Proliferation Of Thyroid Carcinoma Cells Research Articles

Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated.Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression.The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling.We found that cofilin transfection in human MTC cell line TT significantly increased migration (178 ± 44%, p < 0.001), invasion (165 ± 28%, p < 0.01) and proliferation (146 ± 18%, p < 0.001), accompanied by an increase of ERK1/2 phosphorylation (2.23-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (−55 ± 10% migration, p < 0.001, −41 ± 8% invasion, p < 0.001, −17 ± 3% proliferation, p < 0.001). These results have been confirmed in primary cells cultures obtained from human MTCs.The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (−37 ± 8%, p < 0.001 and −31 ± 16%, p < 0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (−57 ± 13%, p < 0.01), but not on cell proliferation, were completely abolished in cells transfected with cofilin.In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

Overexpression of long non-coding RNA LINC00982 suppresses cell proliferation and tumor growth of papillary thyroid carcinoma through PI3K-ATK signaling pathway.

Long non-coding RNAs (lncRNAs) have been widely reported that involved in human cancers, including papillary thyroid carcinoma (PTC). The present study aims to investigate the biological role of LINC00982 in PTC. The mRNA expression of LINC00982 in human PTC tissues was detected using qPCR. Moreover, Kaplan–Meier method was performed to analyze the internal relevance between LINC00982 expression and overall survival (OS) rate of patients with PTC. In addition, gain- and loss-of-functions assays were performed to detect the effects of LINC00982 on the cell proliferation and migration in PTC cells. Furthermore, western blot assay was used to measure the alteration expression levels of apoptosis relative proteins and the relative protein involved phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. Finally, a xenograft model was used to analyze the antitumor role of LINC00982 in vivo. Here, we found that LINC00982 was decreased in human PTC tissues. Patients with decreased LINC00982 expression levels had a reduced OS (P=0.0019) compared with those with high LINC00982 expression levels. Overexpression of LINC00982 suppressed the proliferation and migration of BHT101 and B-CPAP cells and promoted cell apoptosis. Knockdown of LINC00982 promoted the proliferation and migration of BHT101 and B-CPAP cells and induced cell apoptosis. Moreover, in vivo assay showed that overexpression of LINC00982 could suppress the growth of PTC. Finally, LINC00982 could regulate the activity of PI3K/AKT signaling pathway in vitro and in vivo. Taken together, our findings demonstrated that overexpression of LINC00982 could suppress cell proliferation and induce cell apoptosis by regulating PI3K/AKT signaling pathway in PTC.

Parthenolide Inhibits the Proliferation of MDA-T32 Papillary Thyroid Carcinoma Cells in Vitro and in Mouse Tumor Xenografts and Activates Autophagy and Apoptosis by Downregulation of the Mammalian Target of Rapamycin (mTOR)/PI3K/AKT Signaling Pathway.

BackgroundThis study aimed to examine the effects of the sesquiterpene lactone, parthenolide, on migration, autophagy, and apoptosis of MDA-T32 human papillary thyroid carcinoma cells in vitro and in mouse tumor xenografts.Material/MethodsCell proliferation and viability of MDA-T32 human papillary thyroid carcinoma cells were determined by MTT assay, and cell migration was studied using a transwell assay. Fluorescence microscopy using acridine orange (AO) and ethidium bromide (EB) staining evaluated apoptosis. Transmission electron microscopy was used to study the effects of parthenolide on autophagy, and Western blot examined the levels of autophagy-associated proteins, including Bax, Bcl-2, and LC3-ll. Mice (n=10) were injected with 5×106 MDA-T32 cells subcutaneously into the left flank, and xenograft tumors were grown for six weeks. Control untreated mice (n=5) were compared with treated mice (n=5) given parthenolide three times per week.ResultsParthenolide resulted in a dose-dependent reduction in viability and cell migration of MDA-T32 cells, with a half-maximal inhibitory concentration (IC50) of 12 μM. AO and EB staining showed that parthenolide induced cell apoptosis and electron microscopy identified autophagosomes in MDA-T32 cells. Parthenolide induced increased expression of the autophagocytic proteins, LC3-II and beclin-1, had a dose-dependent inhibitory effect on the mTOR/PI3K/AKT cascade in MDA-T32 cells and inhibited the growth of the mouse xenograft tumors in vivo.ConclusionsParthenolide inhibited the growth and migration of MDA-T32 human papillary thyroid carcinoma cells in vitro and mouse tumor xenografts and activated autophagy and apoptosis by downregulation of the mTOR/PI3K/AKT signaling pathway.

Silencing of long noncoding RNA RP11-476D10.1 enhances apoptosis and autophagy while inhibiting proliferation of papillary thyroid carcinoma cells via microRNA-138-5p-dependent inhibition of LRRK2.

The distant metastasis in papillary thyroid carcinoma (PTC) is a major threat for PTC patients. Moreover, the involvement of long noncoding RNAs (lncRNAs) in the regulation of PTC progression has been extensively investigated. The aim of this study was to underscore whether lncRNA RP11-476D10.1 affects the proliferation, apoptosis and autophagy of PTC cells. Initially, we determined that lncRNA RP11-476D10.1 and LRRK2 were highly expressed in PTC cells. Meanwhile, through experimentation, miR-138-5p was confirmed to bind with lncRNA RP11-476D10.1 and LRRK2. It was also revealed that lncRNA RP11-476D10.1 downregulated the miR-138-5p expression, thereby upregulating the LRRK2 expression. After that, PTC cells were transfected with siRNA against RP11-476D10.1, or inhibitor or mimic of miR-138-5p to evaluate the influence of lncRNA RP11-476D10.1 on the PTC cell proliferation, apoptosis, and autophagy in vitro and on the tumor formation ability in vivo. The results showed that silenced lncRNA RP11-476D10.1 or overexpressed miR-138-5p enhanced the apoptosis and autophagy of PTC cells while reducing cell proliferation, with increased levels of Bax, LC3B, and Beclin1 and decreased Bcl-2 level were observed. The inhibitory role of silenced lncRNA RP11-476D10.1 role in the PTC development was further verified by the reduced tumor formation ability in nude mice. Our results demonstrated that lncRNA RP11-476D10.1 could bind to miR-138-5p and promote LRRK2 expression. Moreover, the silencing of lncRNA RP11-476D10.1 may inhibit the development of PTC, highlighting a novel insight for the development of superior therapeutic targets for PTC treatment.

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.

Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation

Abstract Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178 ± 44%, p The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (−37 ± 8%, p In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

Mortalin (GRP75/HSPA9) Promotes Survival and Proliferation of Thyroid Carcinoma Cells.

We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.

Peptide P11 suppresses the growth of human thyroid carcinoma by inhibiting the PI3K/AKT/mTOR signaling pathway.

Thyroid carcinoma is the most common endocrine malignancy, and the incidence of thyroid carcinoma is increasing in recent decades. CYYGQSKYC (P6), a nonapeptide with anti-lymphangiogenic effect by its binding to VEGFR-3 and selectively inhibiting VEGF-C binding to VEGFR-3, could suppress the migration and invasion of cancer cells. LSPPRYP (P9) acts as an effective bFGF/FGFR antagonist and inhibits the growth of the murine melanoma B16-F10 cells. In order to increase the anti-tumor effects of P6 and P9, we connected P6 with P9 via a flexible linker Gly-Gly-Gly (GGG) to reconstruct a novel peptide P11, CYYGQSKYCGGGLSPPRYP. In the present study, the mechanism of action of peptide P11 on the growth of human thyroid carcinoma cells both in vitro and in vivo was determined. Our results showed that peptide P11 inhibited the proliferation, viability, migration, and invasion of human thyroid carcinoma cells. Peptide P11 increased the apoptosis and decreased the protein levels of p-PI3K, p-AKT, and p-mTOR in human thyroid carcinoma cells. In addition, P11 could effectively inhibit the growth of human thyroid carcinoma xenograft tumors in nude mice. In conclusion, peptide P11 could inhibit the growth of human thyroid carcinoma by inhibiting the PI3K/Akt/mTOR signaling pathway. Novel peptides can be designed and applied for the treatment of various types of cancer.

SAT-566 Cofilin Is a Mediator of RET-Promoted Medullary Thyroid Carcinoma Cell Migration, Invasion and Proliferation

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells, accounting for 5% -10% of thyroid cancers. In all inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell motility, proliferation and survival, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178.5±44%, p<0.001), invasion (194±27%, p<0.05) and proliferation (145.9±18%, p<0.001), accompanied by an increase of ERK1/2 phosphorylation (1.8-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (-55±10% migration, p<0.001, -40.7±8% invasion, p<0.001, -17±2.6% proliferation, p<0.001). The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (-30.8±11%, p<0.01 and -31±16%, p<0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (-70±18%, p<0.001) and proliferation (-29.4±9% , p<0.01) were completely abolished in cells transfected with cofilin. In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

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

Wnt5a might regulate the invasion and proliferation of papillary thyroid carcinoma cells through receptor tyrosine kinase-like orphan receptor 2

Objective To detect the effect of receptor tyrosine kinase-like orphan receptor 2 (ROR2) low-expression combining Wnt5a over-expression on the proliferation and invasion abilities of papillary thyroid carcinoma cells. Methods The proliferation ability of papillary thyroid carcinoma cells with ROR2 low-expressed combining Wnt5a over-expressed was detected by methyl thiazol tetrazolium (MTT). The clone formation ability of papillary thyroid carcinoma cells with ROR2 low-expressed combining Wnt5a over-expressed was detected by colony formation assay. The invasion ability of papillary thyroid carcinoma cells with ROR2 low-expressed combining Wnt5a over-expressed was detected by Transwell. Results MTT assay results showed that the A values detected on the fourth day of the K1 and BCPAP cells of the control groups (0.452±0.005, 0.432±0.003) were significantly lower than those of the Wnt5a groups (0.563±0.004, 0.567±0.006) but higher than those of the Si-ROR2 groups (0.404±0.003, 0.389±0.005) (t=-30.026, -34.857, 14.258, 12.773; P=0.000). The A values detected on the fourth day of the K1 and BCPAP cells of the co-stained groups (0.501±0.005, 0.496±0.003) were distinctively lower than those of the Wnt5a groups (t=-16.771, -19.545; P=0.000). The colony formation assay results showed that the numbers of K1 and BCPAP cell clones of the control groups (101.00±5.43, 112.00±4.11) were distinctively lower than those of the Wnt5a groups (156.00±4.34, 168.00±4.55) but higher than those of the si-ROR2 groups (62.00±4.45, 68.00±3.99) (t=-13.704, -15.819, 9.622, 13.304; P=0.000). The clone numbers of K1 and BCPAP cells of the co-stained groups (121.00±4.44, 128.00±3.89) were significantly lower than those of the Wnt5a groups (t=-9.764, -11.574; P=0.001, 0.000). The Transwell assay demonstrated that the numbers of K1 and BCPAP cells passing through the basement membrane of the control groups [(52.90±2.36), (68.40±1.01) cells] were significantly lower than those of Wnt5a groups [(164.30±4.35), (87.70±1.28) cells] but higher than those of si-ROR2 groups [(31.30±2.17), (39.60±1.34) cells, t=-38.829, -20.502, 11.533, 29.728; P=0.000]. The numbers of K1 and BCPAP cells passing through the basement membrane of the co-stained group [(83.50±1.42), (65.90±2.11) cells] were distinctively lower than those of the Wnt5a groups (t=-31.986, -15.300; P=0.000). Conclusion Wnt5a may regulate the proliferation and invasion of thyroid papillary carcinoma cells through ROR2 signaling. Key words: Wnt5a; Receptor tyrosine kinase-like orphan receptor 2; Papillary thyroid carcinoma; Cell proliferation; Cell invasion

Molecular mechanism of microRNA-145 targeting protein kinase Bγ in regulating growth and metastasis of thyroid cancer cells

Objective To investigate the expression of microRNA (miRNA)- 145 and protein kinase Bγ (Akt3) in thyroid carcinoma and the molecular mechanism of miRNA-145 targeting Akt3 inhibiting the proliferation and invasion of thyroid carcinoma cells. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of microRNA-145 and Akt3 in 49 fresh thyroid carcinoma tissues and adjacent tissues. The relationship between the expression of microRNA-145 and Akt3 was analyzed. TPC-1 thyroid cancer cell line was used as the research object. The effects of microwave irradiation on the growth, invasion and metastasis of thyroid cancer cells were detected by methyl thiazol tetrazolium (MTT) and Transwell invasion assays. The expression levels of microwave irradiation-145 and Akt3 were detected by Western blotting. Results The results of FQ-PCR showed that the relative expression levels of miRNA-145 in thyroid carcinoma and adjacent tissues were 1.65±0.33 and 3.13±0.53, and those of Akt3 were 2.96±0.85 and 0.83±0.32, respectively. The difference was statistically significant (P=0.000). The relative expression levels of microRNAs-145 in TPC-1 cells and primary thyroid follicular cells were 1.91±0.57 and 3.47±0.59, and those of Akt3 were 3.62±1.04 and 0.76±0.32, respectively. There was statistically significant difference (P=0.000). The increase of A570 in TPC-1 cells transfected with anti-miR-145 was the fastest, and that in TPC-1 cells transfected with analogues of miR-145 was the slowest (P=0.000). As compared with TPC-1 cells without transfection and TPC-1 cells transfected only with liposome 2000, the number of invasive cells transfected with anti-miR-145 increased significantly (P=0.000), while the number of invasive cells transfected with micR-145 analogues decreased significantly (P=0.000). The expression levels of p-Akt and Akt-3 protein in miR-145-mimics-transfected and non-transfected TPC-1 cells were significantly lower than those in TPC-1 cells transfected with anti-miR-145. Conclusion MiR-145 can target down-regulate the expression of Akt3 to inhibit the proliferation and invasion of thyroid cancer cells. Key words: Thyroid cancer; MicroRNA-145; Protein kinase Bγ; Proliferation; Metastasis

MiR-214 regulates papillary thyroid carcinoma cellproliferation and metastasis by targeting PSMD10.

MicroRNAs (miRNAs) have important effects on cancer occurrence and development by adjusting gene expression. The aim of the present study was to examine the role of miR-214 in papillary thyroid carcinoma cell proliferation and metastasis, and its molecular mechanisms. miR-214 was demonstrated to be markedly downregulated in papillary thyroid carcinoma tissues and cells compared with normal, and this was significantly associated with lymph node metastasis, tumor size and TNM stage. Upregulation of miR-214 significantly decreased cell proliferation, and promoted cell apoptosis and cell cycle arrest in papillary thyroid carcinoma cell lines in vitro. By contrast, downregulation of miR-214 resulted in the opposite effects. In addition, miR-214 mimics significantly decreased papillary thyroid carcinoma cell migration and invasion, which was correlated with decreased expression levels of matrix metallopeptidase (MMP)-2 and MMP-9. Restoration of miR-214 expression in papillary thyroid carcinoma cells decreased the activities associated with epithelial-mesenchymal transition (EMT). Furthermore, proteasome 26S subunit non-ATPase 10 (PSMD10) was predicted to be a target of miR-214. Experimental results demonstrated that miR-214 negatively regulated PSMD10 expression by targeting its 3′ untranslated region directly. Knockdown of PSMD10 reduced papillary thyroid carcinoma cell clone formation, migration and invasion, most likely by repressing glycogen synthase kinase (GSK)-3β/β-catenin and AKT signaling. Finally, a negative correlation was observed between the expression levels of miR-214 and PSMD10 in papillary thyroid carcinoma tissues. Taken together, these data suggested that miR-214 might be a candidate target for the treatment of papillary thyroid carcinoma.

LncRNA LINC00673 induces proliferation, metastasis and epithelial-mesenchymal transition in thyroid carcinoma via Kruppel-like factor2.

The incidence of thyroid cancer has increased in the past decades; however, the underlying molecular mechanisms of thyroid cancer tumorigenesis remain unknown. Using sequencing technology, long intergenic non-protein coding RNA 673 (LINC00673) was identified to be upregulated in several tumor tissues. However, the biological role of LINC00673 in thyroid carcinoma has yet to be determined. In this study, 60 matched pairs of thyroid tumor tissue and normal tissue were selected for study using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to validate previous findings; then, clinicopathologic features of the tissues were analyzed. Proliferation, colony formation, migration and invasion assays were performed, and epithelial-mesenchymal transition (EMT)- associated phenotypes were investigated following transfection with small interfering RNA to determine the specific role of LINC00673 in thyroid carcinoma cell lines (TPC1, KTC-1 and BCPAP). The study revealed that long non-coding RNA LINC00673 was significantly upregulated in thyroid cancer tissues compared with paired adjacent non-tumor tissues using RT-qPCR and that high expression of LINC00673 is was associated with larger tumor size and lymph node metastasis in the validated cohort. Knockdown of LINC00673 inhibited cell proliferation and metastasis, whereas, LINC00673 overexpression had the opposite effect. The results showed that LINC00673 may influence EMT and the expression of Kruppel-like factor 2 (KLF2). Notably, KLF2 is considered a tumor suppressor gene in a variety of tumors. Finally, knock down of KLF2 enhanced thyroid carcinoma cell proliferation, and invasion and migration. In this study, the function of LINC00673 in promoting the proliferation and metastasis of thyroid carcinoma cell lines was identified, and LINC00673 may act as a novel therapeutic target for treating thyroid carcinoma.

MicroRNA-1266 suppresses papillary thyroid carcinoma cell metastasis and growth via targeting FGFR2.

To explore the effects of microRNA-1266 (miR-1266) on metastasis and growth in papillary thyroid carcinoma cells and to provide therapeutic targets for papillary thyroid carcinoma. By quantitative Real-time polymerase chain reaction (PCR), miR-1266 expression level in 38 pairs of papillary thyroid carcinoma tissues and three breast cancer-derived cell lines was examined. After transfection with miR-1266 mimics, the effects of miR-1266 over-expression on cell proliferation, invasion and migration were analyzed. Further, we employed several databases for the target gene prediction. Dual-luciferase activity assay was performed to verify whether FGFR2 was the direct target gene of miR-1266. Western blotting was conducted to detect protein levels. MiR-1266 was significantly downregulated in papillary thyroid carcinoma tissue samples and cell lines. Over-expression of miR-1266 in papillary thyroid carcinoma cells significantly attenuated the cell proliferation, invasion, and migration. Dual-luciferase report assay and Western blotting confirmed that FGFR2 was a target gene of miR-1266. Furthermore, up-regulation of FGFR2 partially reversed the suppressive effects of miR-1266 over-expression on cell growth and progression. miR-1266 could inhibit cell proliferation and progression of papillary thyroid carcinoma via targeting FGFR2. Our findings might provide a new target for the diagnosis and treatment of papillary thyroid carcinoma.

Telomerase reverse transcriptase induced thyroid carcinoma cell proliferation through PTEN/AKT signaling pathway

Thyroid carcinoma is the most common endocrine malignant tumor in the world, and so, there is a requirement to develop novel molecular targets for thyroid cancer diagnosis and treatment. Telomerase reverse transcriptase (TERT) was revealed to promote cell proliferation in a number of types of cell. To evaluate whether and how TERT functioned on papillary thyroid cancer (PTC) cell proliferation, the present study constructed TERT over-expression [recombined (r)TERT plasmid group] and interference [small interfering RNA (si)-TERT group] models by liposome transfection respectively to study the molecular mechanisms. The transfection efficiency was first detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting to analyze TERT levels compared with the negative control (NC) and control groups. Then MTT and carboxyfluorescein diacetate succinimidyl ester assays were performed to determine living cell proliferation and total cell proliferation respectively. Propidium iodide assay was used to detect alterations in cell cycle progression. RT-qPCR and western blotting were performed to detect associated factor variation. The results demonstrated that, following the generation of TERT overexpression or silencing PTC cells, the living cells and also total cell proliferation increased significantly in the rTERT group, and decreased significantly in siTERT group, when compared with the NC and control groups. The cell cycle was accelerated in the rTERT group, and blocked in the G1/S transition in the siTERT group. The mRNA and protein levels of P27, P53 and phosphatase and tensin homolog (PTEN) decreased significantly in the rTERP group and increased in the siTERP group, while cyclin dependent kinase 2 and Cyclin D1 increased significantly in the rTERP group and decreased in the siTERP group. The expression of cell division cycle 25A did not alter significantly. The protein levels of β-catenin and retinoblastoma were also unaltered. Protein kinase B (AKT) was detected once activated by TERT, and there were increased phosphorylated (p)-AKT protein levels in the rTERT group, and decreased p-AKT protein levels in the siTERT group. In conclusion, TERT could induce thyroid carcinoma cell proliferation mainly through the PTEN/AKT signaling pathway.

LncRNA SNHG12 promotes the proliferation and metastasis of papillary thyroid carcinoma cells through regulating wnt/β-catenin signaling pathway.

To investigate the expression and role of long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) in papillary thyroid carcinoma (PTC). The relative expression levels of lncRNA SNHG12 (hereinafter referred to as SNHG12) in 42 pairs of PTC tissues and para-carcinoma tissues were detected via quantitative reverse transcription polymerase chain reaction (qRT-PCR). SNHG12 specific interference sequences were designed and synthesized. The relative expression level and transfection efficiency of SNHG12 in PTC cells were detected via qRT-PCR. After the interference in SNHG12 expression, the change in cell proliferation capacity was detected via methyl thiazolyl tetrazolium (MTT) assay, the change in cell cycle distribution was detected via flow cytometry, the changes in cell migration and invasion capacities were detected via Transwell assay and wound healing assay, and the changes in expressions of molecular markers of Wnt/β-catenin pathway were detected via Western blotting. The pulmonary metastasis model of nude mice was established, and the changes in migration and invasion capacities of tumor cells were studied via the in-vivo experiment after the interference in SNHG12 expression. The results of qRT-PCR showed that the SNHG12 expression was up-regulated in 30 pairs of PTC tissues and cells. The results of MTT assay showed that the cell proliferation capacity was inhibited after the interference in SNHG12. The results of flow cytometry showed that the cell cycle progression was blocked in G1-G0 phase after the knockdown of SNHG12 expression. The results of Transwell assay and Western blotting showed that the interference in SNHG12 could inhibit the invasion and metastasis capacities of tumor cells through influencing the Wnt/β-catenin signaling pathway. Metastatic tumor model of nude mice showed that SNHG12 could affect the invasion and metastasis of tumor cells in vivo. The SNHG12 expression is relatively high in PTC tissues and cells. In-vivo/in-vitro experiments prove that SNHG12 can promote the proliferation and metastasis of PTC cells through influencing the Wnt/β-catenin signaling pathway.

TBX3 promotes proliferation of papillary thyroid carcinoma cells through facilitating PRC2-mediated p57KIP2 repression.

The T-box transcription factor TBX3 has been implicated in the patterning and differentiation of a number of tissues during embryonic development, and is overexpressed in a variety of cancers; however, the precise function of TBX3 in papillary thyroid carcinoma (PTC) development remains to be determined. In the current study, we report downregulation of TBX3 in PTC cells delays the G1/S-phase transition, decreases cell growth in vitro, and inhibits tumor formation in vivo. We identified p57KIP2 as a novel downstream target that serves as the key mediator of TBX3's control over PTC cell proliferation. Reduced expression of TBX3 resulted in increased p57KIP2 level, while knockdown of p57KIP2 rescues the cell-cycle arrest phenotype. In clinical PTC specimens, the expression of TBX3 is markedly upregulated and significantly correlated with advanced tumor grade, but negatively correlated with the expression of p57KIP2. Mechanism investigation revealed that TBX3 directly binds to the CDKN1C gene promoter region, the coding gene of p57KIP2, and represses its transcription. Furthermore, recruitment of main components of the PRC2 complex as well as class I histone deacetylases, HDAC1 and HDAC2, is required for TBX3 to fulfill the transcriptional repression function. Our findings illustrate the previously unknown function and mechanism in cell proliferation regulation by the TBX3-p57KIP2 axis and provide evidence for the contribution of the PRC2 complex and HDAC1/2. Targeting of this pathway may present a novel and molecular defined strategy against PTC development.

LncRNA HOTTIP promotes papillary thyroid carcinoma cell proliferation, invasion and migration by regulating miR-637.

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy. Besides, increasing evidence has demonstrated that long non-coding RNA (lncRNA) HOTTIP played a crucial role in cancer pathogenesis. MiR-637-mediated Akt1 was involved in cell growth, invasion and migration in various malignancies. This study was aimed to investigate the potential biological effect and regulatory mechanism of HOTTIP on cell proliferation, invasion and migration in PTC. Expression of HOTTIP, miR-637 and Akt1 were determined by quantitative RT-PCR (qRT-PCR) and western blotting in PTC tissues, normal tissues, PTC cells (TPC-1 and HTH83) or non-tumor thyroid cells (Nthy-ori 3-1). Cell proliferation, invasion and migration following HOTTIP knockdown were investigated in PTC cells. The target of HOTTIP was validated by RNA immunoprecipitation (RIP) and pull-down assay. Moreover, a xenograft model was performed. HOTTIP was upregulated in human PTC tissues and PTC cell lines. In addition, HOTTIP knockdown inhibited the proliferation, invasion and migration in vitro together with in vivo tumorigenesis of PTC cells. Additionally, HOTTIP knockdown downregulated Akt1 expression and suppressed cell proliferation, invasion and migration in PTC cells by regulating miR-637. In contrast, miR-637 inhibitor reversed above-mentioned tendencies caused by HOTTIP knockdown. HOTTIP is a potential oncogene in PTC and may serve as a therapeutic target for malignancies.

Synergistic cytotoxicity of the dipeptidyl peptidase-IV inhibitor gemigliptin with metformin in thyroid carcinoma cells.

The influence of the dipeptidyl peptidase-IV inhibitor, gemigliptin alone or in combination with metformin on survival, proliferation, and migration of thyroid carcinoma cells was investigated. SW1736 and TPC-1 human thyroid carcinoma cells were used. Gemigliptin and metformin caused cell death in a dose-dependent manner. In cells treated with both gemigliptin and metformin, compared with metformin alone, all of the combination index values were lower than 1.0, suggesting synergistic cytotoxicity of two agents. Cell viability, the percentage of viable cells, ATP levels, and mitochondrial membrane potential decreased; however, cytotoxic activity, and the protein levels of cleaved PARP, phospho-Akt and phospho-AMP-activated protein kinase (AMPK) increased. Administration of wortmannin, but not compound C, further decreased cell viability, and further increased cytotoxic activity. Moreover, compared with control, cell proliferation and migration as well as the protein levels of p53, p21, vascular cell adhesion molecule-1 (VCAM-1), and phospho-extracellular signal-regulated kinase (ERK) 1/2 decreased. The decrement of matrix metalloproteinase-2 and matrix metalloproteinase-9 protein levels was cell specific. Our results demonstrate that gemigliptin induces cytotoxic activity, and has a synergistic activity with metformin in inducing cytotoxicity via regulation of Akt and AMPK in thyroid carcinoma cells. Furthermore, gemigliptin augments the inhibitory effect of metformin on proliferation and migration through involvement of matrix metalloproteinase-2, matrix metalloproteinase-9, p53, p21, VCAM-1, and ERK in thyroid carcinoma cells.