J82 human bladder cancer cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS; Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at 37°C in a cell culture incubator with 5% CO₂.

Invitrogen BLOCK-iT RNAi Designer (rnaidesigner.lifetechnologies.com/rnaiexpress) was used for obtaining the following target sequence from AQP1 gene: 5′-GCTGTACTCATCTACGACTTC-3′ (724–744). The pSuper RNAi System manual was followed for short hairpin (sh)RNA design (22). The primers designed are as follows: Sh-AQP1 forward, 5′-GATCCCCGCTGTACTCATCTACGACTTCTTCAAGAGAGAAGTCGTAGATGAGTACAGCTTTTTA-3′; and reverse, 5′-AGCTTAAAAAGCTGTACTCATCTACGACTTCTCTCTTGAAGAAGTCGTAGATGAGTACAGCGGG-3′; Sh-AQP1 negative control forward, 5′-GATCCCCGCCAGCTTAGCACTGACTCTTCAAGAGAGAGTCAGTGCTAAGCTGGCTTTTTA-3′; and reverse, 5′-AGCTTAAAAAGCCAGCTTAGCACTGACTCTCTCTTGAAGAGTCAGTGCTAAGCTGGCGGG-3′.

Primers were synthesized at GenePharma Co., Ltd (Shanghai, China), dissolved and diluted to 1 µg/µl. Reverse primer (5 µl) and forward primer (5 µl) were mixed and annealed at 95°C for 4 min, 70°C for 10 min and then allowed to cool to 4°C. The resulting double stranded DNA and the plasmid pSUPER-retro-puro (Cell BioLabs, Inc., San Diego, CA, USA) were then double digested by Bgl II and Hind III restriction enzymes at 37°C overnight, ligated together and transferred into E.coli DH5α. Colony PCR was performed to identify the colonies harboring the recombinant plasmid. The reaction system included: 23 µl double distilled H₂O, 1 µl reverse primer, 1 µl forward prime rand 25 µl Taqmix. Pre-denaturation occurred at 95°C for 6 min, denaturation occurred at 95°C for 20 sec, annealing occurred at 55°C for 30 sec and extension occurred at 72°C for 30 sec, for 30 cycles. Primers were then purified, sequenced at GenePharma Co., Ltd and used to transfect 293FT cells. Prior to transfection, log phase 293FT cells were collected and seeded (2×10⁵ cells) to 6 well plates. After 24 h incubation at 37°C, cells were transfected with liposome Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, cells were harvested and interference analysis was performed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The resulting recombinant plasmid was designated pSUPER-retro-puro-shAQP1 and was transferred to 293FT cells using calcium phosphate co-precipitation. At 48 h post-transfection, the culture supernatant containing recombinant virus was collected and the virus titrations were determined using multiple proportion dilutions. J82 cells were then seeded to T25 cell culture flask. Polybrene was added to the culture supernatant collected above to a final concentration of 4 µg/µl. The culture media for J82 cells were replaced by 5 ml culture supernatant collected above and the cells were cultured in a 37°C incubator for 24 h. The transfection was repeated 3 times at 3 h intervals followed by replacement of the current media with DMEM supplemented with 10% FCS. After 48 h, 0.5 µg/ml puromycin was added to the culture for screening of positive clones and culture amplification. This was to establish a J82 cell line (designated as J82-shAQP1) whose expression of AQP1 gene is inhibited. Another J82 cell line transfected with the empty plasmid pSUPER-retro-puro was used as the control (designated as J82-ctrl).

The transcription of AQP1 gene was analyzed using RT-qPCR analysis and quantified using the 2^−ΔΔCq method (23). RT-qPCR was performed according to the manufacturer's protocol (Bio-Rad Laboratories, Inc., Hercules USA). Total RNA from J82 cells was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). GAPDH was used as an internal control. RT was performed using PrimeScript RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China). CFX96 Real Time PCR detection system (Bio-Rad Laboratories, Inc.) with SYBRGreen ІІ (Bio-Rad Laboratories, Inc.) was used for qPCR. Primers used were as follows: AQP-1 forward, 5′-ACCCGCAACTTCTCAAAC-3; and reverse, 5′-AGGCCAAGCCTCCTCTAT-3′; GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′, and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. Thermocycling conditions were as follows: Pre-denaturation at 94°C for 1 min, denaturation at 94°C for 20 sec, annealing at 55°C for 20 sec, extension at 72°C for 20 sec, for 40 cycles.

Cell viability was assessed using an MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). J82-shAQP1 cells and J82-ctrl cells (2×10³ cells/plate) were seeded into 96 well plates and incubated for 24 h in culture media with or without MMC (1 mg/ml). A total of 200 µl MTT (5 mg/ml) was then added to each well and cells were incubated for a further 4 hat 37°C, and 150 µl dimethyl sulfoxide was added to each well and incubated at 37°C for 5 min. A Versamax microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) was used for reading optical density at 490 nm.

Apoptosis was measured using flow cytometry with propidium iodide (PI) and the annexin V-fluorescein isothiocyanate (FITC) staining kit (BD Biosciences, Franklin Lakes, NJ, USA). J82-shAQP1 and J82-ctrl cells (2×10³ cells/plate) were seeded onto 96 well plates and incubated at 37°C for 24 h in DMEM with or without 1 mg/ml MMC. Then, the cells were detached and rinsed with DMEM and cold PBS twice. Cells were then centrifuged at 300 × g for 5 min and resuspended in 1× binding buffer at 1×10⁶ cells/ml. In total, 100 µl of the cell buffer solution (1×10⁵ cells) was transferred to a round bottom tube and incubated with 5 µl of PI and annexin V-FITC for 15 min in the dark. Finally, 400 µl of 1× binding buffer was added to each sample tube and analyzed using a BD FACSCalibur cytometer (BD Biosciences) according to the manufacturers protocol. Data were analyzed using FlowJo software (version 7.2.5; FlowJo LLC, Ashland, OR, USA).

For Western blot analysis, J82-shAQP1 and J82-ctrl cells (2×10³ cells/plate) were seeded into 96 well plates and incubated at 37°C for 24 h in DMEM with or without 1 mg/ml MMC. Total cellular proteins were extracted in EBC lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.5% NP40; Sigma-Aldrich; Merck KGaA) containing a proteinase inhibitor cocktail (100 µg/ml lysozyme, 10 µg/ml aprotinin and 10 µg/ml lepupetin; Sigma-Aldrich; Merck KGaA), as described previously (24). Protein concentrations were measured using Bradford protein assay. A total of 30 µg protein samples were then subjected to SDS-PAGE (12% gel). Following electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, where they were subsequently blocked by TBST and 5% non-fat dry milk at 4°C for 2 h, followed by incubation with primary antibodies at 4°C for 12 h. The primary antibodies used were: Anti-AQP-1 (1:100; cat. no. BM5035, Wuhan Boster Biological Technology, Ltd., Wuhan, China), anti-proliferating cell nuclear antigen (PCNA; 1:5,000; cat. no. sc7907; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-Bcl-2 associated X protein (Bax; 1:2,000; cat. no. ab32503; Abcam, Cambridge UK), anti-B cell lymphoma 2 (Bcl-2; 1:500; cat. no. ab692; Abcam), anti-caspase-3 (1:5,000; cat. no. sc-98758; Santa Cruz Biotechnology, Inc.) and anti-β-actin (1:1,000; cat. no. sc-130656; Santa Cruz Biotechnology, Inc.). Following incubation with corresponding primary antibody at 4°C overnight, the membranes were washed and incubated with either horseradish peroxidase-conjugated-goat anti-rabbit immunoglobulin G secondary antibody (1:2,000; cat. no. ab6721; Abcam) for 1 h at 4°C and developed with electrochemiluminescence (ECL) Western Blotting Substrate (BD Biosciences). Data was analyzed using Quantity One 1D image analysis software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Each experiment was performed ≥3 times and analyzed using SPSS (v19; IBM Corp., Armonk, NY, USA) was used for statistical analysis of the data collected. A two-tailed Student's t-test was adopted for comparison of the expressions of AQP-1 mRNA between J82-ctrl and J82-shAQP1 cells, whereas comparisons among groups were assessed using one-way analysis of variance with a Student-Newman-Keuls post-hoc test. Data were presented as the mean ± standard deviation. P<0.05 was considered to indicate as statistically significant difference.

The expression of AQP-1 mRNA was detected by RT-qPCR. As presented in Fig. 1, the expression of AQP-1 mRNA in J82-shAQP1 cells decreased significantly and was only 36% of that in J82-ctrl cells. In addition, the expression of GAPDH mRNA in the 2 cell types remained consistent. GAPDH is a commonly used loading control for RT-PCR as the GAPDH gene is a housekeeping gene often stably and constitutively expressed in the majority of tissues and cells.

As presented in Table I, there was a significant decrease in cell viability for J82-ctrl cells following treatment with MMC (P<0.05). The cell viability was also significantly decreased in J82-shAQP1 cells compared with that in J82-ctrl cells (P<0.05). Treatment of J82-shAQP1 cells with MMC further decreased cell viability, which was significantly decreased compared with that in J82-ctrl cells treated by MMC (P<0.01). A similar association was also observed in cell apoptosis. As presented in Fig. 2, when cells were treated with MMC, J82-shAQP1 cells exhibited a significantly increased level of cell apoptosis compared with J82-ctrl cells (P<0.05).

As presented in Fig. 3, significantly decreased expression of AQP-1 was observed in J82-shAQP1 cells compared with J82-ctrl cells (P<0.05). Treatment of J82 cells with MMC also significantly decreased AQP-1 expression (P<0.05). Additionally, AQP-1 expression was decreased in J82-shAQP1 cells treated with MMC compared with J82-ctrl cells treated by MMC and was decreased more as compared with J82 cells without MMC treatment.

To further assess how MMC treatment and inhibition of AQP-1 expression affected J82 cell viability and apoptosis, the expressions of PCNA, Bcl-2, Bax and Caspase-3 in J82-shAQP1 cells and J82-ctrl cells were measured. PCNA, Bcl-2, Bax and Caspase-3 are cell proliferation- and apoptosis-associated proteins. As presented in Fig. 3, PCNA was most highly expressed in J82-ctrl cells and was decreased significantly by MMC treatment. Significantly decreased expression of PCNA was also observed in J82-shAQP1 cells compared with J82 cells and was further decreased by MMC treatment (P<0.05). Significantly decreased expression of PCNA was observed between J82-shAQP1 cells treated with MMC (P<0.01) and J82-ctrl cells treated with MMC (P<0.05), compared with the untreated control.

In contrast with PCNA expression, an opposite expression pattern was observed for caspase-3. As presented in Fig. 3, the expression of caspase-3 in J82-shAQP1 cells was significantly increased compared with that in J82-ctrl cells (P<0.05), suggesting recombinant virus transfection of J82 cells increased caspase-3 expression. Treatment of J82 cells with MMC also increased caspase-3 expression. Of all the conditions tested, the highest expression level of caspase-3 was observed in J82-shAQP1 cells treated with MMC.

In addition, the Bcl-2/Bax ratio was also measured. As presented in Fig. 3, the highest Bcl-2/Bax ratio was observed in J82 cells and was significantly decreased following MMC treatment and recombinant virus transfection (P<0.05). The Bcl-2/Bax ratio in J82-shAQP1 cells was further decreased by MMC treatment. A significantly decreased Bcl-2/Bax ratio was observed in J82-shAQP1 cells treated with MMC compared with that in J82 cells treated with MMC (P<0.05).