TMA chips that covered 68 bladder cancer tissue specimens and 54 adjacent normal bladder cancer specimens were purchased from Shanghai Outdo Biotech Co., Ltd. The present study was approved by the ethics committee of Beijing Chao-Yang Hospital, Capital Medical University (approval number 2017-ke-47) and was conducted in accordance with the Declaration of Helsinki. Patients gave written consent for their information to be stored on the hospital database and to be used in research. Immunohistochemical staining was performed according to the instructions of the S-P kit (OriGene Technologies, Inc.). The TMAs were firstly fixed with 4% paraformaldehyde and blocked with 10% goat serum for 30 min at room temperature. Then, the TMAs were stained with BLM primary antibody (cat. no. NBP1-89929; Novus Biologicals, Ltd.) at a dilution of 1:100 overnight at room temperature. Negative and positive controls (by omission of the primary antibody and IgG-matched serum) were included in each test.

The BLM protein staining scores were evaluated by two experienced pathologists and the concordance between the two pathologists was excellent. The whole field of the section was scored and intensities of staining were grouped as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining. The cut-off value used to separate patients into high and low BLM expression groups was ≥2.

Human bladder cancer cell lines (J82 and 5637) were obtained from China Infrastructure of Cell Line Resources. Cells were cultured routinely in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a standard culture conditions (5% CO₂ at 37°C).

Cells were transfected with 20 nM negative control [short interfering (si)RNA-CON] or BLM siRNA (siRNA-BLM) (sense: 5′-CCCACUACUUUGCAAGUAATT-3′, antisense: 5′-UUACUUGCAAAGUAGUGGGAA-3′) according to the manufacturer's instructions (Ambion; Thermo Fisher Scientific, Inc.). Briefly, 15×10⁴ cells were seeded and cultured in 6-well plates and medium was changed to Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) at ~70% confluence. Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) and siRNA-CON or siRNA-BLM was diluted into Opti-MEM separately and incubated for 5 min at room temperature then mixed and incubated for 20 min to allow the formation of lipid-siRNA complex. After 6 h incubation, the medium was changed to RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.). After 48 to 72 h, the cells were harvested and used for experiments.

Cell viability was assessed by the Cell Counting Kit-8 (CCK-8) (Beyotime Institute of Biotechnology) for three times independently. Briefly, the cells was plated into a 96 well plate and treated with corresponding agents for the indicating time. CCK-8 reagent (10 µl/well) was added to each well and incubated for 2 h. Then the cell viability was determined by using a micro-plate reader at a 450 nm wavelength (Multiskan; Thermo Fisher Scientific, Inc.).

A total of 4×10⁴ cells were cultured in 96-well plates and exposed to 50 µM EdU solution (Guangzhou RiboBio Co., Ltd.) for 4 h at 37°C and then fixed in 4% formaldehyde overnight at 4°C and permeabilized in 0.5% Triton X-100 for 10 min. Cells were incubated with 100 µl Apollo reaction cocktail for 30 min at room temperature in the dark and DNA was stained with Hoechst 33342 (100 µl/well) for 30 min at room temperature in the dark. The stained cells were analyzed using a fluorescent microscope (6 fields were selected randomly; magnification, ×40).

A total of 1×10⁶ cells were fixed with pre-cooled 70% ethanol and treated with 10 µg/ml RNase (Sigma-Aldrich; Merck KGaA), then the cells incubated at 37°C for 30 min before staining with 50 µg/ml propidium iodide (PI) for 30 min at 4°C (Invitrogen; Thermo Fisher Scientific, Inc.). Cell cycle distribution was analyzed using a Gallios flow cytometer (Beckman Coulter, Inc.) and ModFit software version 3.2 (Verity Software House) was utilized for cell cycle analysis.

The experiment was performed following the protocols described by the kit manufacturer (BD Biosciences). A total of 5×10⁵ cells were harvested and re-suspended in binding buffer at a concentration of 1×10⁶ cells/ml. 100 µl of the single cells suspension was mixed with 5 µl of Annexin V-FITC and 5 µl of PI and further incubated for 15 min at room temperature in the dark. Finally, 400 µl of binding buffer was added to the mixture and the cells were analyzed by a Gallios flow cytometer (Beckman Coulter, Inc.). The FlowJo software (BD Biosciences; Version 7.6) was used to analyze the early apoptosis rate, late apoptosis rate and total apoptosis rate respectively. The cells undergoing early apoptosis were defined as Annexin V-FITC-positive/PI-negative, and the late apoptotic cells were defined as Annexin V-FITC-positive/PI-positive. The total apoptosis rate was the percentage of early + late apoptotic cells.

The transfected cells were seeded into a 96-well plate at a density of 5×10⁴ cells per well. Then the cells were treated with different concentrations of cisplatin (0, 2, 4 and 8 µmol/l) for different periods of time (12, 24 and 48 h). The IC₅₀ value of cisplatin in the J82 and 5637 was calculated at 24 h by probit regression. Cisplatin was obtained from Qilu Pharmaceutical Co., Ltd.

Total RNA was extracted from 5×10⁵ cells using TRIzol reagent (Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's instructions. RT-qPCR was performed according to the manufacturer's instructions (Taq Plantinum PCR MasterMix, Tiangen Biotech Co., Ltd.). The primers were designed using the software Primer Premier (BLM: forward 5′-GGATCCTGGTTCCGTCCGC-3′, reverse 5′-CCTCAGTCAAATCTATTTGCTCG-3′, β-actin: forward: 5′-TGACGTGGACATCCGCAAAG-3′, reverse: 5′-TCTTCATTGTGCTGGGTGCC-3′). The PCR program included a cycle of 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing/extension at 60°C for 32 sec. All samples were normalized against the internal control (β-actin) and analyzed using the 2^−ΔΔCq method (22).

Cells were lysed with RIPA buffer (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 0.25% sodium deoxycholate) and quantified using the BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts (40 µg) protein were separated on a 10% SDS-polyacrylamide gel and electrotransferred to PVDF membranes (MilliporeSigma). Membranes were immersed in blocking buffer with 10% fat free milk for 1 h at room temperature. The blocked PVDF membrane was incubated with antibodies against BLM (1:1,000; cat. no. 2742; Cell Signaling Technology, Inc.) for 3 h at room temperature. Following incubation with the primary antibodies, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies for 1 h (1:5,000; cat. nos. 31466 and PA174421; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature. Protein bands were detected by using enhanced chemiluminescence. β-actin (1:1,000; cat. no. 3700; Cell Signaling Technology, Inc.) was used as the loading control. Proteins bands were visualized using an ECL reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the immunoblots were quantified using ImageJ software (version 3.0; National Institutes of Health).

Two groups were compared using Mann Whitney test if data were non-parametric or using unpaired Student's t-test if data were parametric. Multiple groups were compared using one-way analysis of variance, followed by Sidak's or Tukey's multiple comparisons test. Survival curves were estimated using the Kaplan-Meier analysis and were compared using the log-rank test. A Cox regression model was used to identify prognostic factors for survival of patients with bladder cancer. The contingency table presented in Table I was analyzed using a chi-squared test or Fisher's exact test when the expected count in <20% of the cells of the analyzed contingency table is 5 or fewer (i.e. age, gender, differentiation, muscle invasion and lymph node metastasis). P<0.05 was considered to indicate a statistically significant difference.

A total of 68 bladder cancer and 54 adjacent healthy bladder tissues were analyzed in the present study. The patient demographics are summarized in Table I. The expression of BLM in cancer tissues was assessed using immunohistochemistry. Bladder cancer tissues expressed a high level of BLM, while the adjacent healthy tissue expressed a low level of BLM (Fig. 1). When the immunostaining scores of BLM were further compared between cancerous tissue and adjacent healthy tissue, a significantly higher expression level of BLM was found in the cancerous tissues in all patients (P<0.0001) or in patients with bladder cancer of grade II or higher (P<0.001; Fig. 1B and C). These results suggested that the expression of BLM was significantly higher in bladder cancer tissues than in adjacent normal tissues.

Survival analysis was then performed for the patients with bladder cancer. In the low BLM expression group (non-staining group and weak staining group), 33 of the 49 (67.35%) patients survived, while in the high BLM expression group (moderate staining group and strong staining group), 8 of the 19 (42.10%) patients survived. There was a significant difference in the survival rate between the high and low BLM expression groups (P=0.013; Fig. 1D).

In Cox regression analysis, two prognostic factors for the overall survival of patients with bladder cancer were identified using univariate analysis: The American Joint Committee on Cancer (AJCC) stage (I–II vs. >II, P=0.008) and TNM stage (I/II vs. III/V, P=0.001). However, the BLM expression level (low vs. high) was not a statistically significant prognostic factor (P=0.981). Furthermore, multivariate analysis revealed that the AJCC (P=0.030) and TNM stage (P=0.008) were significant independent predictors of the poor survival of patients with bladder cancer (Table II). Additionally, a nomogram of the median survival time was drawn, containing BLM expression, age and sex (data not shown).

To investigate the role of BLM in bladder cancer, the expression of BLM was first assessed in two bladder cancer lines (J82 and 5637). As shown in Fig. 2A and B, both cell lines expressed BLM. The mRNA and protein expression level of BLM in the J82 cells was lower compared with that in the 5637 cells.

Subsequently, both bladder cancer cell lines were transfected with a control siRNA (siRNA-CON) or a BLM-specific siRNA (siRNA-BLM). As shown in Fig. 2C and D, both the mRNA and protein expression levels of BLM were markedly decreased in these two cell lines (P<0.0001) following transfection with siRNA-BLM.

The cell cycle was analyzed to identify whether cell cycle perturbation occurs after the silencing of BLM expression. The results of cell cycle assay (Fig. 3) revealed that both cell lines transfected with siRNA-BLM were arrested in the G₀G₁ phase (P<0.001). In the siRNA-BLM group, the J82 cells in the S phase were significantly decreased compared with the siRNA-CON group (P<0.0001) and the 5637 cells in the G₂M phase were significantly decreased compared with the siRNA-CON group (P<0.05). These results indicated that the silencing of BLM expression led to G₁ arrest in the bladder cancer cells.

It was hypothesized that cell proliferation was inhibited as there were fewer in the S phase. To verify this hypothesis, a cell proliferation assay was performed. The numbers of EdU-labeled J82 and 5637 bladder cancer cells transfected with siRNA-CON were 335.25±97.79 and 118.71±19.20 in each field, respectively. In addition, the numbers of EdU-labeled J82 and 5637 bladder cancer cells transfected with siRNA-BLM were 185±24.90 and 76.34±16.45, respectively (P<0.05). Therefore, the silencing of BLM significantly decreased bladder cancer cell proliferation (Fig. 4).

To examine cell functions in response to BLM knockdown in the J82 and 5637 cells, cell apoptosis assay was performed. The results revealed that cell apoptosis was significantly increased following transfection with siRNA-BLM compared with the siRNA-CON group (P<0.05, Fig. 5).

Previous studies have demonstrated that the RecQ helicase level is negatively associated with genomic instability induced by DNA damaging agents in various type of cells (21,23). As platinum-containing anticancer drugs are widely used in intravesical instillation, cisplatin was selected to induce DNA damage in the present study. To determine the cytotoxicity of cisplatin in bladder cancer cell lines, the J82 and 5637 cells were first treated with various concentrations of cisplatin (0, 2, 4 and 8 µmol/l) for different periods of time (12, 24 and 48 h). The results revealed that cisplatin significantly increased the death of the J82 and 5637 cells in a concentration- and time-dependent manner (Fig. 6A and B). The IC₅₀ of cisplatin to J82 and 5637 at 24 h were (10.44±1.00) µmol/l and (6.91±0.32) µmol/l respectively. Subsequently, the siRNA-BLM-siRNA-CON-transfected J82 and 5637 cells were treated with cisplatin at the IC₅₀ concentration for 24 h. As shown in Fig. 6C and D, the silencing of BLM significantly sensitized the J82 and 5637 cells to cisplatin; the death rate of the siRNA-BLM-transfected cells was significantly higher than that of the control group (P<0.05).