Downregulation of miR-200c stabilizes XIAP mRNA and contributes to invasion and lung metastasis of bladder cancer

Abstract

Our previous studies have demonstrated that XIAP promotes bladder cancer metastasis through upregulating RhoGDIβ/MMP-2 pathway. However, the molecular mechanisms leading to the XIAP upregulation was unclear. In current studies, we found that XIAP was overexpressed in human high grade BCs, high metastatic human BCs, and in mouse invasive BCs. Mechanistic studies indicated that XIAP overexpression in the highly metastatic T24T cells was due to increased mRNA stability of XIAP that was mediated by downregulated miR-200c. Moreover, the downregulated miR-200c was due to CREB inactivation, while miR-200c downregulation reduced its binding to the 3'-UTR region of XIAP mRNA. Collectively, our results demonstrate the molecular basis leading to XIAP overexpression and its crucial role in BC invasion.

Keywords: CREB inactivation; XIAP; bladder cancer; invasion/metastasis; mir-200c.

Figure 1.

XIAP expression is positively correlated with bladder cancer invasion. (a) After 180 days treatment with or without BBN, the bladder tissues from indicated mice were analyzed by H&E staining; (b) IHC was also employed to evaluate XIAP expression in bladder tissues from indicated mice; and (c) the optical density was calculated as described in ‘Materials and Methods’. The results are presented as mean ± SD from at least triplicate experiments and an asterisk (*) indicates a significant difference (P < 0.05). (d) Human urothelial carcinoma tissue array was employed to evaluate XIAP expression.

Figure 2.

XIAP mRNA is stabilized in invasive bladder cancer cells. (a) Western Blot was applied to compare the protein expression levels of XIAP between the T24 and T24T (top two panels), β-Actin was used as internal control. RT-PCR was used to compare the mRNA level of xiap between these two cell lines (bottom two panels) and gapdh was used as internal control. The details of total cell protein extracts and mRNA obtained are described in ‘Material and Methods’. (b) Wild-type xiap promoter-driven luciferase reporter was co-transfected together with pRL-TK into T24 and T24T cells. Twenty-four hours post-transfection, the transfectants were extracted to evaluate luciferase activity. TK was used as internal control. The results were presented as xiap promoter activity relative to T24 cell, and each bar indicates mean±SD from three independent experiments. An asterisk (*) indicates a significant difference (P < 0.05). (c) XIAP mRNA stabilities were evaluated by RT-PCT in presence of Act D in T24 and T24T cells. (d) The cell extracts from T24T (nonsense) and T24 (shNucleolin) transfectants were subjected to Western blot as indicated, knockdown nucleolin can increase XIAP protein expression level. (e) xiap-3ʹUTR luciferase reporter was co-transfected together with pRL-TK into T24 and T24T cells, respectively. Twenty-four hours post-transfection, the transfectants were extracted to evaluate luciferase activity. TK was used as internal control. The results were presented as xiap-3ʹUTR activity relative to T24 cells, and each bar indicates mean±SD from three independent experiments. An asterisk (*) indicates a significant difference (P < 0.05).

Figure 3.

miR-200c binds to XIAP-3ʹUTR to decrease XIAP mRNA stability and inhibit cell invasion. (a) Potential miRNAs binding site in XIAP 3ʹ-UTR region. (b) Real-time PCR was applied to compare the expression level of these potential miRNAs in T24 and T24T cells. (c) Real-time PCR was used to identify the expression level of miR-200c in T24T (miR-200c) cells. (d) West blot (WB) was applied to detect the protein level of XIAP after miR-200c was overexpressed in T24T cells. (e) Real-time PCR was used to identify the expression level of miR-200c in T24 cells stably transfected with miR-200c inhibitor. (f) WB was applied to detect the protein level of XIAP after miR-200c inhibitor was transfected in T24 cells. (g) XIAP mRNA stabilities were evaluated by RT-PCT in presence of Act D in T24 and T24T cells. (h) T24T (vector) and T24T (miR-200c) cells were seeded to control or Matrigel inserts for trans-well invasion assay following the manufacturer’s instructions. The images were captured using an inverted microscope. (i) The invasion rate was normalized with the insert control according to the manufacturer’s instruction. An asterisk (*) indicates a significant difference between T24T (vector) and T24T (miR-200c) cells (P < 0.05). The values shown are mean ± SD from three independent experiments.

Figure 4.

p-CREBcontributes tomiR-200ctranscription and inhibits XIAP expression and cancer cell invasion of bladder cancer cells. (a) T24 and T24T cells were co-transfected withmiR-200c promoter-driven luciferase reporter and pRL-TK. Twenty-four hours after transfection, the cells were extracted and luciferase activity was determined. The results are presented as miR-200c promoter activity relative to T24 cells with normalized by internal TK activity. An asterisk (*) indicates a significant difference between the paired cells (P < 0.05). The bars are mean ± SD from three independent experiments. (b) Potential transcription factors binding site in miR-200c promoter region. (c) WB applied to detect the transcription factors, CREB, SMAD4, and ETS1 expression between T24 and T24T cells. GAPDH was used as internal control. (d) XIAP expression level was evaluated using Western Blot after knockdown of CREB in T24 cells. (e) miR-200c expression level was evaluated using real time-PCR after knockdown of CREB in T24 cells. (f) T24 (nonsense) and T24 (shCREB) cells were seeded to control or Matrigel inserts for transwell invasion assay following the manufacturer’s instructions. The images were captured using an inverted microscope. (g) The invasion rate was normalized with the insert control according to the manufacturer’s instruction. An asterisk (*) indicates a significant difference between T24/Nonsense and T24/shCREB cells (p < 0.05). The values shown are mean ± SD from three independent experiments.

Figure 5.

MiR-200c and XIAP are critical for T24T cell lung metastasis. (a and c) Representative images of lungs and lung surface metastatic foci as indicated are shown after fixation in a neutral-buffered formalin/Bouin’s fixative solution; histologic appearance of lung metastases: H&E-stained sections (H&E, ×40 and ×100). (b and d) The lung metastatic tumor number in the group mice that were injected with T24T (nonsense) vs. T24T (shXIAP), T24T (vector) vs. T24T (miR-200c), respectively.