The human PCa DU145 cells and RWPE-1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA); serum-free keratinocyte medium (K-SFM) was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA); the RPMI-1640 medium, fetal bovine serum (FBS) and polyvinylidene fluoride (PVDF) membrane were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Primary antibodies against connective tissue growth factor (CTGF, cat. no. sc-14939 were purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA, while, cysteine-rich angiogenic inducer 61 antibodies (CYR61, cat. no. 14476, 1:1,000), B-cell lymphoma 2 antibodies (Bcl-2, cat. no. 2872, 1:1,000), Bcl-2-associated X protein antibodies (Bax, cat. no. 2774, 1:1,000), caspase 3 antibodies (cat. no. 9662, 1:1,000), GAPDH antibodies (cat. no. 5174, 1:1,000) and secondary antibodies (cat. no. 7076, 1:1,000) were obtained from Cell Signaling Technology Inc., (Danvers, MA, USA); MTT and enhanced chemiluminescence (ECL) Plus reagent were obtained from Eli Lilly & Co., (Indianapolis, IN, USA); Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from Vazyme Biotech Co., (Nanjing, China). YAP-small interfering RNA (YAP-siRNA, cat. no. sc-38637), control (Con)-siRNA (cat. no. sc-37007) and the siRNA Transfection Reagent (cat. no. sc-29528) were obtained from Santa Cruz Biotechnology, Inc.

Human PCa DU145 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin. RWPE-1 cells were cultured in 10 ml complete SFM containing 5 ng/ml recombinant epidermal growth factor (CYT-217, ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA), 50 µg/ml bovine pituitary extract (CC023; macgene.bioon.com.cn/), 100 IU/ml penicillin and 100 IU/ml streptomycin. Cells were incubated in standard cell culture conditions (5% CO₂, 95% humidity) at 37°C. Human PCa DU145 cells were passaged every 2–3 days, and RWPE-1 cells were passaged every ~5 days.

Human PCa DU145 cells were plated in a 6-well plate 1 day prior to transfection, the transfection assay was performed once the cells reached 60–70% confluence. DU145 cells (2×10⁵ cells/well) were transfected with YAP-siRNA or Con-siRNA (1 µg) using 30 µl transfection reagent plus and incubated at 37°C for 6 h in 5% CO₂ incubator, according to the manufacturer's protocol. A total of 24 h following transfection, the transfected DU145 cells were used for subsequent experiments, and were collected for protein analysis after 72 h incubation.

Total RNA was extracted from human PCa DU145 and RWPE-1 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. To determine the mRNA expression levels within the cells, RT was performed using PrimeScript™ RT reagent kit (Takara Bio Inc., Otsu, Japan) according to the manufacture's protocol. For qPCR, SYBR^® Premix Ex Taq™ II (Takara Bio Inc.) was used and all reactions were performed in triplicate with the following conditions: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 72°C for 30 sec, 78°C for 1.5 min for 35 cycles, after which samples were stored at 4°C. GAPDH was used as an internal control. The 2^−ΔΔCq method was performed to analyze the relative amounts of each transcript (20). The qPCR primers are presented in Table I.

Total cellular protein from human PCa DU145 and RWPE-1 cells was extracted using radioimmunoprecipitation assay buffer (P0013B, Beyotime Biotechnology, Nanjing, China) and SDS-PAGE analysis was performed to separate the total protein. Protein concentration was determined by bicinchoninic protein assay kit. Proteins (25 µg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gels and were then transferred to a polyvinylidene membrane. The membrane was blocked with 5% skim milked for 1h at room temperature. Subsequently, the membrane was blotted overnight at 4°C with the following primary antibodies: CTGF (1:1,000 dilution), CYR61 (1:1,000 dilution), Bcl-2 (1:1,000 dilution), Bax (1:1,000 dilution), caspase 3 (1:1,000 dilution) and GAPDH (1:2,000 dilution). The membrane was then incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulin G secondary antibodies (1:5,000 dilution) at room temperature for 2 h. Protein bands were observed using enhanced chemiluminescence Plus reagent prior to imaging and analysis.

Cell proliferation was detected using an MTT assay. YAP-siRNA and Con-siRNA were transfected into DU145 cells. Subsequently, log-phase human PCa DU145 cells were harvested with 0.25% trypsin and seeded in 96-well plates (5×10³ cells/well). After 24 h incubation at 37°C with 5% CO₂, MTT was added to the cell culture medium and incubated for 4h at 37°C. Then DMSO was employed for formazan crystals dissolving. Optical density was detected at 490 nm using a spectrophotometer. Each experiment was repeated in triplicate and data were displayed as the mean ± standard deviation.

To detect alterations in cell apoptosis, an Annexin V-FITC apoptosis detection kit was utilized. DU145 cells were transfected with YAP-siRNA or Con-siRNA for 24 h. Following transfection, cells were rinsed with cold PBS. Cells (5×10⁵ cells/well) were then labeled with Annexin V-FITC and propidium iodide, according to the manufacturer's protocol. Flow cytometry (BD Biosciences, Franklin Lakes. NJ, USA) was used for cell apoptosis analysis. Version 2.5 WinMDI (Purdue University Cytometry Laboratories; www.cyto.purdue.edu/flowcyt/software/Catalog.htm) was applied for data analysis. Each test was repeated in triplicate.

All tests were performed in triplicate. Data are displayed as the mean ± standard deviation. SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) was applied for all statistical analyses. A Student's t-test was used to analyze the difference between the two groups. P<0.05 was considered to indicate a statistically significant difference.

Firstly, the mRNA and protein expression levels of YAP were detected in human PCa DU145 cells via RT-qPCR and western blotting, respectively. As demonstrated in Fig. 1, the mRNA and protein expression levels of YAP were significantly higher in human PCa DU145 cells compared with in normal prostate epithelial RWPE-1 cells. Based on these results, the expression levels of YAP were significantly increased in human PCa DU145 cells.

To investigate the role of YAP in human PCa DU145 cells, a stable YAP-silenced DU145 cell line was generated using YAP-siRNA. The results of RT-qPCR, compared with the blank group, indicated that the expression levels of YAP within YAP-siRNA-transfected cells were markedly decreased, whereas Con-siRNA did not affect YAP expression. Reduced YAP protein expression was also revealed by western blotting in the YAP-siRNA group (Fig. 2). These data indicated that YAP-siRNA may effectively inhibit YAP expression.

To investigate the effects of YAP expression on DU145 cell proliferation, YAP-siRNA and Con-siRNA were transfected into DU145 cells, and an MTT assay was performed. The results of the present study demonstrated that compared with the blank and control group, cell proliferation was markedly inhibited in YAP-siRNA-transfected DU145 cells. Furthermore, colony formation ability of cells was measured by MTT assay. The results indicated that YAP knockdown significantly suppressed the DU145 cell colony-forming ability (Fig. 3). These results indicated that YAP downregulation reduced the proliferation and colony-forming ability of DU145 cells.

The observed growth inhibition in YAP-siRNA-transfected DU145 cells was investigated using an Annexin V-FITC apoptosis detection kit for cell apoptosis via flow cytometry. The results revealed that YAP knockdown significantly induced DU145 cell apoptosis compared with in the control group (Fig. 4).

CTGF and CYR61 are downstream genes of YAP in the Hippo signaling pathway, and are regulated by YAP proteins. It has been reported that CTGF and CYR61 serve important roles in promoting cell proliferation, migration and invasion in cancer (21). Following transfection of DU145 cells with YAP-siRNA, the mRNA and protein expression levels of CTGF and CYR61 genes were significantly reduced compared with in Con-siRNA-transfected cells (Fig. 5).

Western blotting and RT-qPCR were performed to further explore the mechanism of YAP knockdown-induced cell apoptosis through the expression levels of the apoptosis-associated proteins Bax, Bcl-2 and caspase 3. The results of the present study suggested that the Bcl-2/Bax ratio was markedly lower in DU145 cells transfected with YAP-siRNA compared with in cells transfected with Con-siRNA. As expected, the expression levels of caspase 3 were notably decreased following YAP-siRNA transfection (Fig. 5).