Bladder cancer and paired adjacent normal bladder tissues used for immunohistochemistry were collected from 74 bladder cancer patients who underwent surgical resection in The First Affiliated Hospital of China Medical University from 2008 to 2015. None of patients underwent chemotherapy, radiotherapy or adjuvant treatment before surgery. The present study was approved by the Ethics Committee of the China Medical University. Informed consent was obtained from all patients. Twenty pairs of fresh bladder cancer and adjacent non-tumor bladder tissues used for quantitative PCR were collected from The First Affiliated Hospital of China Medical University and snap frozen in liquid nitrogen until use. The patients had not received any therapy before admission.

Immunohistochemistry for TRPM7 expression in bladder cancer tissues was performed using standard methods. Sections were deparaffinized in xylene and hydrated in a graded ethanol series. The sections were then processed in 10 mmol/l citrate buffer (pH 6.0) and heated at 120°C for 5 min to retrieve the antigen. After that, sections were soaking in 3% hydrogen peroxide for 20 min, which served as a blocking agent for endogenous peroxidase activity. After being rinsed in phosphate-buffered saline (PBS; pH 7.2), 10% goat serum was applied for 1 h at room temperature to block non-specific reactions. Sections were incubated with anti-TRPM7 goat polyclonal antibody (diluted 1:50; ab729; Abcam, Cambridge, MA, USA) overnight at 4°C. Horseradish peroxidase-conjugated anti-goat IgG was used as a secondary antibody. After washing, the peroxidase reaction was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) chromogen solution. Finally, the sections were counterstained with hematoxylin, dehydrated and coverslipped. All the slides were evaluated independently by two pathologists using a conventional semi-quantitative scoring system according to a previously defined scoring system (16). Briefly, the intensity of the staining in each section was assessed as very strongly positive (3), moderately positive (2), weakly positive (1), or negative (0). The percentage of positive tumor cells were scored as <5% (0), 5–20% (1), 21–50% (2) and >50% (3) of cells. The final score was calculated by multiplying the percentage and the intensity score. A score of ≥4 was defined as high TRPM7 expression and scores of <4 were defined as low TRPM7 expression.

Human bladder cancer cell lines (BIU87, 5637 and T24) were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) (both from HyClone, Logan, UT, USA) at 37°C in 5% CO₂ incubator. We obtained TRPM7 siRNA and the negative control siRNA (NC_siR) from GenePharma Co. Ltd. (Shanghai, China). Before transfection, cells were plated overnight and grew to 30–50% confluency. According to the manufacturer's instructions, the siRNAs were transfected into 5637 and T24 cells using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA). The sequences of siRNAs are shown in Table I. The efficiency of siRNA was determined by the protein and mRNA levels of TRPM7 in the 48-h post transfected cells.

BIU87, 5637 and T24 cells (5×10⁴ cells/ml) were seeded on 12-mm coverslips for 24 h, fixed with 4% paraformaldehyde for 30 min, and then permeabilized for 20 min with 0.1% Triton X-100 solution and blocked in normal bovine serum for 30 min at room temperature. Cells were incubated overnight at 4°C with anti-TRPM7 (1:50; Abcam) antibody in 2% bovine serum albumin (BSA; BioShop, Burlington, ON, Canada), 2% FBS and 0.2% fish gelatin (Sigma-Aldrich, St. Louis, MO, USA). The cells were then incubated with FITC-labeled secondary antibody for 2 h at room temperature. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI) (2 µg/ml). Fluorescence was visualized with a confocal microscope (Carl Zeiss Inc., Gottingen, Germany).

Total RNA was isolated from bladder cancer and adjacent non-tumor bladder tissues with TRIzol reagent (Invitrogen). cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis kit (Roche, Mannheim, Germany). Then, SYBR-Green Real-Time PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used. Real-time PCR was performed using Thermal Cycler Dice™ Real-Time System TP800 (Takara, Tokyo, Japan). Amplification conditions consisted of 2 min at 50°C for reverse transcription, 5 min at 95°C for Taq activation followed by 45 cycles of 94°C for 40 sec, one cycle of 58°C for 20 sec and elongation at 72°C for 30 sec. The sequence of primers designed for TRPM7 and GAPDH are listed in Table I. GAPDH served as the internal control for mRNA determination of TRPM7. Results were normalized to GAPDH. The relative gene expression was calculated using 2^−ΔΔCt.

Cells were lysed by in ice-cold cell RIPA buffer. Protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Total cellular protein (30 µg) was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) filter membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) buffer for 2 h at room temperature and incubated overnight at 4°C with a specific primary antibody against TRPM7 (ab85016; 1:500 dilution; Abcam) and mouse-anti-human actin (1:2,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as an internal control. Next, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG (1:2,500 dilution; Santa Cruz Biotechnology, Inc.), and signals were developed using Western Blotting Luminol Reagent (Gene Company Ltd., Hong Kong).

Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Tokyo, Japan). The cells (3×10³ cells/well) were seeded into 96-well plates. At time points of 0, 6, 12, 24, 36 and 48 h, 10 µl of CCK-8 was added/well and was incubated at 37°C for 1 h. The absorbance was measured at 450 nm using a plate reader (Model 680; Bio-Rad Laboratories, Hercules, CA, USA) to determine the number of viable cells. All of the experiments were performed three times.

Cells (5×10⁵ cells/well) were seeded into 6-well plates. After the cells were grown to 80% confluency, wounds were created by scraping the cells with a 100-µl pipette tip. The 6-well plates were incubated at 37°C and a microscope was used to observe the migrated distance every 12 h.

The invasion and migration of cells were measured using 8-µm pore-size Transwell chambers with or without Matrigel (BD Biosciences, San Diego, CA, USA). Cells (1×10⁵ cells/well) were seeded into the top chamber with the serum-free medium while the bottom chambers were filled with 500 µl complete RPMI-1640 medium. After 24 h of incubation, the cells remaining on the upper side were carefully removed with cotton swabs, and those cells that had migrated to the lower side were fixed and stained with 1.0% crystal violet. The cells were quantified by counting all of the cells that had migrated through the membrane in five random fields under a light microscope (magnification, ×200). The mean value was calculated from data obtained from three separate chambers.

For apoptosis analysis, after transfection for 48 h, cells were collected, washed with PBS, and stained with FITC/Annexin V Apoptosis Detection Kit I (BD Biosciences), and analyzed by flow cytometric analysis.

Cells were trypsinized and washed in ice-cold PBS, and then fixed in ice-cold 75% ethanol in PBS overnight. PI/RNase staining buffer (BD Biosciences) was added, and the cells were incubated at 4°C for 30 min. Cell cycle profiles were analyzed using a FACSCalibur flow cytometer (BD Biosciences).

All data are presented as means ± standard deviation (SD) and analyzed using SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, USA). t-test was used to determine the significance of differences in multiple comparisons. Survival curves were estimated by Kaplan-Meier analysis and compared by the log-rank test. All tests performed were two-sided. P<0.05 was considered statistically significant.

To characterize the expression of TRPM7 in human bladder cancer tissues, we firstly examined the mRNA and protein levels of TRPM7 in 20 pairs of human bladder cancer, and the adjacent non-tumor bladder tissues by qRT-PCR and western blotting. As shown in Fig. 1A and B, the mRNA and protein levels of TRPM7 were significantly increased in the bladder cancer tissues compared with levels in the paired adjacent non-tumor bladder tissues (P<0.05). Next, we performed immunohistochemical analysis to assess the protein expression and subcellular localization of TRPM7 in 74 paraffin-embedded bladder cancer tissues (Fig. 1C). High TRPM7 expression was detected in 47 (63.5%) of the 74 bladder cancer tissues and in 16 (25.80%) of the 62 adjacent non-tumor tissues (P<0.05). These data suggest that TRPM7 is overexpressed in bladder cancer tissues and may be a potential biomarker for bladder cancer.

To investigate the potential role of TRPM7 in bladder cancer, the relationship between TRPM7 expression level and clinicopathological factors was analyzed and is summarized in Table II. As shown, TRPM7 overexpression was associated with recurrence (P<0.01), and metastasis (P=0.021) of patients with bladder cancer. However, TRPM7 exhibited no significant association with other clinicopathological characteristics, such as age, sex, histologic grade, tumor stage and multiplicity (all P>0.05).

The prognostic value of TRPM7 was evaluated by Kaplan-Meier analysis. The survival curves indicated that high TRPM7 expression was significantly associated with poor survival of bladder cancer patients. Patients with TRPM7 high expression had worse prognoses than those with low expression of TRPM7 (Fig. 2) (P<0.05).

Furthermore, univariate and multivariate Cox regression analyses were applied to the clinicopathological characteristics in regards to TRPM7 expression levels. As shown in Table III, multiplicity (P=0.017), recurrence (P<0.01), metastasis (P=0.049) and TRPM7 expression (P<0.01) were all significantly related to bladder cancer patient poor survival. Multivariate analysis showed that high TRPM7 expression was independently associated with the poor prognosis of bladder cancer patients (P=0.035) (Table IV).

To examine the mRNA and protein levels of TRPM7 in BIU87, 5637 and T24 bladder cancer cell lines, RT-PCR and western blotting were carried out, respectively. Fig. 3A shows that the levels of TRPM7 mRNA in 5637 and T24 cells (normalized to GAPDH, 1.38±0.7, 1.45±0.6; P<0.05) were significantly higher than that noted in the BIU87 cells (0.75±0.4; P<0.05). Western blotting demonstrated that TRPM7 protein levels (normalized to GAPDH) were also higher in the 5637 and T24 cells (1.25±0.41, 1.36±0.49; P<0.05) compared to that noted in the BIU87 cells (0.58±0.32) (Fig. 3B). TRPM7 protein levels were further determined in bladder cancer cell lines using immunofluorescent staining. As shown in Fig. 3C, higher levels of TRPM7 protein were observed in the 5637 and T24 cells compared to that noted in the BIU87 cells in cell culture, and the TRPM7 protein trend was to localize around the nuclear membrane in the 5637 and T24 cells. The fluorescence intensity in the 5637 and T24 cells was 25 or 30% higher than that in the BIU87 cells (P<0.05). Our results provide evidence that TRPM7 channels are highly expressed in bladder cancer cells with a high degree of malignancy, and enrichment of TRPM7 is around the nuclear membrane.

To investigate the role of TRPM7 in bladder cancer, TRPM7 was knocked down using RNAi method. T24 cells were transfected with three siRNAs targeting TRPM7. As illustrated in Fig. 4, the mRNA (Fig. 4A) and protein (Fig. 4B) expression levels of TRPM7 were both significantly inhibited by TRPM7_siR1.

We transfected the 5637 and T24 cells with TRPM7_siR1 and NC_siR. Compared with NC_siR and control groups, flow cytometric analysis revealed that downregulation of TRPM7 significantly induced apoptosis (Fig. 5A). Statistical analysis of apoptosis is shown in Fig. 5B. CCK-8 assay was carried out to assess the effects of TRPM7 knockdown on T24 and 5637 cell proliferation. The CCK-8 assay showed that after downregulation of TRPM7 with TRPM7_siR1, T24 and 5637 cells exhibited a significant decrease in cell proliferation compared with that noted in the control siRNA group (Fig. 5C). Flow cytometric cell cycle analysis revealed that 5637 and T24 cells were blocked in the G2/M phase after transfection with TRPM7_siR1 (Fig. 6A and B).

We next investigated the effect of TRPM7 on cell motility with wound-healing and Transwell assays. After being incubated with physical-wound and cultured in serum-free medium to exclude the interference of proliferation, the percentage of wound closure at 48 h was significantly lower in the TRPM7_siR1-treated cells than that noted in the control cells (Fig. 7A and B). Transwell assays showed that the silencing of TRPM7 measurably inhibited cell migration and invasion in the Transwell assays (Fig. 7C and D). All the data support that TRPM7 stimulates bladder cancer cell migration and invasion.