The present study included 40 patients (aged 49–82 years) diagnosed with localized (n=23) or metastatic (n=17) prostate carcinomas between January 2005 and August 2009 at our institution (Department of Urology at the Second Hospital of Lanzhou University, China). None of the patients had received treatment at the time of sampling. Detailed clinical data regarding these patients are provided in Table I. Tumors were staged using the updated staging system by the American Joint Committee on Cancer (AJCC) and the Union Internationale Contre le Cancer (UICC) (2009 UICC) on cancer TNM classification and graded by Gleason scores. Tumors with a Gleason score of ≥7, distant-stage diseases (including T3, T4, N1 or M1) or pretreatment prostate-specific antigen (PSA) score of ≥20 ng/ml were defined as aggressive PCa, while all others were classified as non-aggressive. Twenty hyperplasia prostate patients served as controls. The Ethics Committee of the Lanzhou University approved the present study. Prior to the study, written informed consent was obtained from all individuals.

The human prostate adenocarcinoma cancer cell lines (PC3, DU145 and LNCaP) and the normal prostate epithelial cell line RWPE2 were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO₂.

For the immunohistochemical staining, 4-μm paraffin sections of the tissue samples were autoclaved in citrate buffer at 121°C for 15 min for antigen retrieval, and then incubated with the anti-RANK, -OPG or -RANKL antibody (Santa Cruz) (1:50 dilution in signal stain antibody diluent) at 4°C overnight. Sections were incubated with the biotinylated secondary antibody and HPR-streptavidin for 30 min at room temperature, respectively, followed by a 3,3′-diaminobenzidine plus reaction (Zhongshan Golden Bridge Biotechnology, Co.). Finally, the sections were counterstained with hematoxylin. Appropriate positive controls (human breast cancer) and negative controls (human normal gastric mucosa) were run concurrently. As another negative control, sections processed in an identical fashion except for the replacement of the primary antibody with normal rabbit serum were also included. Immunoreactive cells were scored as follows: 0, positive cell staining ≤1%; 1, positive cell staining >1–25%; 2, positive cell staining >25–50%; 3, positive cell staining >50–75%; 4, positive cell staining >75% (Fig. 1). Low- and high-intensity signals were scored based on the staining intensity (negative=0; weak=1; moderate=2; and strong=3). The immunoreactive score was calculated by multiplying the percentage of immunoreactive cells by the staining intensity. Scores were considered negative (0–1) and positive (≥1).

In vitro Matrigel invasion assays were performed in Transwell permeable supports with porous filters (8-μm pore size) according to the manufacturer’s instructions (Corning, USA). Briefly, the upper surface of the filter was coated with 500 ng/ml Matrigel (Sigma). Cells with or without siRNA of RANK suspended in the medium were added onto the upper surface of the Transwell (2×10⁵). RANKL and/or OPG (R&D Systems) were added to the lower chamber at final concentrations between 0 and 50 μg/ml. After a 24-h incubation in medium containing chemokines, the filter was removal from the chamber. Cells that has passed through the Matrigel and attached to the lower surface of the filter were fixed with methanol and stained with H&E. Cells in 15 randomly selected microscopic fields (x200) per filter were counted.

Total RNA extracted using TRIzol reagent (Invitrogen) from the PCa cell lines was reverse transcribed to cDNA using the Omniscript RT kit with the oligo(dT) primer (Qiagen, USA). Subsequently, PCR reactions were performed using a PCR kit (Bio-Rad, USA) as follows: denaturation at 95°C for 10 min, 30 cycles consisting of 95°C for 30 sec and 68°C for 1 min and an extra incubation at 68°C for 1 min. The following primers were used to amplify RANK and β-actin: RANK forward, 5′-CAG GGA TCG ATC GGT ACA GT-3′ and reverse, 3′-GTT TGA GAC CAG GCT GGG TA-5′; β-actin forward, 5′-GTG GGG CGC CCC AGG CAC CA-3′ and reverse, 3′-CTC CTT AAT GTC ACG CAC GTA TTC-5′. The amplified PCR products were analyzed by electrophoresis on a 2.5% agarose gel containing ethidium bromide.

To detect RANK protein expression in the PCa cells, equal amounts of protein (50 μg) obtained from the cell extracts were separated on 10% SDS-PAGE under non-reducing conditions. The separated proteins were elec-troblotted onto PVDF membranes and blocked for 1 h at room temperature with Tris-buffered saline containing 5% non-fat milk. The membranes were then probed with a 1:500 dilution of the purified primary antibodies at room temperature for 2 h, followed by incubation with secondary antibodies for 1 h at room temperature. Antibody-protein complexes were detected by the ECL-Plus chemiluminescent system according to the manufacturer’s instructions.

OPG and sRANKL levels in the prostate tissues and serum were measured using the ELISA kit (R&D Systems) according to the manufacturer’s instructions.

To deplete RANK expression in the PCa cells, we designed 3 different RANK siRNA (siRNA1, siRNA2 and siRNA3) duplexes with BLOCK-iT™ RNAi Designer software and synthesized them through a service provided by Invitrogen. PCa cells were transfected with those siRNAs using Lipofectamine 2000 following the manufacturer’s instruction. siRNA duplexes used in the present study had the following sequences: siRNA1 sense, (5′-3′) CCUGGACCAACUGUACCUU and antisense, (5′-3′) AAGGUACAGUUGGUCCAGG; siRNA2 sense, (5′-3′) GGAGAAGACAUUUCCAGAA and antisense, (5′-3′) UUCU GGAAAUGUCUUCUCC; siRNA3 sense, (5′-3′) GCAAAUC CACACCUCCUUU and antisense, (5′-3′) AAAGGAGGUGU GGAUUUGC; control sense, (5′-3′) UAGCGACUAAACACA UCAAUU and antisense, (5′-3′) UUAUCGCUGAUUUGUGU AGUU.

The data are presented as means ± SEM of the indicated number of experiment. Statistical analyses were performed by Student’s t-test and analysis of variance (ANOVA). Chi-square and Fisher exact tests were performed to determine the correlation between RANKL/RANK/OPG expression and clinicopathological parameters. Pearson’s test and line regression were used for correlation analysis. The value of P<0.05 was considered to indicate a statistically significant result.

The morphometric analysis of the immunohistochemical staining (Fig. 1) showed that RANK, OPG and RANKL were not significantly expressed in hyperplasia prostate, while their expression levels were increased to 50, 45 and 52.5%, respectively in the PCa tissues. As shown in Table I, there was a positive correlation between the expression of RANK, RANKL and OPG and the aggressiveness of tumors, based on Gleason score (≥7), distant-stage disease or pretreatment PSA score (≥20 ng/ml). The expression frequency of RANK, RANKL and OPG in the metastatic PCa cases was significantly higher than that in the localized and locally advanced cases. We also observed that the expression of RANK, OPG and RANKL in the tissues of PCa patients with bone metastasis was more intense than that in the patients with lymph node metastasis, suggesting a potential selective involvement of these molecules in bone metastasis.

RANKL exists in both sRANKL and membrane-bound forms. OPG exists only in soluble form. In order to determine if sRANKL and OPG have any effect on prostate tumor metastasis, we next quantified the amounts of sRANKL and OPG by ELISA in the human PCa tissue samples. As shown in Table II, OPG and sRANKL levels in the prostate tumor tissues were ~10-fold of the levels in the BPHs (P<0.01). The levels of sRANKL and OPG in aggressive and metastatic tumors were increased 2.01- and 1.8-fold compared with levels in the non-aggressive and localized/locally advanced tissues, respectively. Consistent with the immunohistochemical studies, ELISA assays detected higher levels of OPG and sRANKL in the tissues of PCa patients with bone metastasis than the levels in patients with lymph node metastasis (Table II).

To further assess the role of sRANKL and OPG in prostate tumor metastasis, we detected sRANKL and OPG levels in serum by ELISA. As shown in Table III, serum sRANKL levels in PCa patients were the same as that in the BPH cases. However, the serum levels of OPG showed a 10-fold increase in PCa over BPH. A significant increase in OPG levels was also observed in aggressive PCa samples and those of bone metastasis.

As shown in Fig. 2, a positive correlation was observed between serum and tissue OPG level. However, the same analyses did not reveal a correlation between serum and tissue RANKL.

RANK is the only receptor of RANKL and the central player of the RANKL/RANKL/OPG signaling network. Since we found that the levels of sRANKL and OPG in prostate tumor tissues were significantly correlated with malignancy, we investigated the RANK expression in different human prostate carcinoma cell lines. Our RT-PCR and western blot analyses detected RANK expression at both the mRNA and protein levels in all 3 human prostate carcinoma cell lines: LNCaP, DU145 and PC3. In contrast, low levels of RANK RNA and protein were detected in the normal prostate epithelial RWPE2 cell line (Fig. 3). The data are consistent with the tumor sample analyses, suggesting that increased RANK signaling may play an important role in PCa development and progression.

To investigate the effect of sRANKL and OPG on PCa invasion, PCa cells which express RANK were seeded on Matrigel-coated Transwell and treated with different concentrations of sRANKL with or without OPG (2.5, 10 or 50 μg/ ml, respectively) for 24 h. The concentrations were chosen according to our preliminary study in which we found that 2.5 μg/ml sRANKL increased PCa invasion (data not shown). As shown in Fig. 4, treatment with 2.5 μg/ml RANKL significantly increased the number of invaded cells when compared to the PBS group (P<0.01). This was particularly true in the PC3 cells. Furthermore, the RANKL effect was suppressed by the decoy receptor OPG with a maximum effect observed at 10 μg/ml. In contrast, this phenomenon was not detected in the normal prostate epithelial RWPE2 cells (Fig. 4).

RANK plays important roles in tumor invasion and metastasis. To evaluate the effect of direct knockdown of RANK expression on RANKL-induced PCa cell invasion, we examined cell invasion after transfection of RANK siRNA in DU-145, PC-3 and LnCaP cells. We found that siRNA2 caused an 80% decrease in RANK protein expression as compared to siRNA1 and siRNA3 (Fig. 5). Consistently, RANK-siRNA-2 decreased cell invasion induced by RANKL by 60, 34 and 52%, respectively, in the PC-3, DU-145 and LNCaP cells (Fig. 5B).