Wild-type p53-induced phosphatase (Wip1) is an established oncogene and is associated with development of multiple forms of human cancer. However, the expression and role of Wip1 in human bladder transitional cell carcinoma (TCC) remains to be elucidated. In the present study, immunohistochemistry demonstrated that Wip1 was overexpressed in bladder TCC tissues compared with corresponding normal bladder tissues in 106 bladder TCC cases (P<0.0001). Furthermore, high expression levels of Wip1 were significantly associated with increasing tumor size (P=0.002), pathological grade (P=0.025), clinical T stage (P=0.001) and lymph nodal metastasis (P=0.003). Kaplan-Meier survival analysis identified that patients with high Wip1 expression levels exhibited a lower overall survival time (P<0.0001), and Cox proportional hazards regression model analysis demonstrated that Wip1 expression was an independent prognostic factor in patients with bladder TCC (P=0.025). In addition, downregulation of Wip1 expression by transfection with small interfering RNA in bladder cancer cells inhibited cell proliferation, invasion and migration (P<0.05), along with the upregulation of p53 protein levels (P<0.05). These findings suggest that Wip1 may function as a potential prognostic marker and therapeutic target in bladder cancer.
Bladder cancer is the second most common genitourinary malignancy worldwide (
Wip1 was first identified as a phosphatase that is induced by p53 in response to ionizing radiation (
The purpose of the present study was to determine the levels of Wip1 expression in patients with bladder cancer and the association between Wip1 expression and clinicopathological features. The effects of Wip1 knockdown on proliferation, invasion and migration in T24 (human bladder carcinoma cell line) cells and the underlying signaling pathways were also evaluated.
A total of 106 formalin-fixed paraffin-embedded bladder TCC tissues and corresponding normal bladder tissues were collected from patients at the Department of Urology, Beijing Friendship Hospital, Capital Medical University (Beijing, China) who underwent surgical resection between January 2009 and December 2011. All patients were diagnosed with TCC by postoperative pathological examination. No patients were treated with radiotherapy or chemotherapy prior to surgery. Clinical data included age, gender, tumor size, pathological grade, lymph nodal status, tumor-node-metastasis (TNM) classification stage and patient survival time. Tumor staging was based on the seventh edition of the TNM classification system (2009) (
Paraffin-embedded tissue sections (4 µm thick) were deparaffinized, rehydrated and subjected to antigen retrieval by immersion in boiling citric acid buffer (pH 6.0; ZSGB-BIO, Beijing, China) for 10 min. Endogenous peroxidase and non-specific conjugation were blocked by 3% hydrogen peroxide and normal goat serum (ZSGB-BIO, Beijing, China). Sections were incubated with anti-Wip1 antibody (#ab31270; dilution 1:1,000; Abcam, Cambridge, UK) at 4°C overnight. Subsequently, sections were incubated with biotinylated anti-rabbit secondary antibody (#ZB-2010; dilution, 1:400; ZSGB-BIO) and streptavidin-horseradish peroxidase complex (ZSGB-BIO) at room temperature for 10 min. Finally, sections were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin, then dehydrated and mounted. An immunoreactivity score was used to evaluate the staining of Wip1 expression in bladder TCC tissues and normal bladder tissues. Four randomly selected fields of each slide were counted using an inverted light microscope (DMi1; Leica Microsystems GmbH, Wetzlar, Germany). Wip1 expression was quantified according to the staining intensity and the percentage of positive cells. The staining intensity was defined as follows: 0 point, no staining; 1 point, weak staining; 2 points, moderate staining; and 3 points, strong staining. The percentage of positive cells was defined as follows: 1 point, ≤10%; 2 points, 11–50%; 3 points, 51–80%; and 4 points, ≥81%. The final points were defined as the points of the staining intensity multiplied by those of the percentage of positive cells. Wip1 expression was divided into the following categories: No expression, 0–2 points; positive expression, ≥3 points (low expression, 3–6 points and high expression, >6 points). Two pathologists (Department of Pathology, Beijing Friendship Hospital, Capital Medical University) assessed these results independently.
Human bladder T24 cells were purchased from the Cell Resource Center, Peking Union Medical College (Beijing, China) and were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO2. siRNA for Wip1 and negative control siRNA were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China), according to the NM_003620 gene sequence in the National Center for Biotechnology Information database (
Total RNA was extracted with the RNeasy® Mini kit (Qiagen Inc., Valencia, CA, USA) at 24 h post-transfection, according to the manufacturer's protocol. Total RNA (1 µg) was reverse transcribed into cDNA using a PrimeScript™ 1st Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) on an ABI Veriti™ Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was conducted in 20 µl reaction buffer with SYBR® Premix DimerEraser™ (Perfect Real Time) (Takara Biotechnology Co., Ltd., Dalian, China) using the ABI® 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The following primer sequences were used: Wip1 forward, 5′-GGAAGAAACTGGCGGAATG-3′ and reverse, 5′-TGGGAAGTTCTGGCTTATGG-3′; GAPDH, forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. GAPDH was used as the internal control. RT-qPCR was performed under the following conditions: 95°C for 30 sec, 40 cycles at 95°C for 3 sec, 60°C for 30 sec and 72°C for 30 sec. The melting curve was analyzed for each sample. All samples were performed in triplicate. The 2-ΔΔCq method was used to calculate the relative mRNA levels of Wip1 (
Western blotting was performed at 48 h post-transfection. The harvested T24 cells were washed with ice-cold PBS and were lysed in radio immunoprecipitation assay lysis buffer containing phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China) at 4°C for 30 min. The cells were subsequently centrifuged at 12,000 × g at 4°C for 10 min, and the supernatant was taken and protein concentration detected using the BCA protein assay kit (Beyotime Institute of Biotechnology). Total protein (40 µg) was separated by 10% SDS-PAGE, then transferred to a nitrocellulose filter membrane. Following blocking with 5% non-fat dry milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Anti-Wip1 antibody (#ab31270; dilution, 1:800; Abcam), anti-P53 antibody (#ab179477; dilution, 1:1,000; Abcam) and anti-GAPDH antibody (#ab181602; dilution, 1:1,000; Abcam) at 4°C overnight. Subsequently, the membranes were incubated with peroxidase-conjugated affinipure goat anti-rabbit secondary antibody (#ZB-5301; dilution 1:5,000; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) at room temperature for 2 h. Following washing, protein bands were detected using the Bio-Rad ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Co., Ltd, Hercules, CA, USA) using Image Lab™ software (Bio-Rad Laboratories, Co., Ltd.). GAPDH was used as the protein control and all experiments were performed in triplicate.
The T24 cells were plated in 96-well plates at a density of 2,000 cells/well in 100 µl DMEM supplemented with 10% FBS. Following transfection with Wip1-siRNA or negative control siRNA, the cells were cultured for 0, 24, 48 and 72 h. A total of 10 µl 5 mg/ml MTT was added to each well and the cells were incubated at 37°C for 4 h. Next, 100 µl formazan solution was added to each well and the cells were incubated at 37°C for an additional 4 h. The absorption value was measured at 570 nm using a SpectraMax® M3 Multi-Mode Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA). All reactions were performed in triplicate.
The 24-well (8 µm pore) Transwell® cell culture inserts (Corning Inc., Corning, NY, USA) were coated with 50 µl Matrigel™ Basement Membrane Matrix (BD Biosciences, San Jose, CA, USA). Following transfection with Wip1-siRNA or negative control siRNA, 1×105 cells were plated in the upper chamber, and cultured with serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.). DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was placed in the lower chamber. Following incubation at 37°C for 24 h, the cells and medium in the upper chamber were removed with a cotton swab. Polyoxymethylene was added to the lower chamber to fix the cells attached to the lower membrane surface, and 0.1% crystal violet was added to the lower chamber to stain the cells. Following this, crystal violet staining was decolorized using 33% acetic acid. The absorption value was measured at 570 nm to indicate cell invasion. Migration assays were performed using the aforementioned method without Matrigel. All experiments were performed in triplicate.
All statistical analyses were performed using SPSS version 19.0 (IBM SSPS, Armonk, New York, USA). Values were expressed as the mean ± standard deviation. For the clinical characteristics, P-values were calculated using the χ2 test. Survival outcomes were estimated with the Kaplan-Meier method and then compared using the log-rank test. The Cox proportional hazards regression model was used to define factors predicting prognosis and the Student's t-test was used to evaluate the significance of differences between two groups. P≤0.05 was considered to indicate a statistically significant difference.
In the present study, 106 bladder TCC tissues and corresponding normal bladder tissues were analyzed using immunohistochemistry. Wip1 expression was significantly higher in the bladder TCC tissues (99/106, 93.4%) than in the normal tissues (13/106, 12.3%; P<0.0001). In addition, the expression rate of Wip1 in the low and high groups was 48.1 and 45.3%, respectively, in the bladder TCC tissues. In normal tissues, Wip1 was expressed at a low level or not expressed at all. Wip1 staining is presented in
To analyze the correlation between Wip1 expression and clinical data, the patients were divided into low (including no expression) and high expression groups based on immunohistochemical points of bladder TCC tissues. High expression levels of Wip1 were significantly associated with increased tumor size (P=0.002), pathological grade (P=0.025), clinical T stage (P=0.001) and lymph nodal metastasis (P=0.003), but not with gender and age (
To assess the effects of Wip1 on TCC cell proliferation, invasion and migration, Wip1-siRNA or negative control siRNA was transiently transfected into T24 cells. RT-qPCR and western blot analyses demonstrated that Wip1 mRNA and protein expression levels in the Wip1-siRNA-transfected T24 cells were significantly lower compared with the control group (mRNA, P=0.019; protein, P<0.001;
Bladder cancer is a leading global cause of cancer morbidity and mortality (
Wip1, an established oncogene, has been the subject of a number of studies since it was identified in 1997 (
To the best of our knowledge, the present study is the first to investigate the expression of Wip1 in patients with bladder TCC. A total of 106 bladder TCC and corresponding normal bladder tissues were analyzed by immunohistochemistry. The data demonstrated that Wip1 was overexpressed in the bladder TCC tissues, compared with the normal tissues. Furthermore, high expression levels of Wip1 were positively associated with more aggressive tumor characteristics, including tumor size, pathological grade, clinical T stage and lymph nodal metastasis. To determine the correlation between Wip1 expression and prognosis, Kaplan-Meier survival curve and the Cox proportional hazards regression model analyses were implemented. The results revealed that high levels of Wip1 expression led to a shorter overall survival time, and that Wip1 expression was an independent prognostic factor. These results suggest that Wip1 expression may be a potential prognostic marker for patients with bladder cancer.
To assess the effects of Wip1 in bladder cancer, further
In conclusion, the current study demonstrated that Wip1 is overexpressed in patients with bladder TCC and that high levels of Wip1 expression were positively correlated with more aggressive tumor characteristics and poorer prognosis. Downregulation of Wip1 expression inhibited bladder cancer cell proliferation, invasion and migration by activating the p53 pathway in the T24 cells. Therefore, these findings indicate that Wip1 may serve as a potential prognostic marker and therapeutic target in bladder cancer.
Immunohistochemical analysis of Wip1 expression in the bladder TCC tissues and corresponding normal bladder tissues. (A) High expression of Wip1 in bladder TCC tissue. Low expression of Wip1 in (B) bladder TCC and (C) normal bladder tissues. (D) No expression of Wip1 in normal bladder tissue. Magnification, ×200; scale bar, 50 µm. TCC, transitional cell carcinoma.
Kaplan-Meier survival analysis of the overall survival time of patients with bladder transitional cell carcinoma. The overall survival time of patients in the high expression group was significantly lower, compared with patients in the low expression group (P<0.0001 vs. low expression group).
(A) Reverse transcription-quantitative polymerase chain reaction analysis of Wip1 mRNA expression in T24 cells. (B and C) Western blot analysis of Wip1 protein expression levels in T24 cells. (B and D) Western blot analysis of p53 protein expression levels in T24 cells. Wip1 mRNA expression was downregulated in Wip1-siRNA-transfected cells (A, *P<0.05 vs. control). Wip1 protein expression was downregulated in Wip1-siRNA-transfected cells (B and C; *P<0.05 vs. control). P53 protein expression was upregulated in the Wip1-siRNA-transfected cells (B and D; *P<0.05 vs. control). Wip1, wild-type p53-induced phosphatase; siRNA, small interfering RNA.
MTT assay analysis of cell proliferation. Downregulation of Wip1 inhibited T24 cells proliferation at 24, 48 and 72 h, compared with the control group (*P<0.05 and **P<0.0001 vs. control). Wip1, wild-type p53-induced phosphatase; siRNA, small interfering RNA; OD, optical density.
Transwell assay analysis of cell invasion and migration. Downregulation of Wip1 inhibited T24 cell invasion and migration, compared with the control group (*P<0.05 vs. control). Magnification, ×200. Wip1, wild-type p53-induced phosphatase; siRNA, small interfering RNA; OD, optical density.
Association between wild-type p53-induced phosphatase expression levels and the clinicopathological features of patients with bladder transitional cell carcinoma.
Clinicopathological features | Total | Low | High | P-value |
---|---|---|---|---|
Gender | 0.144 | |||
Male | 84 | 49 | 35 | |
Female | 22 | 9 | 13 | |
Age, years | 0.526 | |||
≥60 | 74 | 39 | 35 | |
<60 | 32 | 19 | 13 | |
Tumor size, cm | 0.002 | |||
≥3 | 51 | 20 | 31 | |
<3 | 55 | 38 | 17 | |
Pathological grade | 0.025 | |||
G1 | 21 | 13 | 8 | |
G2 | 56 | 33 | 23 | |
G3 | 29 | 12 | 17 | |
Clinical T stage | 0.001 | |||
Ta+Tis+T1 | 43 | 32 | 11 | |
T2-T4 | 63 | 26 | 37 | |
Lymph nodal metastasis | 0.003 | |||
N0 | 73 | 48 | 25 | |
N1 | 22 | 7 | 15 | |
N2 | 11 | 3 | 8 |
Cox proportional hazards regression model analyses of Wip1 expression and other clinical prognostic factors in patients with bladder transitional cell carcinoma.
Cox regression analysis | ||
---|---|---|
Variable | HR (95% CI) | P-value |
Gender (male/female) | 0.74 (0.41–1.32) | 0.302 |
Age (≥60/<60 years) | 1.06 (0.62–1.79) | 0.842 |
Tumor size (≥3/<3 cm) | 0.91 (0.55–1.51) | 0.709 |
Pathological grade (G1+G2/G3) | 1.79 (0.92–3.48) | 0.087 |
Clinical T stage (Ta+Tis+T1/T2-T4) | 2.49 (1.33–4.64) | 0.004 |
Lymph nodal metastasis (N0/N+) | 3.34 (1.60–6.98) | 0.001 |
Wip1 expression (high/low) | 1.86 (1.02–3.18) | 0.025 |
Wip1, wild-type p53-induced phosphatase; HR, hazard ratio; CI, confidence interval.