BCPAP Cell Lines Research Articles

Abstract 2780: IL13RA2 is differentially regulated in papillary thyroid carcinoma versus follicular thyroid carcinoma

Abstract The interleukin-13 receptor alpha2 (IL13RA2), which is known to overexpressed in glioblastoma multiforme, plays a role in various cellular processes such as cell migration that may contribute to tumor progression. Studies have attributed IL13RA2 to invasion and metastasis in cancers of the ovary, breast, and pancreas but the pathological role of IL13RA2 in thyroid cancer is still unclear. This study aims to evaluate the expression of IL13RA2 in thyroid carcinomas and examine the role of IL13RA2 in progression of papillary thyroid cancer (PTC). We performed IL13RA2 immunochemical staining on tissue microarrays of 137 thyroid carcinomas and observed that IL13RA2 expression was significantly correlated with advanced tumor stage (pT3 / pT4; p=0.001) and regional lymph node metastasis (pN1; p<0.001). Moreover, the staining scores of IL13RA2 were significantly higher in PTC compared to follicular and anaplastic subtypes (p<0.02) and correlated with advanced tumor stage amongst PTC samples (pT3 / pT4; p=0.028). This differential profile of IL13RA2 in PTC was further validated in thyroid cancer cell lines overexpressing IL13RA2 to assay the effects on cell proliferation, cell migration and epithelial-mesenchymal transition (EMT) using CCK-8, transwell migration assay, qRT-PCR and western blot analyses. Knockdown of IL13RA2 in the PTC subtype B-CPAP cell line showed significantly reduced cell viability, cell migration and EMT markers including N-cadherin, Vimentin and Snail. Exogenous overexpression of IL13RA2 in another PTC cell line, K1 increased cell migration and EMT although cell proliferation was not affected. In summary, we demonstrated that IL13RA2 is differentially regulated in PTC and is involved in cell migration by enhancing EMT. The underlying molecular mechanisms on how IL13RA2 drives progression of thyroid cancer remains to be further investigated. Citation Format: Siao Ting Chong, Catherine Y. Kok, Khee Ming Tan, Shou Ping Guan, Siang Hui Lai, Cindy Lim, Jiancheng Hu, Charles Sturgis, Charis Eng, Paula Y. Lam, Joanne Ngeow. IL13RA2 is differentially regulated in papillary thyroid carcinoma versus follicular thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2780.

Novel design of NIR-triggered plasmonic nanodots capped mesoporous silica nanoparticles loaded with natural capsaicin to inhibition of metastasis of human papillary thyroid carcinoma B-CPAP cells in thyroid cancer chemo-photothermal therapy

In the search for developing a biomedicine based nanomaterial for therapeutic applications, here we described a new benign development of Photo-triggered Gold nanodots capped mesoporous silica nanoparticles Au@MSNs loaded with capsaicin (Cap) for photothermal therapy of cancer cells. Electron microscopic techniques (SEM and TEM) studies depict the anisotropic shape of Cap-Au@MSNs with mean size ≈110 nm. The successful amine functionalization and covalent interaction of Au nanodots on the mesoporous silica surface were confirmed from the results of FTIR, XPS and UV–vis spectral analyses, which directly indicates the composition of synthesized mesoporous silica surface. Additionally, Dynamic Light Scattering (DLS) revealed that synthesized cap-AuMSNs were stable with highly negatively charged. Cap-AuMSNs exhibited extraordinary in vitro antitumor activity against the tested twp thyroid cancer cell lines (i.e., FTC-133 and B-CPAP). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay determined that capsaicin and Cap-AuMSNs conferred strong cytotoxicity against the FTC-133 and B-CPAP cell lines. Further, evaluation of the mechanism showed that anticancer activity was achieved by inducing apoptosis in thyroid cancer cells. In addition, we found that such compounds exhibited promising antimetastatic activity and reduced the invasiveness of cancer cells. Hence, we suggesting that these Cap-Au@MSNs can be used as promising candidates for cancer therapy and deserve further investigation.

SAT-572 Morphological and Molecular Alterations in Thyroid Neoplastic Cells Infected by Epstein-Barr Virus May Be Associated to Tumor Evolution

ABSTRACT Title: Morphological and molecular alterations in thyroid neoplastic cells infected by Epstein-Barr virus may be associated to tumor evolution. The Epstein-Barr Virus (EBV) is an ubiquitous human herpes virus and more than 90% of adults, worldwide, have evidence of infection. Since the virus is able to cause a latent and persistent infection, interfering in the metabolism of host cells and triggering a tumorigenic process, it has been considered a human carcinogen by the International Agency for Research on Cancer. EBV has been held responsible for 2% of the malignancies, and is associated to nasopharyngeal carcinoma, gastric cancer, Burkitt’s and Hodgkin’s lymphoma. More recently, our group reported the presence of high EBV viral load in malignant thyroid tumors. Thyroid cancer prevalence has been raising all over the world but its typical evolution is indolent and tumors may even regress. In fact, current guidelines advise small low risk tumors observation without immediate surgical intervention. Looking for the putative clinical role of EBV infection in the evolution of thyroid tumors, we aimed to evaluate the ability of the virus to cause morphological and molecular changes in neoplastic thyroid cell lines TPC-1, BCPAP and 8505C. Viral adaptation for the analysis of the cytopathic effect of the neoplastic thyroid cell lines was performed, followed by viral kinetics of 48 hours for viral load and genes expression analysis of KRAS, NRAS, HRAS and TP53. Comparison of the inoculated cells with non-inoculated control cells showed that all tumor cell lines were permissive to the virus; however, the virus caused cytopathic effect, altering the cell morphology only in the TPC-1 and 8505C. There was hyperexpression of KRAS and NRAS in all cell lines whereas the virus was associated to an initial increase of TP53 that further decreased its expression. We conclude that neoplastic thyroid cell lines are permissive to EBV and that the virus is capable of causing cytopathic effect in both well-differentiated and undifferentiated thyroid cell lines. We also demonstrated that EBV interferes in gene expression in thyroid neoplastic cell lines producing an increase in the expression of the oncogenes KRAS, NRAS and TP53 followed by the loss of expression of the tumor suppressor gene TP53. We suggest that these effects could be related to tumor progression. Keywords: Humam Herpesvirus 4. EBV. Thyroid Cancer. Viral Load. RAS expression.

BRAF V600E mutation and microRNAs are helpful in distinguishing papillary thyroid malignant lesions: Tissues and fine needle aspiration cytology cases

AimsMutations of BRAF oncogene are considered to contribute in the invasiveness and poor clinicopathologic features of papillary thyroid cancer (PTC). As a step towards understanding the underlying molecular mechanisms of this contribution, we aimed to examine the association of four microRNAs' (miR-222, -137, -214, -181b) levels with BRAFV600E and clinicopathological features in PTC tissues and fine needle aspiration (FNA) specimens. MethodsIn total, 56 PTC and 27 benign with multinodular goiter tissue samples, 95 FNA samples, and B-CPAP and HEK293 cell lines were examined. BRAFV600E mutation was examined in PTC tissues and FNA samples. Expression of microRNAs was assessed by real-time quantitative reverse transcription-PCR. Key findingsThe frequency of BRAFV600E in PTC tissues and FNA samples “suspicious for PTC” was 41.1 and 36.8%, respectively. MiR-222, -137, -214, and -181b were significantly upregulated in PTC tumors (P < 0.05) and in B-CPAP cell line (P < 0.001). In FNA, the expressions of miR-222, -181b and -214 were significantly elevated in patients suspected for PTC (P < 0.05), while there was no significant difference in miR-137. After adjustment for age and sex, miR-181b was associated with an increased risk of bearing BRAFV600E mutation (OR: 1.27; 95% CI: 1.01–1.61; P = 0.045) and risk of lymphovascular invasion (OR: 1.66; 95% CI: 1.01–2.72; P = 0.045); miR-137 was associated with the risk of larger tumor size (OR: 1.31; 95% CI: 1.04–1.65; P = 0.022); miR-222 was related to increase in extracapsular invasion (OR: 1.28; 95% CI: 1.04–1.57; P = 0.018). SignificanceUpregulation of miR-222, -214 and -181b has been confirmed in PTC tumors, FNA samples and cell line. MiR-137 upregulation has been confirmed in PTC tumors and cell line, but not in FNA samples. MiR-222, -137 and -181b showed an association with the degree of malignancy in PTC tumors.

64Cu and fluorescein labeled anti-miRNA peptide nucleic acids for the detection of miRNA expression in living cells

MiRNAs are single stranded RNAs of 18–22 nucleotides. They are promising diagnostic and prognostic markers for several pathologies including tumors, neurodegenerative, cardiovascular and autoimmune diseases. In the present work the development and characterization of anti-miRNA radiolabeled probes based on peptide nucleic acids (PNAs) for potential non-invasive molecular imaging in vivo of giant cell arteritis are described. MiR-146a and miR-146b-5p were selected as targets because they have been found up-regulated in this disease. Anti-miR and scramble PNAs were synthesized and linked to carboxyfluorescein or DOTA. DOTA-anti-miR PNAs were then labelled with copper-64 (64Cu) to function as non-invasive molecular imaging tools. The affinity of the probes for the targets was assessed in vitro by circular dichroism and melting temperature. Differential uptake of fluorescein and 64Cu labeled anti-miRNA probes was tested on BCPAP and A549 cell lines, expressing different levels of miR-146a and -146b-5p. The experiments showed that the anti-miR-146a PNAs were more effective than the anti-miR-146b-5p PNAs. Anti-miR-146a PNAs could bind both miR-146a and miR-146b-5p. The uptake of fluorescein and 64Cu labeled anti-miR-146a PNAs was higher than that of the negative control scramble PNAs in miRNA expressing cells in vitro. 64Cu-anti-miR-146a PNAs might be further investigated for non-invasive PET imaging of miR-146 overexpressing diseases.

SIRT4 inhibits the proliferation, migration, and invasion abilities of thyroid cancer cells by inhibiting glutamine metabolism.

BackgroundSIRT4, a protein localized in the mitochondria, is one of the least characteristic members of the sirtuin family. It is known that SIRT4 has deacetylase activity and plays a role in energy metabolism, but little is known about its possible role in carcinogenesis. Recently, several studies have suggested that SIRT4 may function as either a tumor oncogene or a tumor suppressor gene. However, its relationship with thyroid cancer remains unclear.MethodsWe stably overexpressed SIRT4 or silenced its expression in the human thyroid cancer cell line BCPAP by means of lentiviral vectors. We conducted a variety of tests, such as CCK-8, wound healing, migration, and invasion assays, to investigate the role of SIRT4 in the proliferation, migration, and invasion abilities of thyroid cancer cells. We also investigated the effects of SIRT4 overexpression on cell cycle progression and apoptosis of BCPAP cells and studied the role of glutamine metabolism in the effects of SIRT4 on BCPAP cell migration and invasion. Finally, we analyzed SIRT4 expression levels in thyroid cancer specimens by immunohistochemistry and investigated their association with clinicopathological features.ResultsOverexpression of SIRT4 inhibited the proliferation, migration, and invasion abilities of BCPAP thyroid cancer cells, blocked the cell cycle in the G0/G1 phase, and induced apoptosis. Mechanistically, SIRT4 inhibited BCPAP migration and invasion by inhibiting glutamine metabolism. Moreover, we found that SIRT4 protein levels in thyroid cancer tissues were markedly lower than in their non-neoplastic tissue counterparts (P<0.001).ConclusionSIRT4 plays a pivotal role in the growth and metastasis of thyroid cancer cells and could be a potential therapeutic target in thyroid cancer.

Lipase member H is a downstream molecular target of hypoxia inducible factor-1α and promotes papillary thyroid carcinoma cell migration in BCPAP and KTC-1 cell lines.

BackgroundPapillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma, which is associated with a high incidence of lymph-node metastasis. Multiple biomarkers have been identified for the precise diagnosis of PTC at an early stage. However, their role in PTC remains poorly elucidated. Previously, we reported that lipase H (LIPH), a membrane-bound protein, was highly expressed in PTC. This study aimed to fully elucidate the causal role of LIPH in the development of PTC and investigated its relationship with lymph-node metastasis in PTC.Materials and methodsQuantitative reverse transcription PCR and immunohistochemistry were used to measure the mRNA and protein expression levels of LIPH in 45 and 6 pairs of PTC tissues and adjacent normal tissues, respectively. Clinical tissue data of 504 PTC tissues and 60 normal thyroid tissues from The Cancer Genome Atlas database were used to analyze the correlation between LIPH expression level and clinical features in PTC. siRNAs were used to knock down genes, while plasmids were used to overexpress genes. Two PTC cell lines (KTC-1 and BCPAP) were used in subsequent cytological function studies. In addition, a hypoxia stress model was constructed using cobaltous chloride hexahydrate reagent, and the protein expression level of the corresponding biomarkers was measured by Western blotting.ResultsThis study revealed that high expression of LIPH in PTC was closely associated with lymph-node metastasis. Our cellular function experiments indicated that LIPH positively correlated with the malignant behavior of PTC cell lines. We further confirmed the role of LIPH in hypoxia and its relationship with the epithelial–mesenchymal transition pathway in PTC.ConclusionLIPH plays an important role in PTC oncogenesis and development, especially in lymph-node metastasis. It can be regarded as a biomarker for the diagnosis and treatment of PTC in the near future.

The effect of wnt5a on proliferation and invasion of papillary thyroid carcinoma cells

Objective To investigate the effect of Wnt5a expression on the biological behavior of papillary thyroid carcinoma cells. Methods The proliferation of papillary thyroid carcinoma cells with Wnt5a low-expressed or over-expressed was detected by MTT. The clone-forming abilities of papillary thyroid carcinoma cells with Wnt5a low-expressed or over-expressed were detected by colony formation assays. The invasion abilities of papillary thyroid carcinoma cells with Wnt5a low-expressed or over-expressed were detected by Transwell assays. Results The MTT assays demonstrated that A values of Wnt5a over-expressing K1 cells detected on the third and fourth days (0.403±0.003, 0.562±0.005) were significantly higher than those of the control groups (0.371±0.006, 0.454±0.002), and the A values of the Wnt5a-small interfering RNA (siRNA)-2 groups (0.342±0.005, 0.402±0.004) were significantly lower than those of the control groups (t=0.951, 14.206, -6.431, -20.140; P=0.030, 0.000, 0.003, 0.000). Besides, the A values of Wnt5a over-expressing BCPAP cells detected on the third and fourth days (0.454±0.007, 0.603±0.002) were significantly higher than those of the control groups (0.403±0.006, 0.493±0.002), and the A values of the Wnt5a-siRNA-2 groups (0.362±0.005, 0.370±0.003) were significantly lower than those of the control groups (t=0.839, 27.361, -9.092, -29.087; P=0.010, 0.000, 0.001, 0.000). The results of colony formation assays showed that the clone numbers of cell line K1 and BCPAP cells (130.00±4.34, 117.00±3.54) were significantly greater than those of the control groups (88.00±3.44, 86.00±3.48) after over-expressing Wnt5a (t=13.136, 10.816; P=0.000). Whereas, after knocking down Wnt5a, the numbers of K1 and BCPAP cell clones (50.00±3.45, 47.00±4.01) were significantly lower than those of the control groups (t=-13.510, -12.253; P=0.000). The Transwell assays showed that after over-expressing Wnt5a, compared with the control groups [(80.20±1.23), (83.90±1.45) cells], the numbers of cells passing through the basement membrane in each high-power field of K1 and BCPAP [(155.60±2.01), (164.50±2.09) cells] were significantly increased (t=-25.420, -24.881; P=0.000, ). After knocking down Wnt5a, compared with the control groups, the numbers of cell lines K1 and BCPAP across the basement membrane were significantly decreased [(50.80±1.02), (52.30±1.08) cells, t=21.868, 28.938, P=0.000]. Conclusion Over-expression of Wnt5a could promote the proliferation, cloning and invasion of thyroid papillary carcinoma cells. Knockdown of Wnt5a could inhibit the proliferation, cloning and invasion of thyroid papillary carcinoma cells. Key words: Wnt5a; Papillary thyroid carcinoma; Cell proliferation; Cell invasion

Metabolomic Alterations in Thyrospheres and Adherent Parental Cells in Papillary Thyroid Carcinoma Cell Lines: A Pilot Study.

Papillary thyroid carcinoma (PTC), is characterized by a heterogeneous group of cells, including cancer stem cells (CSCs), crucially involved in tumor initiation, progression and recurrence. CSCs appear to have a distinct metabolic phenotype, compared to non-stem cancer cells. How they adapt their metabolism to the cancer process is still unclear, and no data are yet available for PTC. We recently isolated thyrospheres, containing cancer stem-like cells, from B-CPAP and TPC-1 cell lines derived from PTC of the BRAF-like expression profile class, and stem-like cells from Nthy-ori3-1 normal thyreocyte-derived cell line. In the present study, gas chromatography/mass spectrometry metabolomic profiles of cancer thyrospheres were compared to cancer parental adherent cells and to non cancer thyrospheres profiles. A statistically significant decrease of glycolytic pathway metabolites and variations in Krebs cycle metabolites was found in thyrospheres versus parental cells. Moreover, cancer stem-like cells showed statistically significant differences in Krebs cycle intermediates, amino acids, cholesterol, and fatty acids content, compared to non-cancer stem-like cells. For the first time, data are reported on the metabolic profile of PTC cancer stem-like cells and confirm that changes in metabolic pathways can be explored as new biomarkers and targets for therapy in this tumor.

Vitamin D, FOXO3a, and Sirtuin1 in Hashimoto's Thyroiditis and Differentiated Thyroid Cancer.

Background: Protective effects of vitamin D have been reported in autoimmune and malignant thyroid diseases, though little is known about the underlying mechanism. Sirtuin 1 histon deacethylase (SIRT1) links the vitamin D pathway with regulation of transcription factor FOXO3a, a key player in cell cycle regulation and apoptosis. Aim of the present study was to investigate common single nucleotide polymorphisms (SNP's) in FOXO3a gene in respect to thyroid diseases, as well as to evaluate the hypothesis of Sirtuin1-FOXO3a interaction being a mediator of anti-proliferative vitamin D effects.Methods: The SNP's FOXO3a rs4946936/rs4945816/rs9400239 were genotyped in 257 patients with differentiated thyroid carcinoma (DTC), 139 patients with Hashimoto thyroiditis (HT) and 463 healthy controls (HC). Moreover, T-helper cells of HC and papillary thyroid cancer cell line BCPAP were incubated with 1,25(OH)2D3 and/or SIRT1 inhibitor Ex-527 in order to elucidate SIRT1- dependent vitamin D effects on cell proliferation and FOXO3a gene expression in vitro.Results: Patients with DTC tended to carry more often allele C in FOXO3a rs4946936 in comparison to HC (pcorrected = pc = 0.08). FOXO3a rs9400239T and rs4945816C was more frequent in HT in comparison to HC (pc = 0.02 and pc = 0.01, respectively). In both DTC and HT, we could not find a correlation of FOXO3a SNP's with vitamin D status. However, on in vitro level, 1,25(OH)2D3 showed an anti-proliferative effect in both T-helper cells and BCPAP, that was blocked by SIRT1 inhibition (T-helper cells: p = 0.0059, BCPAP: p = 0.04) and accompanied by elevated FOXO3a gene expression in T-helper cells (p = 0.05).Conclusions: FOXO3a rs9400239T and rs4945816C may constitute risk factors for HT, independent of the vitamin D status.This indicates the implication of FOXO3a in pathogenesis of autoimmune thyroid diseases. The dependency of anti-proliferative vitamin D effects on SIRT1 activity further suggests a key role of vitamin D-SIRT1-FOXO3a axis for protective vitamin D effects.

MMP-11 promotes papillary thyroid cell proliferation and invasion via the NF-κB pathway.

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer, and its incidence is on the rise. It has been reported that some matrix metalloproteinases (MMPs) are abnormally expressed in PTC and can be used as diagnostic markers. However, few studies have explored the underlying mechanisms by which MMPs promote tumor progression. In this study, we used microarray analysis to compare the variations of gene expression within the PTC cell populations and their adjacent normal tissues and found that MMP-11 was the most differentially expressed MMP. To investigate the role of MMP-11 in the mediation of thyroid cancer cell development, pEnter-MMP-11 plasmid, and MMP-11 small interfering RNA were applied to up- and downregulate MMP-11 expression of in cultured PTC cell lines K1 and BCPAP. The results suggested that the levels of proliferation and migration of cells transfected with MMP-11 siRNA were significantly reduced, while the levels in MMP-11-plasmid-transfected cells were increased. In terms of the mechanism, experimental data showed that the change in cyclin D1 is consistent with MMP-11 expression, which may explain the changes in proliferation. In addition, Western blot assay was conducted to analyze the p65 and activated (phospho-) p65 protein levels concomitant with MMP-11 adjustments. Variations in intracellular MMP-11 significantly altered the amount of phospho-p65 in thyroid cells, while p65 knockdown did not affect MMP-11 expression. These results suggest that MMP-11 is located upstream of p65 and regulates its activity. Interestingly, the data for the Transwell assay suggested that MMP-11 regulatory migration is also associated with the NF-κB p65 signaling pathway. In conclusion, this report describes the important role of MMP-11 in the regulation of thyroid cell proliferation and migration. Mechanistic studies have shown that cyclin D1 and p65 are important mediators in the processes, which provides a new way to study the mechanism of MMPs promoting the progression of thyroid cancer.

Retracted: MicroRNA‐150‐5p affects cell proliferation, apoptosis, and EMT by regulation of the BRAFV600E mutation in papillary thyroid cancer cells

Papillary thyroid cancer (PTC) is the most common endocrine malignancy. Studies have confirmed an association between microRNA (miRNA) and the BRAFV600E mutation in various cellular biological processes of PTC. This study aimed to clarify the potential relationship between miR-150-5p and the BRAFV600E mutation in PTC. Human PTC cell lines B-CPAP and TPC-1 were transfected with the miR-150-5p mimic, an inhibitor, and the corresponding controls. Then, cell proliferation, viability, and apoptosis were detected by bromodeoxyuridine, trypan blue exclusion, and flow cytometry assays. The expressions of the main factors of cell cycle, epithelial mesenchymal transition (EMT), and DNA mismatch repair were examined by Western blot analysis and a real-time quantitative polymerase chain reaction. Additionally, pc-BRAFV600E was transfected into B-CPAP and TPC-1 cells to determine the relationship between miR-150-5p and BRAFV600E . In addition, the methyl ethyl ketone (MEK)/extracellular signal-regulated kinase (ERK) signal pathway was examined using Western blot analysis. Overexpression of miR-150-5p promoted cell proliferation and viability, inhibited apoptosis, and upregulated cell cycle factor expressions at 50 passages of B-CPAP and TPC-1 cells after transfection. Overexpression of miR-150-5p led to an obvious decrease in E-cadherin expression, but enhanced N-cadherin, Slug and Vimentin, ZEB1, and Snail expression. Moreover, overexpression of miR-150-5p markedly suppressed POLD3, MSH2, and MSH3 expression. Furthermore, BRAFV600E overexpression increased the expression level of miR-150-5p in TPC cells, and overexpression of telomerase reverse transcriptase further enhanced the promoting effect of BRAFV600E on miR-150-5p expression in B-CPAP and TPC-1 cells. Finally, BRAFV600E overexpression activated the MEK/ERK signal pathway in B-CPAP and TPC-1 cells. These data indicated that miR-150-5p promoted cell proliferation, suppressed apoptosis, and accelerated the EMT process by regulation of the BRAFV600E mutation. Our findings will help elucidate the pathogenesis of PTC and identify biomarkers.

Evaluation of the role of platelet derived growth factor in papillary thyroid carcinoma by targeted ultrasound contrast agent

Objective To explore the role of targeted ultrasound contrast agent in the evaluation of angiogenesis in papillary thyroid carcinoma (PTC) in nude mice by constructing platelet derived growth factor receptor α (PDGFRα)-targeted ultrasound contrast agents, and to explore the effect of platelet derived growth factor (PDGF) on angiogenesis in PTC. Methods PDGFRα-targeted ultrasound contrast agents were constructed through biotin-avidin linkage, and blank micro-bubbles were served as control group. After the siRNA-PDGF BCPAP cell line was established, well prepared BCPAP cells or siRNA-PDGF-BCPAP cells were injected subcutaneously into the back of male BABL/C nude mice. The growth of the tumors was observed closely. All tumors of the normal group and the siRNA-PDGF group were examined by PDGFRα-targeted ultrasound contrast agents or blank micro-bubbles, and the dynamic images were analyzed quantitatively by QontraXt software.All mice were sacrificed after ultrasonography. Microvessels density (MVD) in the tumors was counted by CD31 staining and the expressions of vascular endothelial growth factor(VEGF) and PDGF were detected by Western Blot. Results PTCs in nude mice were enhanced well by PDGFRα-targeted ultrasound contrast agent or blank micro-bubbles. The peak of tumors in siRNA-PDGF group was significantly lower than that in the normal group [(43.085±13.244)% vs (57.428±10.952)%, P=0.004]; MVD of the siRNA-PDGF group was significantly less than that of the normal group [(23.200±6.017)strips/view vs (35.000±8.456)strips/view, P=0.012]. The protein expression of PDGF and VEGF in siRNA-PDGF group were significantly lower than those in the normal group, respectively (0.142±0.058 vs 0.269±0.102, P=0.002; 0.096±0.036 vs 0.158±0.072, P=0.016). Conclusions PDGF plays an important role in angiogenesis of PTC, it maybe a new ultrasound molecular imaging method for monitoring tumor angiogenesis. Key words: Ultrasonography; Thyroid neoplasms; Receptor, platelet derived growth factor alpha; Contrast media, Target; Angiogenesis

Long non-coding RNA BANCR regulates cancer stem cell markers in papillary thyroid cancer via the RAF/MEK/ERK signaling pathway

Thyroid cancer is one of the most common malignant tumors of the endocrine system. Among all thyroid cancers, papillary thyroid carcinoma (PTC) is the most common type. The BRAF-activated non-coding RNA (BANCR) is a 693-bp nucleotide transcript which was first identified in melanoma. However, the role of BANCR in the development of thyroid cancer remains unclear. Therefore, the present study investigated the potential involvement of BANCR in the development of thyroid cancer invitro using patient tissue samples and a panel of thyroid cancer cell lines, and invivo using a xenograft mouse model. We observed that BANCR was expressed at a higher level in human thyroid tumor tissues than that noted in the adjacent normal tissues. The expression level of BANCR differed between cultured thyroid cancer cell lines; BANCR expression was lower in the BCPAP cell line than that observed in the CAL-62, WRO and FTC-133 cell lines. Western blot analysis and flow cytometry revealed that overexpression of BANCR in the BCPAP cell line resulted in increased expression of the cancer stem cell markers, LGR5 and EpCAM. Single-clone formation experiments showed that upregulated expression of BANCR in the BCPAP cell line promoted an increase in the number of clones formed. Similarly, in microsphere formation experiments, overexpression of BANCR resulted in increased number and size of microspheres compared with the control cell line. Western blotting experiments showed that BANCR overexpression in BCPAP upregulated the expression of phosphorylated c-Raf, MEK1/2 and ERK1/2. Inhibition of c-Raf via U0126 decreased the expression of LGR5 and EpCAM, as well as phosphorylated levels of c-Raf, MEK1/2 and ERK1/2 in the BCPAP cells, compared to levels in the DMSO controls. In the xenograft mouse model, BANCR overexpression in the thyroid cancer cells significantly increased tumor growth. Taken together, these results suggest that BANCR plays a role in PTC development by regulating the expression of cancer stem cell markers LGR5 and EpCAM via the c-Raf/MEK/ERK signaling pathway. Therefore, BANCR may be used as a novel prognostic marker for PTC.

LRP4 promotes proliferation, migration, and invasion in papillary thyroid cancer

Dysregulation of cell proliferation and death is considered the foundation of the malignant biological characteristics of cancer. In this study, we conducted a comprehensive analysis of a massively parallel whole transcriptome resequencing of paired papillary thyroid cancer and normal thyroid tissues from 19 patients. In addition, we found that LRP4, a member of the low-density lipoprotein receptor-related protein family, is significantly overexpressed in thyroid carcinoma. We demonstrated through quantitative real-time polymerase chain reaction (qRT-PCR) that LRP4 is upregulated in papillary thyroid cancer (PTC) tissues. This observation was also consistent with data analyzed from The Cancer Genome Atlas (TCGA) cohort. Thus, the biological role of LRP4 in the thyroid cancer in the present study was investigated using the PTC cell lines TPC1, BCPAP and KTC-1. In these cell lines, the mRNA level of LRP4 was higher than normal thyroid cancer cell named HTORI3. In vitro experiments demonstrated that LRP4 downregulation significantly inhibited the colony formation, proliferation, migration, and invasion of the three PTC cell lines. Knockdown of LRP4 by small interfering RNA (siRNA) in those cell lines decreased the protein expression of N-cadherin, Enhancer of zeste homolog 2 (EZH2), and Zinc finger E-box-binding home-box 1 (ZEB1). Furthermore, LRP4 knockdown significantly reduced the levels of phosphorylated PI3K in the PTC cell lines. In conclusion, the present study indicated that LRP4 is a gene associated with PTC and might become a potential therapeutic target.

PDLIM5 identified by label-free quantitative proteomics as a potential novel biomarker of papillary thyroid carcinoma

In order to better understand the mechanisms underlying the development of papillary thyroid carcinoma (PTC), and to identify new potential biomarkers, high-resolution label-free mass spectrometry was performed on PTC tissues and adjacent normal thyroid tissues from six patients. In this process, 2788 proteins were identified, out of which 49 proteins presented significant differences between PTC tissues and adjacent normal thyroid tissues. Gene ontology revealed that the majority of these proteins are involved in the catalytic activity and binding. We selected three proteins with differential expressions: PDZ and LIM domain 5 (PDLIM5), PDLIM1 and ALDH1A1; Protein expressions were further verified by RT-PCR and western blot. Among these, expression of PDLIM5 and PDLIM1 was up-regulated, while that of ALDH1A1 was down-regulated in PTC tissues. Next, we confirmed their expression through quantitative dot blot (QDB) technique. We found that knockdown of PDLIM5 expression in the B-CPAP cell line could inhibit the migration, invasion and proliferation of PTC cells. In addition, PDLIM5 knockdown reduced Ras and Phospho-ERK1/2 expression. Thus, we suggested that PDLIM5 promotes PTC via activation of the Ras-ERK pathway. Our research provides new molecular insight into the function of PDLIM5, which may assist in studying the mechanism of PTC. In addition, PDLIM5 could be further explored as a potential candidate for PTC treatment.

Epigenetic Modifications in Thyroid Cancer Cells Restore NIS and Radio-Iodine Uptake and Promote Cell Death.

Epigenetic modifications have been identified as being responsible for the de-differentiation of thyroid tissue and its malignant transformation. Cell proliferation inhibitory effects of the pan-deacetylase inhibitors panobinostat, SAHA and Trichostatin A (TSA), the modulation of the sodium iodide symporter (NIS; SLC5A5), thyroid transcription factor 1 (TTF1), high mobility group A2 (HMGA2), and H19 and their putative targeting miRNAs have been evaluated in vitro. The cell viability was measured in five thyroid cancer cell lines (FTC133, TPC1, BCPAP, 8505C, C643) by real time cell analyzer xCELLigence. Expression of the above mentioned markers was performed by RT-qPCR and Western Blot. Radioiodine up-take was detected by Gamma Counter with I131. Cell viability decreased after treatment in all five cell lines. 10 nM panobinostat; 1 µM TSA or 10 µM SAHA caused a significant over-expression of NIS transcript in all five cell lines, whereas NIS protein was up-regulated in FTC133, BCPAP, and C643 cell lines only. Radioiodine up-take increased in FTC133 and C643 cells after 48 h of treatment with 10 nM panobinostat and 1 µM TSA. A significant down-regulation of the oncogene HMGA2 was detected in all five cell lines; except for TPC1 cells that were treated with 1 µM TSA. In accordance, hsa-let-7b-5p and hsa-let-7f-5p were stable or significantly over-expressed in all of the cell lines, except for TPC1 cells that were treated with 10 µM SAHA. TTF1 was significantly down-regulated in FTC133, BCPAP, and 8505C cells; whereas, TPC1 and C643 showed an up-regulated or stable expression. TTF1 was over-expressed in samples of human anaplastic thyroid cancer; whereas, it was down-regulated in follicular and undetectable in papillary thyroid cancer. H19 was over-expressed after 48 h treatment, except for BCPAP cells that were treated with panobinostat and SAHA. H19 was differently expressed in human anaplastic, follicular and papillary thyroid tumor samples. Deacetylase inhibitors reduced cell viability, restored NIS and H19, and suppressed the oncogenes HMGA2 and TTF1 in thyroid cancer cells.

The AMPK-activator AICAR in thyroid cancer: effects on CXCL8 secretion and on CXCL8-induced neoplastic cell migration.

The AMPK-activator AICAR recently raised great interest for its anti-cancer properties. With specific regard to thyroid cancer, AICAR reduces cancer cell growth, invasion and metastasis. CXCL8, a chemokine with several recognized tumorigenic effects, is abundantly secreted in thyroid cancer microenvironment. The aim of this study was to investigate if AICAR could inhibit the basal and the TNFα-induced CXCL8 secretion in normal human thyroid cells (NHT) and in thyroid cancer cell lines TPC-1 and BCPAP (RET/PTC and BRAFV600e mutated, respectively). The effect of AICAR on basal and CXCL8-induced cell migration was assessed. Cells were incubated with AICAR (0.05, 0.5, 1, 2mM) alone or in combination with TNF-α (10ng/ml) for 24h. CXCL8 concentrations were measured in cell supernatants. Transwell migration assays were performed in NHT, TPC-1 and BCPAP, basally and after treatment with AICAR (2mM) and rh-CXCL8 (50ng/ml) alone or in combination. AICAR dose dependently inhibited the basal secretion of CXCL8 in TPC-1 (F = 4.26; p < 0.007) and BCPAP (F = 6.75; p < 0.0001) but not in NHT. TNFα-induced CXCL8 secretion was dose dependently reduced by AICAR in NHT (F = 9.99; p < 0.0001), TPC-1 (F = 9.25; p < 0.0001) and BCPAP (F = 6.82; p < 0.0001). AICAR significantly reduced the basal migration of TPC-1 and BCPAP but not of NHT. CXCL8-induced cell migration was inhibited in NHT, TPC-1 and BCPAP. This is the first demonstration of the inhibition of CXCL8 secretion exerted by AICAR in TPC-1 and BCPAP indicating that the anti-cancer properties of AICAR are, at least in part, mediated by its ability to reduce the pro-tumorigenic effects of CXCL8.

LncRNA BANCR promotes EMT in PTC via the Raf/MEK/ERK signaling pathway.

Thyroid cancer is one of the most common types of cancer in the endocrine system. Among all types of thyroid cancer, papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Long non-coding RNA (lncRNA) BRAF-activated non-protein-coding RNA (BANCR) is a 688-bp-long nucleotide transcript, which was first identified in melanoma. The function of BANCR in thyroid cancer remains unclear. The aim of the present study was to investigate whether BANCR is involved in the development of thyroid cancer. The results indicated that BANCR expression was increased in thyroid tumors compared with in adjacent normal tissues. Among cancer cell lines, the expression level of BANCR differed: BANCR expression in BCPAP cell lines was lower compared with that in CAL-62, WRO and FTC-133 cell lines. Overexpression of BANCR promoted the migration and invasion of BCPAP cells. Additionally, BANCR mediated epithelial-mesenchymal transition (EMT) by regulating the expression of epithelial (E)-cadherin, vimentin and neuronal (N)-cadherin. Overexpression of BANCR in BCPAP cells decreased the expression of E-cadherin and increased the expression of vimentin, N-cadherin, phospho (p)-c-Raf, p-extracellular-signal-regulated kinase (ERK)/mitogen activated protein kinase (MEK)1/2 and p-ERK1/2. Administration of U0126 inhibitor inhibited the regulation of phosphorylation levels by MEK1/2 and ERK1/2. Additionally, U0126 upregulated the expression of E-cadherin and downregulated the expression of vimentin. Taken together, the results of the present study suggest that BANCR induces EMT in PTC through the Raf/MEK/ERK signaling pathway.

Effects of nuclear factor‑κB on the uptake of 131iodine and apoptosis of thyroid carcinoma cells.

Thyroid carcinoma is primarily treated by surgery combined with radioactive 131iodine (131I) treatment; however, certain patients exhibit resistance to 131I treatment. Previous research indicated that nuclear factor-κB (NF-κB) was associated with resistance to 131I in cancer cells. The present study aimed to investigate the effects of NF-κB on 131I uptake and apoptosis in thyroid carcinoma cells. TPC-1 and BCPAP cell lines were employed as research models in the present study, and the expression of NF-κB was inhibited by RNA interference (RNAi). The ability of TPC-1 and BCPAP cells to uptake 131I was measured and the cell viability was detected by an MTT assay. Finally, the expression of the apoptosis-associated proteins X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1) and caspase-3 in TCP-1 and BCPAP cells was determined by western blotting. Western blotting results demonstrated that the expression levels of NF-κB in TPC-1 and BCPAP cells were successfully downregulated by RNAi (P<0.05), while analysis of 131I uptake revealed no significant alterations in the 131I uptake ability of cells following RNAi (P>0.05). MTT experiments demonstrated that the inhibition of NF-κB expression in combination with radiation (131I treatment) led to a marked reduction in cell viability (P<0.05). Furthermore, western blot analysis revealed that the inhibition of NF-κB expression downregulated the expression levels of XIAP and cIAP1 (P<0.05), while the expression levels of caspase-3 were upregulated, indicating that the observed reduction in cell viability following NF-κB inhibition may be due to an increased level of apoptosis. Although NF-κB inhibition did not affect the 131I uptake of thyroid cancer cells, this inhibition may increase the apoptotic effects of radioactive 131I.