Cell culture and reagents
Human bladder cancer 5637 and T24 cell lines were obtained from Chinese Academy of Science (Shanghai, China) and maintained in Roswell Park Memorial Institute medium–1640 (RPMI–1640; Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and1% penicillin-streptomycin in a humidified 5% CO2 environment at 37°C. Cycloheximide (CHX), the SHH signaling activator SAG (an SMO agonist), the SMO antagonist GDC0449, and the NAE (Nedd8-activating enzyme) inhibitor MLN4924 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at –20°C as stock solutions. G418 was also from Sigma-Aldrich. The recombinant plasmid carrying HA-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 sublines, we subcloned the full-length wild-type human ROC1 cDNA into the pcDNA3.1 vector (Invitrogen, Shanghai, China), named it 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 of the G418-resistant monoclonal cells were selected and expanded under the 100 μg/ml G418-selective medium. The stable cell sublines were named as p-ROC1 or p-CONT. Furthermore, to knockdown ROC1 or 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. ROC1 siRNA sequences were 5’-GACTTTCCCTGCTGTTACCTAA–3’; SUFU siRNA sequences were 5’-GCCATGACAATCGGAAATCTA–3’; and scrambled control siRNA sequences were 5’-ACGTGACACGTTCGGAGAA–3’.
Cell viability and colonogenic assays
Changed cell viability was assessed by using the Cell Counting Kit–8 kit (Beyotime, China) and carried out as described previously [13]. For the colonogenic 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 cell culture dishes and fixed in ice-cold 70% ethanol overnight. One day after, cells were washed twice with ice-cold phosphate buffered saline (PBS) and then stained with propidium iodide (PI; Sigma-Aldrich) solution (20 mg/ml) for 5 min and then analyzed by using a BD FACScan flow cytometer (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 resulted cDNA samples were then amplified in the 7300 Real Time PCR system (Applied Biosystems, Foster City, CA) 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 blotting and co-immunoprecipitation
After 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 (Co-IP) Kit (Cat. #26419) from Thermo Scientific (Waltham, MA, USA) was used according to the manufacturer’s instructions with the following antibodies, i.e., an anti-ROC1 (Abcam, Cambridge, MA, USA), anti-cyclinD1, anti- Cdc25c, anti-SUFU, anti-Gli1, anti-GAPDH, anti-HA (all from Abcam, Hangzhou, China), or anti-Gli2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody.
Immunofluorescence staining
Immunofluorescence staining was used to assess Gli2 expression in cells as described previously [13]. Briefly, cells were grown on coverslips, fixed, and permeabilized, and then 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 (DAPI; 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 particularly, tumor cells were cultured to reach 70% to 80% confluent, harvested and resuspended in PBS, then mixed with Matrigel (Invitrogen) at 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 of 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 xenograftformation and growth were assessed by using the whole-body fluorescence imaging system weekly, with an IVIS Spectrum 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). Three months after nude mice were inoculated with the pROC1 or pCONT tumor cells, the mice were sacrificed and xenografts 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 of 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 in Yancheng First People’s Hospital (Jiangsu, China) between January 2010 and May 2015. The patients were 79 males and 14 females with median age of 67 years (range between 45 to 87 years old), while 43 of them 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 (WHO) 1973 criteria and the American Joint Committee on Cancer (AJCC) 2002 TNM system. This study of human subjects was approved by the Medical Ethics Committee of Yancheng First People’s Hospital with a Permit Number of 2013KY004 and informed consent was obtained from each patient before enrolling into this study.
Paraffin embedded tissue blocks were retrieved from Pathology Department and used for preparation of 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 the Image J software (National Institute of Heath, 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 the Bonferroni t-test after one-way analysis of variance (ANOVA) for multi-group comparison, while two-group comparison was analyzed by using Student’s t-tests and the correlation between ROC1 or SUFU and Gli2 expression was assessed by using the Pearson’s χ2 test. A p < 0.05 was considered statistically significant.