3p21.3 tumor suppressor gene RBM5 inhibits growth of human prostate cancer PC-3 cells through apoptosis

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%.

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.

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 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.

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.

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.

We first analyzed the expression of RBM5 by IHC staining in a collection of prostate cancer and normal tissues in a Chinese cohort. As shown in Figure 1, the RBM5 protein was virtually undetectable in prostate cancer but highly expressed in normal prostatic tissue. The cellular localization of RBM5 is mostly cytoplasmic. Overall, RBM5 was expressed in 10 of 11 (90.9%) normal prostate tissues, and the cytoplasmic/nuclear ratio of the staining was about 50%; while the RBM5 was expressed in two of 12 prostate cancer specimens (16.7%), and the cytoplasmic/nuclear ratio of the staining was about 10%. The difference is statistically significant (P <0.01). These results indicated that RBM5 is less-expressed in clinical prostate cancers, suggesting RBM5 is a promising target for prostate cancer therapy.

As a eukaryotic expression, pcDNA3.1 can transfer the ectopic gene into cells, and express the interest protein in the target cells. In this study, we employed PC-3 cells to investigate the function of RBM5 in prostate cancer cells.

After transfection with pcDNA3.1 vector and pcDNA3.1-RBM5 vector, semi-quantitative RT-PCR and western blot were performed to analyze the expression of RBM5 mRNA and protein. As shown in Figure 2A and 2B, the expression of RBM5 mRNA increased significantly in transfected pcDNA3.1-RBM5 cells compared with transfected pcDNA3.1 vector and mock control cells (treated with lipofectamine 2000 only) (P <0.01). Western blot assay showed a similar and statistically significant increase (P <0.01) (Figure 2C and 2D). The result demonstrated that RBM5 was effectively overexpressed in the pcDNA3.1-RBM5 transfected cells (RBM5), compared with the cells transfected with empty vector pcDNA3.1 (EV).

To explore the effect of RBM5 overexpression on cell growth, MTT assays were performed at 24 h, 48h, and 72 h, respectively, after the transfection. Results showed that there was a significant inhibition of cell proliferation in RBM5 overexpressing PC-3 cells compared with the mock control and EV groups (Figure 3A). Along with the extension of the time, the inhibition rate was increased, and at 72 h, the inhibition rate had been more than 30% (data not shown).

To further explore the effect of RBM5 overexpression on proliferation of PC-3 cells, Immunocytochemistry staining was employed to detect the expression of PCNA after transfected. As shown in Figure 3B, the staining results showed that the lowest numbers of proliferating cells (PCNA positive) were found in pcDNA3.1-RBM5 transfected group. Quantitative analyses of the stained slides were performed and the results are summarized in Figure 3C. The proliferation index was defined as percent of tumor cells stained positively for PCNA. In RBM5 group, the proliferation index was significantly lower (P <0.01), when compared with the control groups.

To determine the contribution of inhibition of cell growth induced by RBM5 overexpression, we employed Rhodamine 123 staining and flow cytometry analysis to determine apoptotic activity in PC-3 cells. Rhodamine 123 can easily be absorbed by mitochondrial membrane, the fluorescence intensity depends on mitochondrial membrane potential, can be used in analysis of apoptosis. As showed in Figure 4B, the fluorescence intensity was significantly decreased in the PC-3 cells treated with pcDNA3.1-RBM5 compared with the control groups, which suggested the dysfunction of mitochondrial membrane potential.

Flow cytometry analysis was used to detect apoptotic cells which are characterized with phosphatidyl serine (PS) translocation on the outer cell membrane. Cells were double-stained with Annexin V and PI after 48 h transfection. The early and the late apoptotic cells were distributed in the Q2 and Q3 regions, respectively. The necrotic cells were located in the Q4 region. Representative raw FCM data are as follows: cells transfected with RBM5 showed a higher proportion of early and late apoptosis (11.16%±2.53% and 1.07%±0.18%) as compared to the control cells (3.49%±1.17% and 0.81%±0.13%) and EV cells (3.55%±0.62 and 0.92%±0.15%), respectively (Figure 4C). The result suggested that overexpression of RBM5 significant increased apoptosis in PC-3 cells.

To investigate potential molecular mechanisms underlying RBM5-induced cell apoptosis, we next examined the expression of some apoptosis-related genes including P53, Bid, Bim, Bad, p-Bad, cleaved-caspase3, and cleaved-caspase9. We observed that the expression of P53, Bim, Bid, p-Bad, cleaved-caspase3, and cleaved-caspase9 proteins was increased significantly when RBM5 was overexpressed compared to that in the control cells (Figure 5). RBM5 might play an import role in the mitochondrial apoptotic pathway.