Prostate tissue acquisition and immunohistochemistry staining (IHC)
Prostate cancer specimens were obtained from patients undergoing prostatectomy in the Second and the Third Affiliated Hospitals of Jilin University, the Gleason scores for which were from grade 7 to grade 8. Normal prostate tissues were obtained from patients undergoing surgery for benign prostatic hyperplasia (BPH) in these hospitals. This study was approved by the Research Ethics Committee of Norman Bethune College of Medicine, Jilin University and was in compliance with the Helsinki Declaration. The tissues were examined by the pathologists to confirm the diagnosis before IHC analyses. All specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4-μm-thick slides. The slides were dewaxed, and the endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide solution in methanol for 20 min. Non-specific binding was prevented by blocking with normal goat serum (1:10) for 10 min. The staining procedure was carried out using an avidin-biotin-peroxidase complex method. The presence of RBM5 was evaluated by staining with rabbit anti-RBM5 antibody (Abcam, Cambridge, MA, USA). After incubation with the primary antibody for 60 min, the slides were incubated with the biotinylated goat anti-rabbit IgG (H+L) (DAKO, Carpinteria, CA, USA) at 37°C for 30 min, followed by incubation with a 1:200 streptavidin-biotin-peroxidase complex (Sigma, St. Louis, MO, USA) for 30 min. Reactive products were visualized with 3,3′-diaminobenzidene (DAB) as the chromogen, and the slides were counterstained with hematoxylin. Sections previously known to express RBM5 were included in each run, receiving either the primary antibody as the positive control, or a mouse IgG as the negative control. The stained slides were analyzed with a microscope at 400× magnification. Cellular brownish staining was scored as positive and the threshold was set at 10%.
Cell culture and transfection
The human prostate cancer cell line PC-3 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and incubated at 37°C with humidified 5% CO2 in IMDM (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Hyclone, UT, USA), 100 unit/mL of penicillin, 100 μg/mL of streptomycin, and 2 mM of L-glutamine. 5 × 105 cells were plated onto 6-well plates 24 h before transfection. The pcDNA3.1-RBM5 and pcDNA3.1 plasmids were provided by Dr. Leslie C. Sutherland from Research Program, Northeast Cancer Centre, Health Sciences North in Canada. PC-3 cells were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with 4 mg DNA according to the manufacturer’s instructions. Transfection media were removed 6 h after transfection and replaced with fresh complete medium containing 10% fetal bovine serum (FBS). Controls included lipofectamine 2000 treated cells and empty vector pcDNA3.1 transfected cells.
Semi-quantitative reverse transcription PCR (RT-PCR) and western blot analysis
PC-3 cells were divided into three groups: (a) control (mock-transfected); (b) EV (transfected with empty vector pcDNA3.1); (c) RBM5 (transfected with pcDNA3.1-RBM5), and cells were harvested after 48 h following transfection and treatment. Overexpression of RBM5 mRNA and protein was confirmed by RT-PCR and western blot.
For semi-quantitative RT-PCR, total RNA was extracted from the cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was generated by reverse transcription of 1 μg RNA samples using a SuperScript pre-amplification system (Promega, Madison, MI, USA). One-tenth of the reverse-transcribed RNA was used in the PCR reaction. The primer sequences were as follows: GAPDH sense: 5′-GAAGGTGAAGGTCGGAGTC-3′ and antisense: 5′-GAAGATGGTGATGGGATTTC-3′; RBM5 sense: 5′-GCACGACTATAGGCATGACAT-3′ and antisense: 5′-AGTCAAACTTGTCTGCTCCA-3′. The PCR products were separated by electrophoresis on 1% agarose gels. The PCR products were visualized by a Tanon-1600 figure gel image processing system and analyzed with a GIS 1D gel image system software (Tanon, Shanghai, China).
For western blot analysis, cells were lysed with HEPES lysis buffer. The cell lysates were then centrifuged for 30 min at 12,000×g and the protein concentrations were determined using the Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Forty micrograms of protein were separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and probed with antibodies of anti-RBM5 (Abcam, Cambridge, MA, USA), anti-Caspase3 (Cell Signaling Technology, Boston, MA, USA), anti-Caspase9 (Cell Signaling Technology, Beverly, MA, USA), anti-Bid, anti-Bim, anti-Bad (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies (Thermo, Waltham, MA, USA) at a dilution of 1:2,000 were then applied for 1 h at room temperature. The protein bands were then detected using an Enhanced Chemiluminescece kit (Pierce Biotechnology Ltd., Rockford, IL, USA). The protein levels were quantified by densitometry using Quantity One software (Bio-Rad).
Cell viability assays
Cell viability was determined using 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The cells were plated onto 96-well plates at a concentration of 1×104 cells per well for 24 h and then transfected with pcDNA3.1 or pcDNA3.1-RBM5 for 24 h, 48 h, and 72 h. The cells were treated with 0.5 mg/mL MTT (Sigma-Aldrich, St. Louis, MO, USA) solution for 4 h. The medium was removed, and 100 mL of dimethylsulfoxide was added to each well. The formazan dye crystals were solubilized for 15 min and the optical density was measured at 570 nm with a Vmax Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability was calculated as a percentage of the control (untreated) values. Experiments were repeated three times with each experiment containing triplicate samples.
Rhodamine123 staining for mitochondria membrane potentials
Mitochondria membrane potentials were assessed by Rhodamine123 (Sigma-Aldrich, St. Louis, MO, USA) staining. After transfected by plasmid for 48 h, PC-3 cells were incubated with 2 μM Rhodamine123 at 37°C for 30 min, washed with phosphate-buffer solution, and examined using a fluorescence microscope (Olympus, Japan) to observe the mitochondria membrane potentials.
Flow cytometry analysis of apoptosis
After transfected by plasmid for 48 h, PC-3 cells were trypsinized and centrifuged, washed, and stained using the Annexin-V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China). The samples were then analyzed for apoptosis by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Annexin-V-FITC(−)/PI(−) was used to indicate cells that had survived, Annexin-V-FITC(+)/PI(−) was used to indicate cells that were in the early stage of apoptosis, and Annexin-V-FITC(+)/PI(+) was used to indicate cells in the late stages of apoptosis or necrosis.
Chi-square test was used to analyze the difference in RBM5 expression between normal and cancerous prostate tissues. Data were presented as mean ± standard deviation (SD) when appropriate. The regression analysis was performed with SPSS software (version 17.0). Comparisons between treatments were made using a paired Student’s t-test, or one-way ANOVA for multiple group comparisons. A P value of <0.05 was considered statistically significant.