Cell culture and reagents
Human bladder cancer 5637 and T24 cell lines were obtained from the Chinese Academy of Science (Shanghai, China) and maintained in Roswell Park Memorial Institute medium-1640 (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin in a humidified 5% CO2 environment at 37 °C. Cycloheximide, the SHH signaling activator smoothened (SMO) agonist (SAG), the SMO antagonist GDC0449, and the Nedd8-activating enzyme inhibitor MLN4924 were purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (Sigma-Aldrich), and stored at -20 °C as stock solutions. G418 was also from Sigma-Aldrich. The recombinant plasmid carrying hemagglutinin (HA)-tagged ubiquitin cDNA was purchased from Invitrogen (Shanghai, China).
Establishment of stable ROC1-overexpressed or -silenced bladder cancer cell lines
To establish a stable ROC1-overexpressed bladder cancer cell subline, we subcloned the full-length wild-type human ROC1 cDNA into the pcDNA3.1 vector (Invitrogen, Shanghai, China), named as pcDNA3.1-ROC1. After DNA sequence confirmation, this recombinant plasmid or pcDNA3.1 vector-only plasmid was transfected into bladder cancer cells using Lipofectamine 2000 (Invitrogen) for 48 h, and the cells were then cultured in G418-selecting cell culture medium at 100 μg/mL for 14 days. After that, individual G418-resistant monoclonal cells were selected and expanded in the 100 μg/mL G418-selective medium. The stable cell sublines were named as p-ROC1 or p-CONT. Furthermore, to knock down ROC1 or suppressor of fused homolog (SUFU) expression, we purchased siRNA oligonucleotides targeting ROC1 or SUFU from Invitrogen (Shanghai, China) and transfected them into bladder cancer cells, according to the manufacturer’s instructions. The ROC1 siRNA sequence was 5ʹ-GACTTTCCCTGCTGTTACCTAA-3ʹ; the SUFU siRNA sequence was 5ʹ-GCCATGACAATCGGAAATCTA-3ʹ; and the scrambled control siRNA sequence was 5ʹ-ACGTGACACGTTCGGAGAA-3ʹ.
Cell viability and colony formation assays
Changed cell viability was assessed by using the Cell Counting Kit-8 kit (Beyotime, China), as previously described [13]. For the colony formation assay, tumor cells were seeded in triplicate into 35-mm culture dishes at a density of 400 cells (for 5637 tumor cells) or 1000 cells (for T24 cells) per well and cultured for 9 days. The cells were fixed and stained with crystal violet in 50% methanol, and the number of cell colonies with more than 50 cells was counted.
Flow cytometry cell cycle distribution assay
Both ROC1-overexpressed and siRNA-transfected bladder cancer cells were detached from the cell culture dishes and fixed in ice-cold 70% ethanol overnight. One day later, the cells were washed twice with ice-cold phosphate-buffered saline (PBS), then stained with propidium iodide (Sigma-Aldrich) solution (20 mg/mL) for 5 min, and finally analyzed by using a BD FACScan flow cytometer (BD Biosciences, San Diego, CA, USA).
Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA was isolated with the Trizol reagent (Invitrogen) and reversely transcribed into cDNA with a PrimeScript Reverse Transcription kit (Takara, China), according to the manufacturers’ protocols. The resultant cDNA samples were then amplified using a 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with the SYBR Green master mix kit (Takara, Dalian, China) for detection of different genes using gene-specific primers (the detailed DNA sequences of each primer used in this study are available upon request). All measurements were performed in triplicate and quantified using the 2-∆∆Ct method.
Western blot and co-immunoprecipitation-Western blot
After the cells were subjected gene transfection or drug treatments, cell lysates were prepared and quantified according to a previous study [14]. The western blot was carried out as described previously [13], while the co-immunoprecipitation kit (Cat. #26419) from Thermo Scientific (Waltham, MA, USA) was used according to the manufacturer’s instructions with the following antibodies: anti-ROC1 (Abcam, Cambridge, MA, USA); anti-cyclin D1, anti-Cdc25c, anti-SUFU, anti-Gli1, anti-GAPDH, and anti-HA (Abcam, Hangzhou, China); and anti-Gli2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Immunofluorescence staining
Immunofluorescence staining was performed to assess Gli2 expression in cells, as described previously [13]. Briefly, cells were grown on coverslips, fixed, and permeabilized, and then they were incubated with a primary antibody against Gli2 (Santa Cruz Biotechnology) followed by incubation with the Alexa 548-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA). Subsequently, the cells were counterstained by using 4,6-diamidino-2-phenylindole (Sigma) and analyzed under a Zeiss LSM500 confocal microscope (Zeiss International, Oberkochen, Germany).
In vivo tumor cell xenograft assay
An orthotopic tumor model of bladder cancer was used. In particular, tumor cells were cultured to reach 70–80% confluency, harvested, resuspended in PBS, and then mixed with Matrigel (Invitrogen) at a 1:1 vol/vol ratio. Next, mice (6-week-old, male, athymic, BALB/C nu/nu; n = 10 per group) were anesthetized by using 40 mg/kg sodium pentobarbital, and a small lower abdominal incision was made to expose the bladder for tumor cell injection. Tumor cells were then injected into the bladder wall using a 28-gauge needle; thereafter, the injection site was pressed with a cotton swab for 30 s, and the skin incision was then closed with the absorbable line. Tumor cell xenograft formation and growth were assessed by using the whole-body fluorescence imaging system weekly, with a Spectrum in vivo imaging system (Promega, Madison, WI, USA) with 470-nm excitation from an MT-20 light source. The emitted fluorescence signal was collected by using appropriate filters on a DP70 CCD camera and processed for contrast and brightness with Paint Shop Pro 8 (Corel, Ottawa, ON, Canada). Twelve weeks after the nude mice were inoculated with the pROC1 or pCONT tumor cells, the mice were sacrificed, and the xenograft tissues were resected. This study protocol was approved by the Animal Care and Use Committee of Yancheng First People’s Hospital (Jiangsu, China) and carried out following the Guidelines for the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research.
Human bladder tissue samples and immunohistochemistry
Bladder cancer tissue specimens were retrospectively collected from 93 bladder cancer patients who were cared for at Yancheng First People’s Hospital (Jiangsu, China) between January 2010 and May 2015. The patients included 79 males and 14 females with a median age of 67 years old (range: 45–87 years old); 43 of the patients underwent a transurethral resection, 12 underwent a partial cystectomy, and 38 underwent a radical cystectomy. Their tumor grade and stage were classified according to the World Health Organization 1973 criteria and the American Joint Committee on Cancer 2002 Tumor, Node, Metastasis system. This study of human subjects was approved by the Medical Ethics Committee of Yancheng First People’s Hospital (Permit Number: 2013KY004), and informed consent was obtained from each patient before enrolling into this study.
Paraffin-embedded tissue blocks were retrieved from the Pathology Department and used for the preparation of the tissue microarray and then immunostained with a primary antibody against ROC1 (Abcam), SUFU (Abcam), Ki67 (Boster, Wuhan, China), or Gli2 (Boster), according to our previous study [13].
Statistical analysis
The western blot band intensities were quantified by using Image J software (National Institutes of Health, Bethesda, MD, USA). The data were expressed as means ± standard error of the mean (SEM) and statistically analyzed by using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). For multi-group comparisons, the Bonferroni t-test was used after one-way analysis of variance. Meanwhile, for two-group comparisons, the Student’s t-test was used. The correlation between ROC1 or SUFU and Gli2 expression was assessed by using Pearson’s χ2 test. A p value < 0.05 was considered statistically significant.