ABCG2/BCRP gene expression is related to epithelial–mesenchymal transition inducer genes in a papillary thyroid carcinoma cell line (TPC-1)

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   Figure 1

   Characterization of TPC-1 parental cells and the TPC-1 MITO-resistant subline. (A) Representatives images of cell stained for ABCG2/BCRP, nestin, and CD133 in both before and after drug selection (20× magnification). (B) Relative expression levels of mRNA for the ABCG2/BRCP gene. Data are representative of three independent experiments. Data shown represent the mean±s.d. for three independent experiments for mRNA expression, ***P<0.001. (C) Mean intensity of flourescence (MIF) of TPC-1 parental cells vs the TPC-1 MITO-resistant subline. Bars represent mean±s.d. from three separate experiments (*P<0.05).
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   Figure 2

   Relative expression of protein and mRNA transcription factors: ZEB1, TWIST, and SNAIL1 in TPC-1 parental cells and the TPC-1 MITO-resistant subline. (A) qRT-PCR analysis shows a significant higher expression at the mRNA level ZEB1 and TWIST in the TPC-1 MITO-resistant subline. Significant changes were observed in ZEB1 (***P<0.001) and TWIST (**P<0.01) genes. No significant change in SNAIL1 gene expression was found. Bars represent mean±s.d. from three separate experiments. TPC-1 cells were used as a control. (B) Representative images of cells stained for ZEB1 (20× magnification), TWIST1, and SNAIL1 (40× magnification) by immunocytochemistry both before (TPC-1 parental cells) and after mitoxantrone selection (TPC-1 MITO-resistant subline).
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   Figure 3

   Relative expression of mRNA and protein for N- and E-cadherin in TPC-1 parental cells and the TPC-1 MITO-resistant subline. (A) qRT-PCR analysis shows a significant increase in expression of N-cadherin in the TPC-1 MITO-resistant subline (twofold change, **P<0.01). Bars represent mean±s.d. from three separate experiments. TPC-1 parental cells were used as a control. (B) Representative image of cells stained for N-cadherin (20×magnification) and E-cadherin (20× magnification) by immunocytochemical staining both before and after drug selection.
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   Figure 4

   Relative expression of mRNA and protein for ITGB1, ITGB1BPA, and vimentin in TPC-1 parental cells and the TPC-1 MITO-resistant subline. (A, B and C) qRT-PCR analysis shows a significant downregulation of the expression of ITGB1 and ITGB1P1 (twofold change, *P<0.05). No significant change in the expression of the vimentin gene was observed in selected cells. Bars represent mean±s.d. from three separate experiments. TPC-1 parental cells were used as a control. (D) Representative image for vimentin (20× magnification) stained by immunocytochemistry both before (TPC-1 parental cells) and after mitoxantrone selection (TPC-1 MITO-resistant subline).
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   Figure 5

   Effect of knockdown of ZEB1 on EMT inducer genes (E-cadherin and vimentin). (A) qRT-PCR analysis showed a significant increase in the expression of the mRNA for the E-cadherin gene (***P<0.001). (B) Representative image of cells positively stained for E-cadherin protein showing nuclear. The arrows indicate staining at 48 h of ZEB1 silencing (20× magnification). (C) qRT-PCR analysis showed a significant reduction of expression at 24 and 48 h of silencing of ZEB1 for vimentin mRNA (**P<0.01 and ***P<0.001). (D) Representative image showed a reduction of expression of protein at 48 h of silencing of ZEB1 (20× magnification). Bars represent mean±s.d. from three separate experiments. The TPC-1 MITO-resistant subline transfected with siRNA control was used as a control.
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   Figure 6

   Effect of knockdown of ZEB1 on the relative expression of mRNA for ABCG2/BCRP, N-cadherin, ITGB1, and ITGB1BP1 at 24, 48, and 72 h. (A) Knockdown of ZEB1 resulted in upregulation of ABCG2/BCRP gene expression at 24 h (**P<0.01). This increment was decreased at 48 and at 72 h, a reduction of expression was detected (***P<0.001). (B) Downregulation of N-cadherin was detected at 24 h of ZEB1 silencing (***P<0.001) and was maintained at 48 and 72 h (**P<0.01). (C) The relative mRNA expression of ITGB1 showed an upregulation at 24 h (*P<0.05), 48 h (**P<0.001), and 72 h (***P<0.001). (D) The relative mRNA expression of ITGB1BP1 gene showed an upregulation of gene expression at 72 h (***P<0.001). Bars represent mean±s.d. from three separate experiments. The TPC-1 MITO-resistant subline transfected with siRNA was used as a control.
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   Figure 7

   Analysis of survivin (BIRC5) gene expression, cellular migration in TPC-1 parental cells. The TPC-1 MITO-resistant subline, and the TPC-1 MITO-resistant subline trasnsfected with siRNA ZEB1. (A) The relative mRNA expression of survivin gene showed upregulation in selected cells (TPC-1 MITO-resistant subline cells; twofold change, *P<0.05). (B) The wound-healing assay showed significantly faster migration in the TPC-1 MITO-resistant subline compared with TPC-1 parental cells (*P<0.05). The images were obtained by phase-contrast microscope (100×). (C) Transient ZEB1 knockdown resulted in downregulation of BIRC5 gene expression at 48 h (**P<0.01) and at 72 h (***P<0.001). (D) The wound-healing assay showed a significant decrease in the closed wound area in the TPC-1 MITO-resistant subline transfected with siRNA ZEB1 compared with the TPC-1 MITO-resistant subline transfected with siRNA control (*P<0.05). Phase-contrast microscope images (×100). Bars represent mean±s.d. from four separate experiments.
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   Figure 8

   Transwell invasion ability of TPC-1 parental cells and TPC-1 MITO-resistant subline cells. (A and B) The ability to invade surrounding extracellular matrigel was analyzed at 48, 72, and 92 h of cell culture in complete medium. The TPC-1 MITO-resistant subline cells showed a high capacity for invasion at 92 h (*P<0.05) compared with TPC-1 parental cells used as a control. Bars represent mean±s.d. from four separate experiments.
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   Figure 9

   Gene expression of EMT inducer genes (ZEB1, SNAIL1, and TWIST1). E-cadherin, N-cadherin, and ABCG2/BCRP from human thyroid carcinoma (papillary tumors and poorly differentiated/anaplastic tumours). (A, B, C and D) The relative mRNA expression of ABCG2/BCRP, SNAIL1, TWIST1, and ZEB1 showed a higher increase in expression in tumors from patients with aggressive stages (III and IV) in comparison with those with the more benign stages (I and II). (E and F) The relative mRNA expression of E- and N-cadherin genes showed a significant increase in expression in tumors from patients with benign stages (I and II), in contrast a decrease in expression of both genes was detected in tumors from patients with more aggressive stages (III and IV). Data shown represent the mean±s.d. *P<0.05, **P<0.01, and ***P<0.001. The tumor tissue was compared with normal thyroid tissue.

• © 2014 Society for Endocrinology