Avaliação da ocorrência de apoptose em células foliculares da tiróide de rato após estimulação seguida de supressão do hormônio tireotrófico endógeno

Objective: In this study, the levels of pituitary TSH in rats were modulated by pharmacological treatment in vivo to evaluate the possible cell loss through apoptosis during TSH supression of thyroid gland. Methods. Male Winstar rats (~220gr body weight-bw) were given methimazole (MTZ) 0.03% in drinking water for three weeks. Then the animals received tyroxine (T4) 20mg/100g bw/day ip for one (T4-1d), two (T4-2d), three (T4-3d) or five (T4-5d) days while the control group (MTZ) was not treated with T4. After perfusion of the rats with physiologic serum (0.09%), the thyroids were surgically dissected and weighed. DNA were quantified to verify cell loss and the DNA value measured the espectrophotometer set at A595 was compared to the normal curve of salmon sperm DNA. Results. The table show the thyroid weight (T) and the DNA concentration of T4 treatment groups and the control group (MTZ). Mean ±SD. On themorphological analysis of the tissue by optical and electron microscopy (OM, EM), the follicules of the rats treated with MTZ present hight tyrocytes with closed lumens whith gradual reappearance of lumens and reduction of cell height were observed in the tissue of the groups treated with T4. In some of their lumens, particularly in those subjected to prolongedtreatment, electrondense structures containing cellular residue were seen. The fragments containing intensely condensed cromatine, cell structure residue (apoptotic bodies) or even cells with cromatine condensed in a half-moon shape (apoptotic cells) sporadically appear in the basal follicular region of the T4 groups. Conclusion. In the thyroids of animalstreated with T4, there is a reduction of DNA and the presence of morphological structures suggesting apoptosis. In addition with the hypoplasia of the folicular cells, the cell loss by apoptotic process could be contributing to the shrinkage in the rat thyroid gland submitted to TSH supression.Objetivo: Neste estudo foram modulados os níveis de TSH hipofisário de ratos por tratamento farmacológico in vivo, para avaliação da possível perda celular por apoptose durante a involução da glândula tiróide por supressão de TSH. Métodos: Ratos Winstar machos (~220g peso corporal pc) foram tratados com metimazol (MTZ) 0,03% na água de beber por três semanas. Após este período receberam tiroxina (T4) 20 mg/100 g pc/dia intraperitonealmente por 1(T4-1d), 2 (T4-2d), 3 (T4-3d) e 5 (T4-5d) dias e o grupo controle (MTZ) sem tratamento com T4. Foi realizada a quantifição de DNA para verificação da perda celular e o valor obtido na A595 no espectofotômetro foi comparada à curva padrão de DNA de esperma de salmão. Resultados: O peso da tiróide (T)/100g de pc do animal e os valores de DNA calculados em mg/100 g de pc, Média ± SE, e a variação da quantidade de DNA (%) e m comparação ao grupoMTZ. Na análise morfológica do tecido à microscopia óptica e eletrônica (MO e ME), os folículos dos ratos tratados com MTZ apresentam tirócitos altos com lúmens fechados e se observam reaparecimento gradativo dos lúmens e redução variada da altura das células nos tecidos com tratamento com T4. E m alguns lúmens, dos grupos tratados com T4 nota-se a presença de estruturas eletrondensas contendo resíduos de organelas, sendo mais freqüente nos tratamentos prolongados. Na região basal dos folículos aparecem esporadicamente células envolvendo fragmentos densos contendo cromatina intensamente condensada e resíduos de organelas (corpos apoptóticos) ou ainda células com cromatina condensada em meia-lua (células apoptóticas) nos grupos que receberam tratamento com T4. Conclusão: Nas tiróides dos animais tratados com T4 ocorre redução de DNA e observam-se estruturasmorfológicas de apoptose à MO e ME . A perda celular por processo de apoptose contribui juntamente com a hipoplasiadas células foliculares na involução da glândula tiróide de ratos submetidos a supressão de TSH

Down-regulation of 14q32-encoded miRNAs and tumor suppressor role for miR-654-3p in papillary thyroid cancer

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOPapillary thyroid carcinoma (PTC) is the most prevalent malignant neoplasia of the thyroid gland. A fraction of PTC cases show loss of differentiation and aggressive behavior, with radioiodine therapy resistance and metastasis. Although microRNAs (miRNAs) emerged as promising molecular markers for PTC, their role in the loss of differentiation observed during PTC progression remains to be fully understood. We performed the large-scale analysis of miRNA expression during PTC progression in BRAFT1799A-transgenic animals (Tg-Braf) and thyroid Cancer Cell lines and identified the marked downregulation of several miRNAs from the region 14q32. Data from The Cancer Genome Atlas (TCGA) confirmed the global downregulation of miRNAs from the 14q32 region in human PTC. The regulatory network potentially suppressed by these miRNAs suggests that key cancer-related biological processes such as cell proliferation, adhesion, migration and angiogenesis. Among the downregulated miRNAs, we observed that miR-654-3p levels decrease with long-term PTC progression in Tg-Braf mice and inversely correlate with EMT. The in vitro restoration of miR-654-3p decreased cell proliferation and migration and induced reprogramming of metastasis-related genes, suggesting a tumor suppressor role for this miRNA. In conclusion, we show global downregulation of 14q32-encoded miRNAs in an in vivo model of PTC progression. The potential circuitry in which these miRNAs are involved suggests that these miRNAs could play a key role in the pathophysiology of PTC and therefore be relevant for the development of new therapeutic strategies.Papillary thyroid carcinoma (PTC) is the most prevalent malignant neoplasia of the thyroid gland. A fraction of PTC cases show loss of differentiation and aggressive behavior, with radioiodine therapy resistance and metastasis. Although microRNAs (miRNAs)8695979607FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO11/52051-2; 2011/50732-2308527/2013-5; 458505/2014-

MicroRNA Deregulation in Anaplastic Thyroid Cancer Biology

Anaplastic thyroid cancer (ATC) is among the most lethal types of cancers, characterized as a fast-growing and highly invasive thyroid tumor that is unresponsive to surgery and radioiodine, blunting therapeutic efficacy. Classically, genetic alterations in tumor suppressor TP53 are frequent, and cumulative alterations in different signaling pathways, such as MAPK and PI3K, are detected in ATC. Recently, deregulation in microRNAs (miRNAs), a class of small endogenous RNAs that regulate protein expression, has been implicated in tumorigenesis and cancer progression. Deregulation of miRNA expression is detected in thyroid cancer. Upregulation of miRNAs, such as miR-146b, miR-221, and miR-222, is observed in ATC and also in differentiated thyroid cancer (papillary and follicular), indicating that these miRNAs’ overexpression is essential in maintaining tumorigenesis. However, specific miRNAs are downregulated in ATC, such as those of the miR-200 and miR-30 families, which are important negative regulators of cell migration, invasion, and epithelial-to-mesenchymal transition (EMT), processes that are overactivated in ATC. Therefore, molecular interference to restore the expression of tumor suppressor miRNAs, or to blunt overexpressed oncogenic miRNAs, is a promising therapeutic approach to ameliorate the treatment of ATC. In this review, we will explore the importance of miRNA deregulation for ATC cell biology

MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells

Abstract\ud \ud Background\ud Tumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, usually degrading extracellular matrix. Despite significant progress in the knowledge about migration and invasion, there is much more to elucidate about their regulatory mechanisms, especially in cancer cells. MicroRNAs (miRs) were recently described as important regulators of migration. Differential expression of miRs in cancer is frequently associated with progression, invasion and metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed and positively correlated to the degree of malignancy.\ud \ud \ud Methods\ud This study aimed to investigate the role of miR-146b-5p on the migratory and invasive behaviors of thyroid cells, using a non tumor rat thyroid follicular cell line (PCCl3) transfected with the miR-146b-5p genomic region, and two PTC cell lines (TPC-1 and BCPAP, bearing distinct oncogenic backgrounds), which express high levels of miR-146b-5p, after miR-146b inhibition by antagomiR and miR-146b overexpression by mimics-miR. Migration and invasion were studied by time-lapse and transwell assays (with and without Matrigel®). Gelatin degradation assays were also employed, as well as F-actin staining.\ud \ud \ud Results\ud Migration and invasion of PCCl3 were increased 2-3x after miR-146b-5p overexpression (10X) and large lamellipodia were evident in those cells. After miR-146b-5p inhibition, TPC-1 and BCPAP migration and invasion were significantly reduced, with cells showing several simultaneous processes and low polarity. Gelatin degradation was inhibited in TPC-1 cells after inhibition of miR-146b-5p, but was unaffected in BCPAP cells, which did not degrade gelatin. The inhibition of miR-146b-5p in PCCl3 also inhibited migration and invasion, and additional (exogenous) overexpression of this miR in TPC-1 and BCPAP cells increased migration and invasion, without effects on cell morphology or gelatin degradation. The overexpression of SMAD4 in BCPAP cells, a validated target of miR-146b-5p and key protein in the TGF-β signaling pathway, inhibited migration similarly to the effects observed with the antagomiR 146b-5p.\ud \ud \ud Conclusions\ud miR-146b-5p positively regulates migration and invasion of thyroid normal and tumor follicular cells (independently from their original mutation, either BRAF or RET/PTC), through a mechanism that involves the actin cytoskeleton but not an increased capacity of matrix degradation.This research was supported by Fundação de Amparo à Pesquisa do Estado\ud de São Paulo (FAPESP, Grants # 2011/18936-7 and #2012/03990-9),\ud Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES,\ud PNPD), Conselho Nacional de Desenvolvimento Científico e Tecnológico\ud (CNPq grants #307452/2012-3 and #448052/2014-8) and Pró-Reitoria de\ud Pesquisa da Universidade de São Paulo (NAPMiR)

The Highly Expressed FAM83F Protein in Papillary Thyroid Cancer Exerts a Pro-Oncogenic Role in Thyroid Follicular Cells

Thyroid cancer is the most common endocrine cancer with predominant prevalence of papillary thyroid cancer (PTC) histotype. MAPK signaling genetic alterations are frequent in PTC, affecting more than 80% of cases. These alterations constitutively activate MAPK signaling cross-regulating different pro-oncogenic pathways. However, additional molecular alterations associated with thyroid cancer are not completely understood. In this extent, the new family of proteins named FAM83 (FAMily with sequence similarity 83) was recently identified as mediator of oncogenic signaling in different types of cancer. Here we report FAM83F as a novel highly expressed protein in PTC. We evaluated FAM83F levels in 106 PTC specimens, 34 goiter, and 41 adjacent non-tumoral human thyroid, and observed FAM83F cytoplasmic overexpression in 71% of PTC (76 of 106) while goiter tissues showed nuclear positivity and normal thyroid showed no staining by immunohistochemistry. Moreover, TSH-induced goiter and BRAFT1799A-induced PTC animal models also showed FAM83F activation. In vitro, we generated a stable thyroid cell line PCCL3 with FAM83F overexpression and observed that FAM83F deregulates thyroid follicular cell biology leading to loss of thyroid differentiation genes such as Sodium-Iodide Symporter (NIS), reactivation of stem cell markers such as LIN28B and SOX2, induction of cell migration and resistance to doxorubicin-induced apoptosis. Moreover, FAM83F activates MAPK signaling through interaction with BRAF and RAF while impairs TGFβ antiproliferative signaling transduction. In this study, we showed FAM83F as a new pro-oncogenic protein overexpressed in thyroid cancer that modulates thyroid follicular cell biology and differentiation through cross-regulation of MAPK and TGFβ signaling

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.CNPqCNPqFAPESP [01/09401-0]FAPESPEuropean CommunityEuropean Community [242095]Spanish Ministry of Science and InnovationSpanish Ministry of Science and Innovation [SAF2009-07760

MicroRNAs miR-146-5p e let-7f como ferramenta de prognóstico para o carcinoma papilífero de tiroide: relato de caso

Papillary thyroid cancer (PTC) is the most incident histotype of thyroid cancer. A certain fraction of PTC cases (5%) are irresponsive to conventional treatment, and refractory to radioiodine therapy. The current prognostic factors for aggressiveness are mainly based on tumor size, the presence of lymph node metastasis, extrathyroidal invasion and, more recently, the presence of the BRAFT(1799A) mutation. MicroRNAs (miRNAs) have been described as promising molecular markers for cancer as their deregulation is observed in a wide range of tumors. Recent studies indicate that the over-expression of miR-146b-5p is associated with aggressiveness and BRAFT(1799A) mutation. Furthermore, down-regulation of let-7f is observed in several types of tumors, including PTC. In this study, we evaluated the miR146b-5p and let-7f status in a young male patient with aggressive, BRAFT(1799A)-positive papillary thyroid carcinoma, with extensive lymph node metastases and short-time recurrence. The analysis of miR-146b-5p and let-7f expression revealed a distinct pattern from a cohort of PTC patients, suggesting caution in evaluating miRNA expression data as molecular markers of PTC diagnosis and prognosis. Arq Bras Endocrinol Metab. 2012;56(8):552-7O carcinoma papilífero (PTC) é o histotipo mais prevalente de câncer de tiroide. Cerca de 5% dos casos são refratários ao tratamento convencional e à radioiodoterapia. Os fatores prognósticos para agressividade mais utilizados atualmente são o tamanho do tumor, a presença de metástases linfonodais ao diagnóstico, a presença de invasão extratiroideana e, mais recentemente, a presença da mutação BRAFT1799A. A análise de perfil de expressão de microRNAs (miRNA) mostra que esses pequenos RNAs são marcadores moleculares promissores para o câncer, por apresentarem desregulação de sua expressão em uma ampla gama de tumores, includindo o PTC. Estudos recentes revelam a associação entre o aumento da expressão do miRNA e miR-146b-5p e a presença da mutação BRAFT1799A como um fator de pior prognóstico no PTC. Além disso, observa-se a diminuição da expressão de let-7f em diversos tipos de tumores, incluindo tumores tiroideanos. Neste relato de caso, realizamos a quantificação da expressão de miR-146b-5p e let-7f em um paciente jovem, de sexo masculino, apresentando PTC positivo para a mutação BRAFT1799A com extensas metástases linfonodais ao diagnóstico e recidiva precoce. A análise da expressão de miR-146b-5p e let-7f mostrou um padrão diferente do observado em um grupo de pacientes PTC, sugerindo a necessidade de cautela na interpretação da expressão de miRNAs como marcador molecular no diagnóstico e prognóstico de PTC. Arq Bras Endocrinol Metab. 2012;56(8):552-7Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (Fapesp)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (Capes

Descrição de um novo foco endêmico de esquistossomose mansônica no Estado de São Paulo, Brasil

A new endemic focus of schistosomiasis mansoni in the town of Bebedouro, S. Paulo, Brazil, is described. Two hundred and twenty-one cases are analyzed according to sex, age, and origin (autochthonous or not), the factors involved in the emergence of the focus are discussed and the necessity for control measure is emphasized.Foi descrito novo foco endêmico de esquistossomose mansônica situado na Cidade de Bebedouro, Estado de São Paulo, Brasil. Foram analisados 221 casos diagnosticados da doença segundo a idade, sexo e origem (autóctone ou não), bem como foram discutidos os fatores envolvidos no aparecimento do foco, alertando-se para a necessidade de medidas de controle