes específicos da tireóide foi menor no tecido não-nodular que no tecido normal controle. Expressões de TPO e NIS foram extremamente baixas no tecido nodular. Verificou-se lúmen folicular aumentado com grandes vesículas de retículo endoplasmático, e detectou-se forte marcação de TG no citoplasma e fraca no lúmen folicular. No tecido não-nodular observou-se forte positividade de NIS intracelular e, TPO e TSHR na membrana plasmática. O acompanhamento em longo prazo dos pacientes mostrou adequado desenvolvimento, apesar de um deles ter recebido tratamento tardio. CONCLUSÕES: A mutação IVS30+1G>T não só promove alterações no retículo endoplasmático, como alterações na expressão de genes específicos da tireóide. A evolução clínica destes pacientes reforça o conceito da influência do meio ambiente, como o aporte nutricional de iodo, no fenótipo final.
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Adolescente , Criança , Feminino , Humanos , Masculino , Hipotireoidismo Congênito/genética , Mutação , Nódulo da Glândula Tireoide , Tireoglobulina/genética , Seguimentos , Irmãos , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/patologiaRESUMO
CONTEXT: The expression of sodium iodide symporter (NIS) is required for iodide uptake in thyroid cells. Benign and malignant thyroid tumors have low iodide uptake. However, previous studies by RT-PCR or immunohistochemistry have shown divergent results of NIS expression in these nodules. OBJECTIVE: The objective of the study was to investigate NIS mRNA transcript levels, compare with NIS and TSH receptor proteins expression, and localize the NIS protein in thyroid nodules samples and their surrounding nonnodular tissues (controls). DESIGN: NIS mRNA levels, quantified by real-time RT-PCR, and NIS and TSH receptor proteins, evaluated by immunohistochemistry, were examined in surgical specimens of 12 benign and 13 malignant nodules and control samples. RESULTS: When compared with controls, 83.3% of the benign and 100% of the malignant nodules had significantly lower NIS gene expression. Conversely, 66.7% of the benign and 100% of malignant nodules had stronger intracellular NIS immunostaining than controls. Low gene expression associated with strong intracellular immunostaining was most frequently detected in malignant (100%) than benign nodules (50%; P = 0.005). NIS protein was located at the basolateral membrane in 24% of the control samples, 8.3% of the benign, and 15.4% of the malignant nodules. The percentage of benign nodules with strong TSH receptor positivity (41.6%) was higher than malignant (7.7%). CONCLUSION: We confirmed that reduced NIS mRNA expression in thyroid malignant nodules is associated with strong intracellular protein staining and may be related to the inability of the NIS protein to migrate to the cellular basolateral membrane. These results may explain the low iodide uptake of malignant nodules.
Assuntos
Carcinoma Papilar/genética , Espaço Intracelular/metabolismo , RNA Mensageiro/análise , Coloração e Rotulagem , Simportadores/genética , Simportadores/metabolismo , Neoplasias da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/genética , Adulto , Idoso , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Membrana Celular/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Transporte Proteico , RNA Mensageiro/metabolismo , Receptores da Tireotropina/metabolismo , Coloração e Rotulagem/métodos , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/metabolismo , Nódulo da Glândula Tireoide/patologia , Distribuição TecidualRESUMO
OBJECTIVE: To evaluate the prevalence of chronic autoimmune thyroiditis (CAT) and iodine-induced hypothyroidism, hyperthyroidism (overt and subclinical), and goiter in a population exposed to excessive iodine intake for 5 years (table salt iodine concentrations: 40-100 mg/kg salt). Design This was a population-based, cross-sectional study with 1085 participants randomly selected from a metropolitan area in São Paulo, Brazil, and conducted during the first semester of 2004. METHODS: Thyroid ultrasound examination was performed in all participants and samples of urine and blood were collected from each subject. Serum levels of thyroid-stimulating hormone, free thyroxine, and anti-thyroid peroxidase (TPO) antibodies, urinary iodine concentration, thyroid volume, and thyroid echogenicity were evaluated. We also analyzed table salt iodine concentrations. RESULTS: At the time the study was conducted, table salt iodine concentrations were within the new official limits (20-60 mg/kg salt). Nevertheless, in 45.6% of the participants, urinary iodine excretion was excessive (above 300 microg/l) and, in 14.1%, it was higher than 400 microg/l. The prevalence of CAT (including atrophic thyroiditis) was 16.9% (183/1085), women were more affected than men (21.5 vs 9.1% respectively, P=0.02). Hypothyroidism was detected in 8.0% (87/1085) of the population with CAT. Hyperthyroidism was diagnosed in 3.3% of the individuals (36/1085) and goiter was identified in 3.1% (34/1085). CONCLUSIONS: Five years of excessive iodine intake by the Brazilian population may have increased the prevalence of CAT and hypothyroidism in subjects genetically predisposed to thyroid autoimmune diseases. Appropriate screening for early detection of thyroid dysfunction may be considered during excessive nutritional iodine intake.
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Meio Ambiente , Iodo/toxicidade , Distúrbios Nutricionais/complicações , Doenças da Glândula Tireoide/epidemiologia , Glândula Tireoide/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil/epidemiologia , Estudos de Coortes , Estudos Transversais , Feminino , Alimentos/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Distúrbios Nutricionais/epidemiologia , Prevalência , Doenças da Glândula Tireoide/etiologiaRESUMO
OBJECTIVE: To extend the molecular analysis of the IVS30+1G>T intronic thyroglobulin (TG) mutation, and to report the eleven year follow-up of the affected patients. METHOSD: Two siblings with severe congenital hypothyroidism with fetal and neonatal goiter, harboring the IVS30+1G>T mutation were included. Nodular and non-nodular thyroid tissue specimens were collected. Specific thyroid genes expression was evaluated by real-timePCR and by immunohistochemistry. RESULTS: In non-nodular tissue specific thyroid genes mRNA were reduced when compared to normal thyroid sample. In the nodule, TPO and NIS expression was very low. Microscopic examinations showed very large follicular-lumina and swollen vesicles of endoplasmatic-reticulum. Strong cytoplasmatic and low follicular-lumen TG immunostaining were detected. Intracellular NIS, membrane TPO and TSHR immunostaining had higher positivity in non-nodular sample. Both patients had a long-term adequate developmental outcome, besides one patient have been lately-treated. CONCLUSIONS: IVS30+1G>T mutation not only lead to very enlarge endoplasmatic-reticulum, but also to alterations of specific thyroid genes expression. The clinical evolution of patients harboring these mutations strengthen the concept of the influence of environment, like iodine nutrition, to determine the final phenotypic appearance.