Normal Thyroid Cells Research Articles

A ceRNA Circuitry Involving the Long Noncoding RNA Klhl14-AS, Pax8, and Bcl2 Drives Thyroid Carcinogenesis.

Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology. SIGNIFICANCE: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.

Understanding Thyroid Cell Stress.

Understanding the regulatory mechanisms that control intracellular stress has fundamental importance since its failure results in cell death. Evidence has emerged indicating that the intracellular signals that are induced in response to diverse stresses include the deoxyribonucleic acid damage response, the unfolded protein response, the mitochondrial and/or endoplasmic reticulum stress responses, and the autophagy signals to degrade dangerous protein aggregates. These signals bring changes to the stressed cells that may support systemic homeostasis or contribute to disease pathology. In normal thyroid cells, both reactive oxygen species (ROS) and antioxidant (AOD) activity is low. An increase in ROS balanced by AOD leads only to mild inflammation, but unopposed increases in ROS lead to a strong inflammatory response and may result in apoptosis. A balance between ROS and AOD is, therefore, needed to maintain thyrocyte homeostasis. This perspective describes how thyroid cells are subjected to multiple insults and how they try to protect themselves using these different cellular responses.

PTEN-knockdown disrupts the morphology, growth pattern and function of Nthy-Ori 3-1 cells by downregulating PAX8 expression.

The incidence of thyroid disorders, which are common endocrine diseases, has rapidly increased in recent years. However, the etiology and pathogenesis of these disorders remain unclear. Phosphatase and tension homolog (PTEN) is a dual-specific phosphatase that is associated with multiple thyroid disorders; however, the role of PTEN in thyroid disorders remains unknown. In the present study, the human thyroid follicular epithelial cell line Nthy-Ori 3-1 was used to determine the role of PTEN in thyroid disorders. PTEN expression was knocked down using a PTEN-specific short hairpin RNA. Western blotting was subsequently used to determine protein expression, the Matrigel tube formation assay and iodide uptake assay were applied for evaluating the morphology and function of thyroid cells. The results showed that PTEN knockdown decreased the protein expression of paired box 8 (PAX8). The morphology and tubular-like growth pattern of thyroid cells were therefore disrupted, and restoration of PAX8 expression reversed these effects. Furthermore, PTEN-knockdown decreased the expression of specific thyroid proteins (thyroglobulin, TG; thyroid peroxidase, TPO; and sodium/iodide symporter, NIS) and inhibited the iodide uptake ability of thyroid cells by downregulating PAX8, suggesting that PTEN deficiency may impair the function of thyroid cells. In conclusion, the present study reported an important function of PTEN in normal thyroid cells and identified the involvement of PAX8. These results may improve understanding of the role of PTEN in the pathogenesis of thyroid disorders.

The Role of Lithium in Management of Endocrine Tumors-A Comprehensive Review.

Background: Epidemiological data reveal that treatment with lithium, a mood stabilizer, is associated with decreased incidence and mortality of certain cancer types, such as melanoma. Therefore, repositioning of lithium as an anticancer agent has emerged as a promising strategy in oncology. Since lithium affects the physiology of several endocrine tissues, the goal of this study was to analyze the role of lithium in the pathogenesis and treatment of tumors of the endocrine system.Methods: The databases of PubMed, EMBASE, MEDLINE, were searched from January 1970 through February 2019 for articles including the keywords “lithium and”—“thyroid cancer,” “thyroid nodule,” “parathyroid adenoma,” “parathyroid carcinoma,” “pituitary adenoma,” “pituitary neuroendocrine tumor,” “neuroendocrine tumor,” “carcinoid,” “adrenal adenoma,” “adrenal carcinoma,” “pheochromocytoma/paraganglioma.” Preclinical in vitro and in vivo studies as well as case series, retrospective cohort studies and prospective trials were selected for the analysis.Results: Treatment with lithium has been associated with a higher prevalence of thyroid enlargement, hypothyroidism and increased calcium levels due to parathyroid adenoma or hyperplasia, as one of the mechanisms of its action is to stimulate proliferation of normal follicular thyroid and parathyroid cells via activation of the Wnt signaling pathway. Supratherapeutic concentrations of lithium decrease the activity of glycogen synthase kinase-3β (GSK-3β), leading to cell cycle arrest in several in vitro cancer models including medullary thyroid cancer (TC), pheochromocytoma/paraganglioma and carcinoid. Growth inhibitory effects of lithium in vivo have been documented in medullary TC xenograft mouse models. Clinically, lithium has been used as an adjuvant agent to therapy with radioactive iodine (RAI), as it increases the residence time of RAI in TC.Conclusion: Patients chronically treated with lithium need to be screened for hypothyroidism, goiter, and hyperparathyroidism, as the prevalence of these endocrine abnormalities is higher in lithium-treated patients than in the general population. The growth inhibitory effects of lithium in medullary TC, pheochromocytoma/paraganglioma and carcinoid were achieved with supratherapeutic concentrations of lithium thus limiting its translational perspective. Currently available clinical data on the efficacy of lithium in the therapy of endocrine tumors in human is limited and associated with conflicting results.

Effect of bone morphogenetic protein-4 on proliferation, apoptosis and migration of thyroid cancer cells and its mechanism

Objective To investigate the effects of bone morphogenetic protein-4 (BMP-4) on the proliferation, apoptosis and migration of thyroid cancer cells through extracellular signal-regulated kinase 1/2 (ERK1/2)/p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. Methods Thyroid papillary carcinoma (TPC)-1 cells (thyroid papillary carcinoma cells) were randomly divided into blank control group (BC), negative control group (NC) and interference group (KD). Cells in BC group were not transfected. NC group cells were transfected with negative control plasmids. KD group cells were transfected with the silenced BMP-4 plasmid. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the BMP-4 mRNA levels. Cell counting kit-8 (CCK-8) was used to measure cell proliferation. Flow cytometry was used to determine cell apoptosis. Transwell chamber and scratch assay was used to determine cell migration capacity. The levels of ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), p38MAPK and p-p38MAPK protein were determined by Western blotting. Results The level of BMP-4 mRNA in TPC-1 cells (5.86±0.42) was higher than that in HT-ori3 cells (normal thyroid cells) (1.00±0.03, t=30.537, P 0.05). Conclusion Silencing BMP-4 can inhibit the proliferation and migration of thyroid cancer cells and promote apoptosis, which may be related to down-regulation of ERK1/2/p38MAPK signaling pathway. Key words: Bone morphogenetic protein-4; Extracellular regulated protein kinase; p38 mitogen-activated protein kinase; Thyroid cancer; Proliferation; Apoptosis; Migration

Role of chemokine receptors in thyroid cancer and immunotherapy.

Inflammation is currently regarded as an essential component of malignancies. It is now known that the tumor microenvironment may profoundly influence the biological behavior of cancer cells and ultimately the patient's outcome. Chemokine and their receptor play a major role in determining the immune phenotype of the cells infiltrating the thyroid tumor microenvironment. Experimental evidence shows that both normal and cancer thyroid cells express specific chemokine receptors. The expression of at least some of these receptors exerts several biological effects, which influence the course of the disease. The present review article will take into account the role of the most studied chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, DARC, CCR3, CCR6 and CCR7) in the context of thyroid cancer. This review will focus on current knowledge provided by in vitro and in vivo studies specifically performed on thyroid cancer including (i) expression of chemokine receptors in normal and cancer thyroid cells; (ii) role of chemokine receptors in affecting the biological behavior of thyroid tumors including the metastatic process; (iii) current knowledge about immunotherapies through targeting of chemokine receptors in thyroid cancer.

MicroRNA-26a-5p inhibits proliferation, invasion and metastasis by repressing the expression of Wnt5a in papillary thyroid carcinoma.

BackgroundThyroid cancer (TC) is considered as the fastest growing malignancy in the human endocrine system, particularly papillary thyroid cancer (PTC). MicroRNAs (miRs) serve as a role in promoting or suppressing tumors in various types of malignant tumor including PTC. This study aims to explore whether microRNA-26a-5p (miR-26a-5p) could affect the proliferation, invasion and metastasis ability of PTC cells by regulating Wnt5a.Materials and methodsThe expression of miR-26a-5p was examined by qRT-PCR in PTC tissue samples (58 cases, mean age 53 years old) and PTC cell lines (K1 and BCPAP). Cell proliferation, invasion and migration were tested with CCK8 assay, colony formation assay, transwell invasion assay and wound healing assay, respectively. Luciferase reporting experiment was used to verify that Wnt5a is a molecular target of miR-26a-5p. The relationship between miR-26a-5p and Wnt5a was analyzed by qRT-PCR and Western blot and was further proved by Pearson’s correlation analysis. Animal (24 nude mice) experiments were used to demonstrate that miR-26a-5p inhibits tumor growth by targeting Wnt5a.ResultsThe expression of miR-26a-5p declined in PTC tissues (P<0.01). The expression of miR-26a-5 was also significantly down-regulated in PTC tissues with advanced TNM stages (P<0.01) and lymph node metastasis (P<0.01) compared with normal thyroid tissues. Compared with normal human thyroid cell line Nthy-ori 3-1, the expression of miR-26a-5p in K1 cells and BCPAP cells were nearly 4.02-fold (P<0.01) and 2.51-fold (P<0.01) reduced. Up regulation of miR-26a-5p inhibited proliferation, colony formation, invasion and migration of PTC cells. MiR-26a-5p negatively regulated Wnt5a expression (r=−0.887, P<0.01), yet Wnt5a overexpression reversed the tumor-suppressive effect of miR-26a-5p in PTC. Animal experiments further verified that miR-26a-5p inhibited PTC growth by targeting Wnt5a.ConclusionOverexpression of miR-26a-5p depresses proliferation, invasion, metastasis of PTC via Wnt5a. Therefore, miR-26a-5p may represent a potentially effective target gene for PTC.

Inhibition of mitochondrial respiration by tigecycline selectively targets thyroid carcinoma and increases chemosensitivity.

The role of mitochondria in cancer and mitochondria-targeted therapy has been gaining attention for its effectiveness and selectivity between cancer and normal cells. In line with this notion, our work demonstrates that inducing mitochondrial dysfunction by tigecycline, a FDA-approved antibiotic, selectively targets thyroid cancer and enhances chemosensitivity. We found that tigecycline inhibited proliferation and induced apoptosis in a panel of thyroid cancer cell lines. Consistently, tigecycline inhibited thyroid cancer growth in mice without causing significant toxicity. The combination of tigecycline with paclitaxel achieved greater efficacy than paclitaxel alone in vitro and in vivo. Mechanistically, tigecycline inhibited mitochondrial respiration and ATP reduction through decreasing mitochondrial membrane potential and inhibiting mitochondrial translation, leading to oxidative stress and damage. In contrast, tigecycline was ineffective in mitochondrial respiration-deficient cells, confirming that tigecycline acts on thyroid cancer via inhibiting mitochondrial respiration. Interestingly, although tigecycline inhibited mitochondrial respiration in both thyroid cancer and normal thyroid cells in a similar manner, tigecycline was more effective in thyroid cancer than normal thyroid cells, suggesting that thyroid cancer cells are more dependent on mitochondrial functions than normal thyroid cells. This was supported by our observations that thyroid cancer cells had higher level of mitochondrial biogenesis compared to normal thyroid cells. Our work is the first to demonstrate that the combination of chemotherapy with tigecycline is a potential sensitizing strategy for thyroid cancer treatment. Our findings also highlight the higher dependence of thyroid cancer cells on mitochondrial functions than normal thyroid cells.

Nobiletin Alone or in Combination with Cisplatin Decreases the Viability of Anaplastic Thyroid Cancer Cell Lines

Aim: Anaplastic thyroid cancer (ATC) is the most aggressive subtype of thyroid cancer, presenting high mortality. Currently, no curative treatments exist and new therapeutic strategies are required. Although nutraceuticals were reported to have anticancer properties, few studies exist on ATC. This study aimed to investigate the anticancer effects of nutraceuticals in ATC cell lines (T235, T238) in comparison with normal thyroid cells (PCCL3).Methods: The IC50 values of isothiocyanates (ITCs: sulforaphane, SFN; phenethyl isothiocyanate, PEITC) and polymethoxylated flavones (PMFs: nobiletin; orange peel extract, OPE) were determined. ITCs decreased ATC metabolic viability more efficiently than PMFs. The effects of PEITC and nobiletin on viability and cell cycle, alone or in combination with conventional drugs, were evaluated.Results: PEITC did not affect viability of normal thyroid and ATC cells, while nobiletin decreased viability in a dose-dependent manner in all cell lines, although cell cycle was not arrested. At 100 μM, nobiletin reduced ATC cell viability as efficiently as conventional drugs, such as cisplatin, while being less toxic to normal thyroid cells. When conjugated with 1 μM cisplatin, the combination decreased viability of T235 cells more efficiently than each compound alone.Conclusion: These results suggest nobiletin as a potential anticancer agent that warrants further investigation in ATC.

Two distinct E3 ligases, SCFFBXL19 and HECW1, degrade thyroid transcription factor 1 in normal thyroid epithelial and follicular thyroid carcinoma cells, respectively.

Thyroid transcription factor 1 (TTF1) regulates the tissue-specific expression of genes. However, the molecular regulation of TTF1 in thyroid normal and carcinoma cells has not been revealed. Here we identify 2 distinct ubiquitin E3 ligases that are responsible for TTF1 degradation in normal thyroid cells and carcinoma cells, respectively. Phorbol myristate acetate induced TTF1 protein degradation in the ubiquitin-proteasome system in both HTori3 thyroid follicular epithelial cells and follicular thyroid carcinoma 133 (FTC133) cells. Lysine 151 residue was identified as a ubiquitin acceptor site within TTF1 in both cell types. Overexpression of E3 ubiquitin protein ligase 1 containing HECT, C2, and WW domain (HECW1) induced TTF1 degradation and ubiquitination in Htori3 cells but not in FTC133 cells. Overexpression of ubiquitin E3 ligase subunit FBXL19 increased TTF1 ubiquitination and degradation in FTC133 cells, but it had no effect on TTF1 levels in Htori3 cells. Overexpression of TTF1 increased thyroglobulin and sodium/iodide symporter mRNA levels, cell migration, and proliferation in HTori3 cells, whereas the effects were reversed by the overexpression of HECW1. This study reveals an undiscovered molecular mechanism by which TTF1 ubiquitination and degradation is regulated by different E3 ligases in thyroid normal and tumor cells.-Liu, J., Dong, S., Wang, H., Li, L., Ye, Q., Li, Y., Miao, J., Jhiang, S., Zhao, J., Zhao, Y. Two distinct E3 ligases, SCFFBXL19 and HECW1, degrade thyroid transcription factor 1 in normal thyroid epithelial and follicular thyroid carcinoma cells, respectively.

AXL Is a Novel Predictive Factor and Therapeutic Target for Radioactive Iodine Refractory Thyroid Cancer.

Papillary thyroid carcinomas (PTCs) have an excellent prognosis, but a fraction of them show aggressive behavior, becoming radioiodine (RAI)-resistant and/or metastatic. AXL (Anexelekto) is a tyrosine kinase receptor regulating viability, invasiveness and chemoresistance in various human cancers, including PTCs. Here, we analyze the role of AXL in PTC prognosis and as a marker of RAI refractoriness. Immunohistochemistry was used to assess AXL positivity in a cohort of human PTC samples. Normal and cancerous thyroid cell lines were used in vitro for signaling, survival and RAI uptake evaluations. 38.2% of human PTCs displayed high expression of AXL that positively correlated with RAI-refractoriness and disease persistence or recurrence, especially when combined with v-raf murine sarcoma viral oncogene homolog B(BRAF) V600E mutation. In human PTC samples, AXL expression correlated with V-akt murine thymoma viral oncogene homolog 1 (AKT1) and p65 nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation levels. Consistently, AXL stimulation with its ligand growth arrest-specific gene 6 (GAS6) increased AKT1- and p65 NF-kB-phosphorylation and promoted survival of thyroid cancer cell lines in culture. Enforced expression or activation of AXL in normal rat thyroid cells significantly reduced the expression of the sodium/iodide symporter (NIS) and the radioiodine uptake. These data indicate that AXL expression levels could be used as predictor of RAI refractoriness and as a possible novel therapeutic target of RAI resistant PTCs.

Exosomes Expressing Thyrotropin Receptor Attenuate Autoantibody-Mediated Stimulation of Cyclic Adenosine Monophosphate Production.

Background: Exosomes or small extracellular vesicles secreted from cells are nanovesicles with a diameter of 40-150 nm, which play a number of roles in both physiologic and pathologic processes. In Graves' disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) on the surface of thyroid follicular epithelial cells and stimulate thyroid growth and thyroid hormone synthesis and secretion via cyclic adenosine monophosphate (cAMP) production. The present study aimed to confirm the existence of TSHR in exosomes secreted from thyroid cells and to define the role of TSHR exosomes in GD. Methods: Exosomes were isolated by differential centrifugation from the culture medium of the human thyroid follicular epithelial cell line (NTHY-ori 3-1) and thyroid carcinoma cell lines (8305C, 8505C, and FTC-133). TSHR expression in cell lysates and exosomes was evaluated by Western blot analysis. In order to study the function of TSHR exosomes, human embryonic kidney (HEK) 293 cells stably expressing TSHR (HEK/TSHR) were established. Using exosomes isolated from both HEK and HEK/TSHR cells, the binding capacity of the M22 human monoclonal autoantibody to TSHR exosomes and their effect on M22-mediated stimulation of cAMP production in HEK/TSHR cells were evaluated. As a positive control for the functional assay, human recombinant TSHR chimera protein capable of binding to TSH was used. Results: TSHR was detected in exosomes from cancer cells as well as normal epithelial cells. An in vitro binding assay showed that alkaline phosphatase-labeled M22 bound to TSHR exosomes in a dose-dependent manner. M22 dose-dependently stimulated intracellular cAMP production in HEK/TSHR cells. The addition of exosomes from HEK/TSHR cells but not those from parental HEK cells significantly ameliorated cAMP production stimulated by treatment with M22 in HEK/TSHR cells. A decoy effect similar to TSHR exosomes was observed for human recombinant TSHR chimera. Conclusions: The results suggest that exosomes expressing TSHR may be secreted from normal and cancerous thyroid cells. In the thyroid gland of patients with GD, TSHR exosomes may exert a decoy effect by sequestering autoantibody, thereby ameliorating autoantibody-mediated activation of thyroid function.

Exogenous Hydrogen Sulfide Regulates the Growth of Human Thyroid Carcinoma Cells.

Hydrogen sulfide (H2S) is involved in the development and progression of many types of cancer. However, the effect and mechanism of H2S on the growth of human thyroid carcinoma cells remain unknown. In the present study, we found that the proliferation, viability, migration, and invasion of human thyroid carcinoma cells were enhanced by 25–50 μM NaHS (an H2S donor) and inhibited by 200 μM NaHS. However, H2S showed no obvious effects on the proliferation, viability, and migration of human normal thyroid cells. Administration of 50 μM NaHS increased the expression levels of CBS, SQR, and TST, while 200 μM NaHS showed reverse effects in human thyroid carcinoma cells. After treatment with 25-50 μM NaHS, the ROS levels were decreased and the protein levels of p-PI3K, p-AKT, p-mTOR, H-RAS, p-RAF, p-MEK1/2, and p-ERK1/2 were increased, whereas 200 μM NaHS exerted opposite effects in human thyroid carcinoma cells. Furthermore, 1.4-2.8 mg/kg/day NaHS promoted the tumor growth and blood vessel formation in human thyroid carcinoma xenograft tumors, while 11.2 mg/kg/day NaHS inhibited the tumor growth and angiogenesis. In conclusion, our results demonstrate that exogenous H2S regulates the growth of human thyroid carcinoma cells through ROS/PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways. Novel H2S-releasing donors/drugs can be designed and applied for the treatment of thyroid cancer.

H2O2 Metabolism in Normal Thyroid Cells and in Thyroid Tumorigenesis: Focus on NADPH Oxidases.

Thyroid hormone synthesis requires adequate hydrogen peroxide (H2O2) production that is utilized as an oxidative agent during the synthesis of thyroxin (T4) and triiodothyronine (T3). Thyroid H2O2 is generated by a member of the family of NADPH oxidase enzymes (NOX-es), termed dual oxidase 2 (DUOX2). NOX/DUOX enzymes produce reactive oxygen species (ROS) as their unique enzymatic activity in a timely and spatially regulated manner and therefore, are important regulators of diverse physiological processes. By contrast, dysfunctional NOX/DUOX-derived ROS production is associated with pathological conditions. Inappropriate DUOX2-generated H2O2 production results in thyroid hypofunction in rodent models. Recent studies also indicate that ROS improperly released by NOX4, another member of the NOX family, are involved in thyroid carcinogenesis. This review focuses on the current knowledge concerning the redox regulation of thyroid hormonogenesis and cancer development with a specific emphasis on the NOX and DUOX enzymes in these processes.

Transcription Factor HOXA9 is Linked to the Calcification and Invasion of Papillary Thyroid Carcinoma

Calcification is important for the diagnosis of papillary thyroid carcinoma (PTC). Runt-related transcription factor 2 (RUNX2), a master transcription factor associated with osteogenic differentiation, is reportedly related to PTC calcification and invasiveness. However, its regulatory role in this process is somewhat uncharacterized. Here, we attempted to identify genes that regulate RUNX2 and clarify its function in PTC carcinogenesis and calcification. The expression of RUNX2-upstream genes was evaluated by real-time PCR in Nthy-Ori 3-1 normal thyroid cells and TPC1 and BHP10-3 PTC cell lines. Luciferase and chromatin immunoprecipitation assays were performed with candidate genes after cloning the RUNX2 promoter. We found that RUNX2 promoter activity was enhanced by homeobox family A9 (HOXA9). Over-expression of HOXA9 was found to enhance alkaline phosphatase activity, mineralization, and in vitro tumour cell migration and invasion, whereas downregulation had the opposite effects. These results indicate that HOXA9, a positive regulator of RUNX2, can enhance calcification, migration, and invasion in PTC. Our data improve the understanding of the molecular mechanisms of microcalcification in PTC as well as tumorigenesis.

MicroRNA-30a suppresses papillary thyroid cancer cell proliferation, migration and invasion by directly targeting E2F7.

microRNA (miRNA/miR)-30a, a tumor-associated miRNA, has been implicated in the tumorigenesis and progression of different types of human cancer. Thyroid cancer is a common endocrine malignancy, of which papillary thyroid cancer (PTC) accounts for ~80–90% of all TC. However, the effect of miR-30a in PTC is yet to be fully elucidated. The TPC-1 human PTC cell line, as well as the normal human thyroid cell line (HT-ori3), were utilized in the current study. The PTC cell line was transfected with a miR-30a mimic. Subsequently, reverse transcription-quantitative polymerase chain reaction was performed to detect the expression of miR-30a and E2F transcription factor 7 (E2F7). Cell proliferation was assessed via a MTT assay and transwell migration and invasion assays were performed to detect the migration and invasion of PTC cells. A dual-luciferase reporter assay was also utilized to clarify the association between miR-30a and E2F7. The results of the current study revealed that miR-30a was significantly downregulated in TPC-1 cells compared with HT-ori3 cells and that the expression of E2F7 was significantly upregulated in PTC cells. The upregulation of miR-30a also inhibited the proliferation, migration and invasion of PTC cells. Furthermore, the luciferase assay revealed that miR-30a binds to the 3′-UTR of E2F7. Additionally, the overexpression of miR-30a decreased E2F7 levels in TPC-1 cells. These results indicate that miR-30a functions as a tumor suppressor in PTC by direct targeting E2F7 and that miR-30a may be a novel therapeutic target for patients with PTC.

High-Resolution Raman Microscopic Detection of Follicular Thyroid Cancer Cells with Unsupervised Machine Learning.

We use Raman microscopic images with high spatial and spectral resolution to investigate differences between human follicular thyroid (Nthy-ori 3-1) and follicular thyroid carcinoma (FTC-133) cells, a well-differentiated thyroid cancer. Through comparison to classification of single-cell Raman spectra, the importance of subcellular information in the Raman images is emphasized. Subcellular information is extracted through a coarse-graining of the spectra at high spatial resolution (∼1.7 μm2), producing a set of characteristic spectral groups representing locations having similar biochemical compositions. We develop a cell classifier based on the frequencies at which the characteristic spectra appear within each of the single cells. Using this classifier, we obtain a more accurate (89.8%) distinction of FTC-133 and Nthy-ori 3-1, in comparison to single-cell spectra (77.6%). We also infer which subcellular components are important to cellular distinction; we find that cancerous FTC-133 cells contain increased populations of lipid-containing components and decreased populations of cytochrome-containing components relative to Nthy-ori 3-1, and that the regions containing these contributions are largely outside the cell nuclei. In addition to increased classification accuracy, this approach provides rich subcellular information about biochemical differences and cellular locations associated with the distinction of the normal and cancerous follicular thyroid cells.

SAT-549 Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Primary Human Thyrocytes

Thyroid gland, as one crucial endocrine organ to synthesize and secrete thyroid hormones directly response to TSH, is indispensable to human health. The secretion of exosomes by FRTL-5 normal thyroid cell line and TPC-1 papillary thyroid cancer cell line has been verified. However, the verification and profiling of exosomes secreted by human thyroid and a possible role for exosomes in thyroid function has not been explored. To investigate whether human thyroid could secrete exosomes, normal thyroid tissue from the surgical thyroidectomy of benign follicular nodules were collected and digested. Primary thyrocytes were cultured with or without TSH, and the medium was collected. Exosomes were isolated via differential ultracentrifugation, observed under transmission electron microscopy, measured by nanoparticle tracking analysis, and confirmed by immunoblot. Further, label-free liquid chromatography (LC)-mass spectrometry (MS)/MS analysis was conducted to investigate the proteomic profiling of thyrocytes exosomes. In the present study, secreted exosomes were verified in primary human thyrocytes. Exosomes exhibited the same shape and size (102.80±36.01 vs. 95.82 ±30.10 nm, p>0.05) as control exosomes after TSH stimulation, but importantly, the total number of exosomes increased significantly after TSH stimulation for 48 h (3.46±0.56×107 vs. 1.98 ±0.20 ×107 nm, p<0.05). The mRNA level of Rab27b, the protein crucial for late endosomal formation, was also increased significantly after TSH stimulation, indicating that TSH activated the biogenesis of exosomes. Overall, 202 unique proteins were identified in thyrocytes exosomes. These included multiple proteins known to be involved in the endosomal pathway, such as annexins, and several cytoskeletal, cytoskeletal binding-proteins and motor proteins. One significant identified protein was thyroglobulin. Among the identified proteins, 77 proteins exhibited a differential expression pattern in TSH exosomes compared with the control exosomes, which mainly participate in cellular process, metabolic process, biological regulation, response to stimulus and regulation of biological process. This study provides direct evidence that human thyroid secretes exosomes. The characterization and proteomic profiling of exosome secreted by primary human thyrocytes provides potential physiological significance of exosomes in thyroid function.

The BRAF-inhibitor PLX4720 inhibits CXCL8 secretion in BRAFV600E mutated and normal thyroid cells: a further anti-cancer effect of BRAF-inhibitors

CXCL8 is a chemokine secreted by normal and thyroid cancer cells with proven tumor-promoting effects. The presence of BRAFV600E mutation is associated with a more aggressive clinical behavior and increased ability to secrete CXCL8 by papillary-thyroid-cancer cells. Aim of this study was to test the effect of the BRAF-inhibitor (PLX4720) on the basal and TNF-α-induced CXCL8 secretions in BRAFV600E mutated (BCPAP, 8305C, 8505C), in RET/PTC rearranged (TPC-1) thyroid-cancer-cell-lines and in normal-human-thyrocytes (NHT). Cells were incubated with increasing concentrations of PLX4720 alone or in combination with TNF-α for 24-hours. CXCL8 concentrations were measured in the cell supernatants. PLX4720 dose-dependently inhibited the basal and the TNF-α-induced CXCL8 secretions in BCPAP (F: 14.3, p < 0.0001 for basal and F: 12.29 p < 0.0001 for TNF-α), 8305C (F: 407.9 p < 0.0001 for basal and F: 5.76 p < 0.0001 for TNF-α) and 8505C (F:55.24 p < 0.0001 for basal and F: 42.85 p < 0.0001 for TNF-α). No effect was found in TPC-1 (F: 1.8, p = 0.134 for basal; F: 1.6, p = 0.178 for TNF-α). In NHT an inhibitory effect was found only at the highest concentration of PLX4720 (F: 13.13 p < 0.001 for basal and F: 2.5 p < 0.01 for TNF-α). Cell migration assays showed that PLX4720 reduced both basal and CXCL8-induced cell migration in BCPAP, 8305C, 8505C and NHT but not in TPC-1 cells. These results constitutes the first demonstration that PLX4720 is able to inhibit the secretion of CXCL8 in BRAFV600E mutated thyroid cancer cells indicating that, at least some, of the anti-tumor activities of PLX4720 could be exerted through a lowering of CXCL8 in the thyroid-cancer-microenvironment.

MicroRNA-510-5p promotes thyroid cancer cell proliferation, migration, and invasion through suppressing SNHG15.

SNHG15 has been suggested to be correlated with clinical progression and prognosis, and function as tumor suppressive long noncoding RNA in thyroid cancer at our previous study. SNHG15 was proposed to be a potential target for miR-510-5p at LncBase Predicted database. Thus, the aim of this study was to explore the relationship between miR-510-5p and SNHG15 in thyroid cancer, and the clinical significance of miR-510-5p in patients with thyroid cancer. In our results, levels of miR-510-5p expression were increased in thyroid cancer tissues and cell lines compared with adjacent normal thyroid tissues and normal thyroid cell line, respectively. There was a statistically negative correlation between SNHG15 expression and miR-510-5p expression in thyroid cancer tissues. Moreover, miR-510-5p directly bound to SNHG15, and negatively regulated SNHG15 expression in thyroid cancer cells. Furthermore, miR-510-5p promoted thyroid cancer cell proliferation, migration, and invasion through suppressing SNHG15. Finally, high miR-510-5p expression was observed in tumor tissues with advanced clinical stage or lymph node metastasis. In conclusion, we provide evidence to support a pivotal role for miR-510-5p in regulating thyroid cancer cell proliferation, migration, and invasion.