Normal Thyroid Cells Research Articles

HOTAIR is a promising novel biomarker in patients with thyroid cancer.

Thyroid cancer (TC) is the most common endocrine malignancy. Lack of effective early diagnostic tools is one of the clinical obstacles for TC treatment. Thus, enhanced comprehension of the molecular changes in TC tumorigenesis is urgently needed to develop novel strategies for the diagnosis and treatment of TC. Long non-coding RNAs (lncRNAs) manage fundamental biochemical and cellular processes in tumorigenesis and development. One of the best-described lncRNAs, HOX transcript antisense RNA (HOTAIR), functions as a regulatory molecule in a wide variety of biological processes, and represses gene expression through recruitment of the chromatin modifying complex. However, the function of HOTAIR in TC remains unclear. In the current study, the expression of HOTAIR is elevated in TC and correlates with metastasis and poor prognosis. Furthermore, the expression of HOTAIR is significantly upregulated in human thyroid carcinoma cells compared with normal human thyroid cells. Furthermore, knockdown of HOTAIR significantly inhibited cell growth and invasion in TPC-1 and SW579 human thyroid carcinoma. In summary, HOTAIR is a promising novel biomarker in patients with TC.

DNA methylation of METTL7A gene body regulates its transcriptional level in thyroid cancer.

DNA methylation is the best-studied epigenetic mechanism for regulating gene transcription and maintaining genome stability. Current research progress of transcriptional regulation by DNA methylation mostly focuses on promoter region where hypomethylated CpG islands are present transcriptional activity, as hypermethylated CpG islands generally result in gene repression. Recently, the DNA methylation patterns across the gene body (intragenic methylation) have increasingly attracted attention towards their role in transcriptional regulation and efficiency, due to the improvement of numerous genome-wide DNA methylation profiling studies. However, the function and mechanism of gene body methylation is still unclear. In this study, we revealed that the methylation level of METTL7A, a seldom studied gene, was downregulated in thyroid cancer compared to normal thyroid cells in vivo and in vitro. Moreover, we determined the methylation level of one CpG site at the exon of the METTL7A gene body impacted the transcriptional activity. Through generating a mutation of this CpG site (CG to CC) of METTL7A exogenous vector artificially in vitro, we observed higher RNA polymerase II recruitment and a declined enrichment of methyl-CpG binding protein-2 in gene body of METTL7A, in papillary thryoid cancer cells (BCPAP) compared to normal thryoid cells. Finally, we revealed that EZH2, a subunit of polycomb repressor complex 2, dominant in thyroid cancer, might be responsible for regulating gene body methylation of METTL7A. Our study depicted the DNA methylation patterns and the transcriptional regulatory mechanism of the gene body in thyroid cancer. Furthermore, this study provides new insight into potential future avenues, for therapies targeting cancer.

Human a-L-fucosidase-1 attenuates the invasive properties of thyroid cancer.

Glycans containing α-L-fucose participate in diverse interactions between cells and extracellular matrix. High glycan expression on cell surface is often associated with neoplastic progression. The lysosomal exoenzyme, α-L-fucosidase-1 (FUCA-1) removes fucose residues from glycans. The FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors. However, the role of FUCA-1 in tumor progression remains unclear. It is speculated that its inactivation perturbs glycosylation of proteins involved in cell adhesion and promotes cancer. FUCA-1 expression of various thyroid normal and cancer tissues assayed by immunohistochemical (IHC) staining was high in normal thyroids and papillary thyroid carcinomas (PTC), whereas it progressively decreased in poorly differentiated, metastatic and anaplastic thyroid carcinomas (ATC). FUCA-1 mRNA expression from tissue samples and cell lines and protein expression levels and enzyme activity in thyroid cancer cell lines paralleled those of IHC staining. Furthermore, ATC-derived 8505C cells adhesion to human E-selectin and HUVEC cells was inhibited by bovine α-L-fucosidase or Lewis antigens, thus pointing to an essential role of fucose residues in the adhesive phenotype of this cancer cell line. Finally, 8505C cells transfected with a FUCA-1 containing plasmid displayed a less invasive phenotype versus the parental 8505C. These results demonstrate that FUCA-1 is down-regulated in ATC compared to PTC and normal thyroid tissues and cell lines. As shown for other human cancers, the down-regulation of FUCA-1 correlates with increased aggressiveness of the cancer type. This is the first report indicating that the down-regulation of FUCA-1 is related to the increased aggressiveness of thyroid cancer.

Cytokine Release and Focal Adhesion Proteins in Normal Thyroid Cells Cultured on the Random Positioning Machine

Background/Aims: Spaceflight impacts on the function of the thyroid gland in vivo. In vitro normal and malignant thyrocytes assemble in part to multicellular spheroids (MCS) after exposure to the random positioning machine (RPM), while a number of cells remain adherent (AD). We aim to elucidate possible differences between AD and MCS cells compared to 1g-controls of normal human thyroid cells. Methods: Cells of the human follicular epithelial thyroid cell line Nthy-ori 3-1 were incubated for up to 72 h on the RPM. Afterwards, they were investigated by phase-contrast microscopy, quantitative real-time PCR and by determination of cytokines released in their supernatants. Results: A significant up-regulation of IL6, IL8 and CCL2 gene expression was found after a 4h RPM-exposure, when the whole population was still growing adherently. MCS and AD cells were detected after 24 h on the RPM. At this time, a significantly reduced gene expression in MCS compared to 1g-controls was visible for IL6, IL8, FN1, ITGB1, LAMA1, CCL2, and TLN1. After a 72 h RPM-exposure, IL-6, IL-8, and TIMP-1 secretion rates were increased significantly. Conclusion: Normal thyrocytes form MCS within 24 h. Cytokines seem to be involved in the initiation of MCS formation via focal adhesion proteins.

RASSF10 is Epigenetically Inactivated and Suppresses Cell Proliferation and Induces Cell Apoptosis by Activating the p53 Signalling Pathway in Papillary Thyroid Carcinoma Cancer

Objectives: We aimed to confirm whether RASSF10 activated the p53 signalling pathway, thereby modulating cell proliferation, migration, invasion, and apoptosis in papillary thyroid carcinoma (PTC) cells. Methods: A total of 108 PTC tissue samples and normal adjacent tissues were obtained. RT-PCR and Western blotting analyses were performed to detect RASSF10 expression, and methylation levels of RASSF10 were estimated by methylation-specific PCR (MSP). We also detected the expression and methylation status of RASSF10 in both a human PTC cell line (K1) and a normal thyroid cell line (FRTL5). After transfection of cells with empty vector pcDNA3.1, pcDNA3.1-RASSF10, p53 siRNA and shRASSF10, Coulter counter, colony-formation, wound healing, Transwell and flow cytometry analyses were performed to examine the role of RASSF10 in cell proliferation, migration, invasion, and apoptosis. Finally, the expression of p53, p21, Bcl-2 and Bax were detected using Western Blotting analyses. Results: RASSF10 expression in PTC tissues was significantly lower and hyper-methylated compared to normal adjacent tissues. In addition, RASSF10 was significantly down-regulated and hyper-methylated in K1 cells compared to FRTL5 cells. In addition, suppressed proliferation and significantly induced apoptosis of K1 cells were observed after transfection with pcDNA3.1-RASSF10 (P < 0.05). Furthermore, RASSF10 activated the p53 signalling pathway and regulated the expression of p53, p21, Bcl-2 and Bax. Furthermore, p53 siRNA could antagonize the effects of RASSF10 in K1 cells. Conclusions: RASSF10 induces apoptosis in PTC cells by activating the p53 signalling pathway, indicating its role as a treatment target for PTC.

C14orf93 (RTFC) is identified as a novel susceptibility gene for familial nonmedullary thyroid cancer

The genetic causes for familial nonmedullary thyroid cancer (FNMTC) remain largely unknown. Through genetic linkage analysis and exome sequencing, C14orf93 (RTFC), PYGL, and BMP4 were identified as susceptibility gene candidates in a FNMTC family. By examining the expression and the oncogenic functions of these candidate genes, PYGL and BMP4 were excluded. We further characterized the functions of the uncharacterized gene RTFC in thyroid cancer. RTFC promotes thyroid cancer cell survival under starving conditions, and thyroid cancer cell migration. The R115Q, V205M and G209D RTFC mutants enhance the colony forming capacity of thyroid cancer cells, and are able to transform normal thyroid cells. In summary, our data suggest the roles of RTFC in thyroid carcinogenesis.

Screening between normal and cancer human thyroid cells through comparative adhesion studies using the Quartz Crystal Microbalance

In this work, the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) was used to distinguish the dynamic cell adhesion behavior of human normal (Nthy) thyroid epithelial cells from poorly differentiated anaplastic carcinoma cells (ARO). The surfaces used to facilitate cell adhesion were bare titanium (Ti), gold (Au) and fibrinogen-coated gold (Fg-Au). The pattern of cell adhesion for both cell lines was that the largest acoustic signals were observed on Ti, followed by Au and last by Fg-Au; in addition, ARO cells always produced smaller acoustic signals than Nthy cells on the same surface and for the same number of cells in suspension. Moreover, the calculated acoustic ratio of energy dissipation over frequency change suggests a higher ability of Nthy cells to spread and potentially form more attachment points on the surface than the ARO cells, something observed in SEM images. Finally, we demonstrated that the application of two surfaces for cell adhesion experiments, one of which is Au and the other either Ti or Fg-Au, can discriminate with accuracy between the two particular cell types and potentially form a platform for differentiation between normal and cancer thyroid cell types.

MiR-30a и lin28b образуют петлю двойной отрицательной связи, регулируемую pik3, способным изменять степень прогрессирования рака щитовидной железы

Background . Our recent results showed that tumor supressor miR-30a is firmly downregulated in Thyroid carcinomas. On the other hand, recent studies showed RNA- and DNA- binding proteins LIN28B and HMGA2 induce EMT, thus playing an important role in dedifferentiation and cancer malignification. Finally, latterly several authors agreed on the importance of a robust activation of PI3K for thyroid cancer emergency and progression. Aim : to study the link between the emergency of LIN28B and HMGA2, miR-30a silencing and PI3K hyperactivation, and to determine their effect on thyroid cancer progression. Methors . MiRNA targets computational predictions were performed with MiRanda algorythm. LIN28B and miR-30a expression vectors were transfected in ATC derived and normal thyroid cell lines; mRNA and protein levels were determined by qPCR, Luciferase and Western Blot. Invasion, proliferation and cell cycle assays were performed in Transwell, cell counter, and FACScan respectively. Results . MiRanda algorythm identified multiple miR30a recognition elements in all LIN28B, HMGA2 and PI3K effectors. LIN28B expression correlated with PI3K activating mutations in ATC derived cell lines. Overexpression of miR30a resulted in LIN28B, HMGA2, and several PI3K effectors silencing, and in an increase in p27(Kip) protein levels. Inversely, LIN28B overexpressing cells showed a decrease in miR30a levels and an increased expression of HMGA2 and PI3K effectors. The general outcome was a significant decrease in invasion and proliferation in miR-30a overexpressing cells and, conversely, an increase in these parameters by LIN28B. Conclusions . These data suggest the existence of a PI3K regulated feedback with a double-negative loop between miR-30a and LIN28B. Here, PI3K activation acts to switch the steady states. Initially, high miR-30a levels repress LIN28B expression. After PI3K is activated, LIN28B is produced and miR-30a is repressed. This state reinforces PI3K hyperactivation. Thus, the feedback implements a tumoral gene expression shift, contributing to thyroid cancer progression.

Mir-30a and lin28b conform a double-negative feedback loop regulated by pi3k modulating thyroid cancer progression

Background. Our recent results showed that tumor supressor miR-30a is firmly downregulated in Thyroid carcinomas. On the other hand, recent studies showed RNA- and DNA- binding proteins LIN28B and HMGA2 induce EMT, thus playing an important role in dedifferentiation and cancer malignification. Finally, latterly several authors agreed on the importance of a robust activation of PI3K for thyroid cancer emergency and progression.Aim: to study the link between the emergency of LIN28B and HMGA2, miR-30a silencing and PI3K hyperactivation, and to determine their effect on thyroid cancer progression.Methors. MiRNA targets computational predictions were performed with MiRanda algorythm. LIN28B and miR-30a expression vectors were transfected in ATC derived and normal thyroid cell lines; mRNA and protein levels were determined by qPCR, Luciferase and Western Blot. Invasion, proliferation and cell cycle assays were performed in Transwell, cell counter, and FACScan respectively.Results. MiRanda algorythm identified multiple miR30a recognition elements in all LIN28B, HMGA2 and PI3K effectors. LIN28B expression correlated with PI3K activating mutations in ATC derived cell lines. Overexpression of miR30a resulted in LIN28B, HMGA2, and several PI3K effectors silencing, and in an increase in p27(Kip) protein levels. Inversely, LIN28B overexpressing cells showed a decrease in miR30a levels and an increased expression of HMGA2 and PI3K effectors. The general outcome was a significant decrease in invasion and proliferation in miR-30a overexpressing cells and, conversely, an increase in these parameters by LIN28B.Conclusions. These data suggest the existence of a PI3K regulated feedback with a double-negative loop between miR-30a and LIN28B. Here, PI3K activation acts to switch the steady states. Initially, high miR-30a levels repress LIN28B expression. After PI3K is activated, LIN28B is produced and miR-30a is repressed. This state reinforces PI3K hyperactivation. Thus, the feedback implements a tumoral gene expression shift, contributing to thyroid cancer progression.

WOMEN IN CANCER THEMATIC REVIEW: Thyroid-stimulating hormone in thyroid cancer: does it matter?

Differentiated thyroid cancer is the most common endocrine malignancy and the incidence is increasing rapidly worldwide. Appropriate diagnosis and post-treatment monitoring of patients with thyroid tumours are critical. Fine needle aspiration cytology remains the gold standard for diagnosing thyroid cancer, and although there have been significant refinements to this technique, diagnostic surgery is often required for patients suspected to have malignancy. Serum thyroid-stimulating hormone (TSH) is higher in patients with malignant thyroid nodules than in those with benign disease, and TSH is proportionally increased in more aggressive tumours. Importantly, we have shown that the pre-operative serum TSH concentration independently predicts the presence of malignancy in subjects presenting with thyroid nodules. Establishing the use of TSH measurements in algorithms identifying high-risk thyroid nodules in routine clinical practice represents an exciting, cost-efficient and non-invasive approach to optimise thyroid cancer diagnosis. Binding of TSH to receptors on thyrocytes stimulates a number of growth promoting pathways both in normal and malignant thyroid cells, and TSH suppression with high doses of levothyroxine is routinely used after thyroidectomy to prevent cancer recurrence, especially in high-risk tumours. This review examines the relationship between serum TSH and thyroid cancer and reflects on the clinical potential of TSH measurements in diagnosis and disease monitoring.

MicroRNA-7 inhibits proliferation, migration and invasion of thyroid papillary cancer cells via targeting CKS2.

The purpose of this study was to examine the expression levels of microRNA-7 (miR-7) in human thyroid papillary cancer and its potential role in disease pathogenesis. The expression levels of different miRNAs were detected by miRNA-microarray analysis in ten thyroid papillary cancer specimens and adjacent normal thyroid cancer tissues. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to determine the expression level of miR-7 in both thyroid papillary cancer tissues and cell lines. To characterize the function of miR-7, MTT assay, colony formation assay, cell migration assay, cell invasion assay, cell cycle assay and cell apoptosis assay were used. Luciferase reporter assays were performed to validate the regulation of a putative target of miR-7, in corroboration with western blot assays. Finally, MTT assay, cell migration assay, cell invasion assay and cell cycle assay were used to indicate the roles of endogenous cyclin-dependent kinase regulatory subunit 2 (CKS2) in thyroid papillary cancer cells. Our results reveal that miR-7 expression was relatively decreased in thyroid papillary cancer specimens and cell lines compared with adjacent normal tissues and normal thyroid cells. Overexpression of miR-7 inhibited cellular proliferation, suppressed cellular migration and invasion, caused a G0/G1 arrest in vitro. Dual-luciferase reporter assays showed that miR-7 binds the 3'-untranslated region (3'-UTR) of CKS2. Western blotting showed that miR-7 negatively regulated CKS2 protein expression. As its downstream genes, cyclinB1 (G2/mitotic-specific cyclin-B1) and cdk1 (cyclin-dependent kinase 1) were regulated by miR-7 and CKS2 axis. Knockdown of CKS2 expression by CKS2-siRNA in TPC1 and K1 cells also significantly suppressed cell proliferation, cell migration and invasion. Our results demonstrated for the first time that miR-7 functions as a tumor suppressor and plays an important role in inhibiting the tumorigenesis through targeting CKS2 in thyroid papillary cancer cells.

Pioglitazone, a PPARγ Agonist, Upregulates the Expression of Caveolin-1 and Catalase, Essential for Thyroid Cell Homeostasis: A Clue to the Pathogenesis of Hashimoto's Thyroiditis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates the expression of multiple target genes involved in several metabolic pathways as well as in inflammation. The expression and cell localization of caveolin-1 (Cav-1), thyroperoxidase (TPO), and dual oxidase (DUOX), involved in extracellular iodination, is modulated by Th1 cytokines in human normal thyroid cells and in Hashimoto's thyroiditis (HT). The objectives of this study were (i) to analyze the PPARγ protein and mRNA expression at the follicular level in HT versus controls in correlation with the one of Cav-1; (ii) to study the effects of Th1 cytokines on PPARγ and catalase expression in human thyrocyte primary cultures; and (iii) to study the effects of pioglitazone, a PPARγ agonist, on thyroxisome components (Cav-1, TPO, DUOX) and on catalase, involved in antioxidant defense. Although the global expression of PPARγ in the whole gland of patients with HT was not modified compared with controls, there was great heterogeneity among glands and among follicles within the same thyroid. Besides normal (type 1) follicles, there were around inflammatory zones, hyperactive (type 2) follicles with high PPARγ and Cav-1 expression, and inactive (type 3) follicles which were unable to form thyroxine and did not express PPARγ or Cav-1. In human thyrocytes in primary culture, Th1 cytokines decreased PPARγ and catalase expression; pioglitazone increased Cav-1, TPO, and catalase expression. PPARγ may play a central role in normal thyroid physiology by upregulating Cav-1, essential for the organization of the thyroxisome and extracellular iodination. By upregulating catalase, PPARγ may also contribute to cell homeostasis. The inhibitory effect of Th1 cytokines on PPARγ expression may be considered as a new pathogenetic mechanism for HT, and the use of PPARγ agonists could open a new therapeutic approach.

Rewiring of the apoptotic TGF-β-SMAD/NFκB pathway through an oncogenic function of p27 in human papillary thyroid cancer.

Papillary thyroid carcinoma (PTC), the most frequent thyroid cancer, is characterized by low proliferation but no apoptosis, presenting frequent lymph-node metastasis. Papillary thyroid carcinoma overexpress transforming growth factor-beta (TGF-β). In human cells, TGF-β has two opposing actions: antitumoral through pro-apoptotic and cytostatic activities, and pro-tumoral promoting growth and metastasis. The switch converting TGF-β from a tumor-suppressor to tumor-promoter has not been identified. In the current study, we have quantified a parallel upregulation of TGF-β and nuclear p27, a CDK2 inhibitor, in samples from PTC. We established primary cultures from follicular epithelium in human homeostatic conditions (h7H medium). TGF-β-dependent cytostasis occurred in normal and cancer cells through p15/CDKN2B induction. However, TGF-β induced apoptosis in normal and benign but not in carcinoma cultures. In normal thyroid cells, TGF-β/SMAD repressed the p27/CDKN1B gene, activating CDK2-dependent SMAD3 phosphorylation to induce p50 NFκB-dependent BAX upregulation and apoptosis. In thyroid cancer cells, oncogene activation prevented TGF-β/SMAD-dependent p27 repression, and CDK2/SMAD3 phosphorylation, leading to p65 NFκB upregulation which repressed BAX, induced cyclin D1 and promoted TGF-β-dependent growth. In PTC samples from patients, upregulation of TGF-β, p27, p65 and cyclin D1 mRNA were significantly correlated, while the expression of the isoform BAX-β, exclusively transcribed in apoptotic cells, was negatively correlated. Additionally, combined ERK and p65 NFκB inhibitors reduced p27 expression and potentiated apoptosis in thyroid cancer cells while not affecting survival in normal thyroid cells. Our results therefore suggest that the oncoprotein p27 reorganizes the effects of TGF-β in thyroid cancer, explaining the slow proliferation but lack of apoptosis and metastatic behavior of PTC.

Altered expression of mir-222 and mir-25 influences diverse gene expression changes in transformed normal and anaplastic thyroidcells, and impacts on MEK and TRAIL protein expression.

Thyroid cancer is the most common endocrine malignancy and accounts for the majority of endocrine cancer-related deaths each year. Our group and others have previously demonstrated dysfunctional microRNA (miRNA or miR) expression in the context of thyroid cancer. The objective of the present study was to investigate the impact of synthetic manipulation of expression of miR-25 and miR-222 in benign and malignant thyroid cells. miR-25 and miR-222 expression was upregulated in 8505C (an anaplastic thyroid cell line) and Nthy-ori (a SV40-immortalised thyroid cell line) cells, respectively. A transcriptomics-based approach was utilised to identify targets of the two miRNAs and real-time PCR and western blotting were used to validate a subset of the targets. Almost 100 mRNAs of diverse functions were found to be either directly or indirectly targeted by both miR-222 and miR-25 [fold change ≥2, false discovery rate (FDR) ≤0.05]. Gene ontology analysis showed the miR-25 gene target list to be significantly enriched for genes involved in cell adhesion. Fluidigm real-time PCR technologies were used to validate the downregulation of 23 and 22 genes in response to miR-25 and miR-222 overexpression, respectively. The reduction of the expression of two miR-25 protein targets, TNF-related apoptosis-inducing ligand (TRAIL) and mitogen-activated protein kinase kinase 4 (MEK4), was also validated. Manipulating the expression of both miR-222 and miR-25 influenced diverse gene expression changes in thyroid cells. Increased expression of miR-25 reduced MEK4 and TRAIL protein expression, and cell adhesion and apoptosis are important aspects of miR-25 functioning in thyroid cells.

Thyroid Hormone Receptor-β (TRβ) Mediates Runt-Related Transcription Factor 2 (Runx2) Expression in Thyroid Cancer Cells: A Novel Signaling Pathway in Thyroid Cancer.

Dysregulation of the thyroid hormone receptor (TR)β is common in human cancers. Restoration of functional TRβ delays tumor progression in models of thyroid and breast cancers implicating TRβ as a tumor suppressor. Conversely, aberrant expression of the runt-related transcription factor 2 (Runx2) is established in the progression and metastasis of thyroid, breast, and other cancers. Silencing of Runx2 diminishes tumor invasive characteristics. With TRβ as a tumor suppressor and Runx2 as a tumor promoter, a compelling question is whether there is a functional relationship between these regulatory factors in thyroid tumorigenesis. Here, we demonstrated that these proteins are reciprocally expressed in normal and malignant thyroid cells; TRβ is high in normal cells, and Runx2 is high in malignant cells. T3 induced a time- and concentration-dependent decrease in Runx2 expression. Silencing of TRβ by small interfering RNA knockdown resulted in a corresponding increase in Runx2 and Runx2-regulated genes, indicating that TRβ levels directly impact Runx2 expression and associated epithelial to mesenchymal transition molecules. TRβ specifically bound to 3 putative thyroid hormone-response element motifs within the Runx2-P1 promoter ((-)105/(+)133) as detected by EMSA and chromatin immunoprecipitation. TRβ suppressed Runx2 transcriptional activities, thus confirming TRβ regulation of Runx2 at functional thyroid hormone-response elements. Significantly, these findings indicate that a ratio of the tumor-suppressor TRβ and tumor-promoting Runx2 may reflect tumor aggression and serve as biomarkers in biopsy tissues. The discovery of this TRβ-Runx2 signaling supports the emerging role of TRβ as a tumor suppressor and reveals a novel pathway for intervention.

Long noncoding RNA H19 competitively binds miR-17-5p to regulate YES1 expression in thyroid cancer.

The long noncoding RNA H19 is overexpressed in many cancers and acts as an oncogene. Here, we investigated the role of H19 in thyroid carcinogenesis and its relation to microRNA miR-17-5p and its target gene YES1. H19 expression was higher in tumor samples and in thyroid cancer cell lines than nontumor tissues and normal thyroid cells. H19 knockdown and ectopic expression in the TPC-1 and NIM thyroid cancer cell lines showed that overexpression of H19 promoted proliferation, migration, and invasion, whereas H19 knockdown reduced cell viability and invasion and induced growth arrest in vitro and in vivo. H19 was identified as a target of miR-17-5p, by Dual-Luciferase Reporter assays and RNA-binding protein immunoprecipitation assays. H19 antagonized the function of miR-17-5p on upregulation of its target YES1 and inhibited miR-17-5p-induced cell cycle progression. Our results suggest that H19 functions as a competitive endogenous RNA (ceRNA) by acting as a sink for miR-17-5p, revealing a potential ceRNA regulatory network involving H19 and miR-17-5p with a role in the modulation of YES1 expression. This mechanism may contribute to a better understanding of thyroid cancer pathogenesis and provide new insights into the treatment of this disease.

Roles of SHARP1 in thyroid cancer.

SHARP1 is a basic helix‑loop‑helix transcription factor involved in various cellular processes, including proliferation and differentiation. The present study assessed the role of SHARP1 in the progression and invasion of thyroid cancer. PCR and western blot analysis demonstrated that in thyroid cancer tissues, SHARP1 was significantly downregulated at the mRNA and protein level compared with that in normal tissues. Furthermore, SHARP1 was downregulated in the TT and TPC‑1 thyroid cancer cell lines compared with a normal thyroid cell line, while it was upregulated in other thyroid cancer cell lines. Overexpression of SHARP1 in TT and TPC‑1 cells significantly inhibited the cell viability, migration and invasion invitro. Furthermore, the protein and mRNA levels of HIF‑1α were found to be decreased in TT and TPC‑1 cells following forced overexpression of SHARP1. In addition, silencing of HIF‑1α reduced the viability, migration and invasion of TT and TPC-1 cells. In conclusion, the present study indicated that SHARP1 acts as a tumor suppressor in thyroid cancer and that its downregulation may contribute to the proliferation, migration and invasion of thyroid cancer cells through mechanisms possibly involving HIF‑1α, suggesting that SHARP1 may be an important therapeutic target for the treatment of thyroid cancer.

High-throughput image analysis in the diagnosis of papillary thyroid carcinoma.

Thyroid cancer is the most common endocrine cancer, and its incidence has been increasing worldwide in the past decades. The increasing demand in medicine for rapid and accurate diagnosis enabled the application of digital imaging analysis in order to increase workflow efficiency and accurate analyses. The present study aimed to automatically differentiate papillary thyroid carcinoma from normal thyroid cells using high-throughput image analysis. Images of cellular specimens were taken with a digital camera and were subsequently analyzed. Other software was used for machine-learning-based cellular diagnostics. The two different classes were correctly identified with high sensitivity and specificity. The data created offers great potential for an automated diagnosis. Diagn. Cytopathol. 2016;44:574-577. © 2016 Wiley Periodicals, Inc.

FRTL-5 Rat Thyroid Cells Release Thyroglobulin Sequestered in Exosomes: A Possible Novel Mechanism for Thyroglobulin Processing in the Thyroid.

Exosomes are 30–100 nm, membrane-bound vesicles containing specific cellular proteins, mRNAs, and microRNAs that take part in intercellular communication between cells. A possible role for exosomes in thyroid function has not been fully explored. In the present study, FRTL-5 rat thyroid cells were grown to confluence and received medium containing either thyroid stimulating hormone (TSH), exogenous bovine thyroglobulin (bTg), or neither additive for 24 or 48 hours followed by collection of spent medium and ultracentrifugation to isolate small vesicles. Transmission electron microscopy and Western blotting for CD9 indicated the presence of exosomes. Western blotting of exosome extract using a monoclonal anti-Tg antibody revealed a Tg-positive band at ~330 kDa (the expected size of monomeric Tg) with a higher density in TSH-treated cells compared to that in untreated cells. These results are the first to show that normal thyroid cells in culture produce exosomes containing undegraded Tg.

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.