The 293 cell line, human normal prostate epithelial RWPE-1 cell line, and human Pca DU-145, PC-3 and LNCaP cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). FH55 [F5682; high performance liquid chromatography (HPLC) purity ≥98%] and LiCl (746460; HPLC ≥99%) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

All cells were placed in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), and cultured with 5% CO₂ at 37°C, under saturated humidity. The cells in logarithmic growth phase were detected.

Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was performed following the manufacturer's protocol. In brief, 2 µl Lipofectamine^® 3000, 40 pmol small interfering (si) RNA targeting URG11 and the negative control (siNC; Shanghai GenePharma Co., Ltd., Shanghai, China) were mixed separately in 50 µl serum-free medium and incubated at room temperature for 15 min. The lipid compounds were diluted in 300 µl serum-free medium and 600 µl medium containing fetal bovine serum (FBS) to produce a 1 ml volume mixture, and incubated with the LNCaP cells at 37°C with 5% CO₂. The URG11-targeting siRNA sequence and NC siRNA were 5′-CAGACGGAUUGCUGUACUU-3′, and 5′-UUCUCCGAACGUGUCACGUTT-3′, respectively. For the expression vectors used to upregulate URG11 (pcDNA3.1-URG11) and the corresponding NC (pcDNA3.1-NC), 5 µl Lipofectamine^® 3000 and 2 µg vector (Shanghai GenePharma Co., Ltd.) were mixed in 125 µl Dulbecco's modified Eagle's medium (DMEM; Corning Inc., Corning, NY, USA) and incubated at room temperature for 5 min. A total of 4 µl Lipofectamine^® 3000 and 125 µl DMEM were mixed, and incubated at room temperature for 5 min. Then, the Lipofectamine^® 3000 + DNA mixture was combined with the Lipofectamine^® 3000 + DMEM solution. After 5 min, the LNCaP cells were treated with the combined mixture at 37°C with 5% CO₂. The cells were transfected for 48 h for subsequent experiments.

The LNCaP cells of each treatment group were collected, and 1×10⁵ cells/well were counted. Following the addition of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) into the Transwell chamber for 6 h at 37°C, the cells was resuspended in serum-free medium and added to the chamber of the Transwell cell culture plate. Following incubation with 5% CO₂ for 24 h at 37°C, the chamber was removed, and cells were fixed in 4% paraformaldehyde for 20 min at 4°C and washed once with PBS. The cells were then stained with 0.1% crystal violet for 10 min at room temperature, washed once with PBS, and observed under a light microscope. Images of the cells were captured and the number of cells that had passed through the membrane was counted in 5 fields using a light microscope at magnification, ×200. The number of cells per field was calculated, and experiments were repeated 3 times.

LNCaP cells in the logarithmic growth phase (~1×10⁹ cells/well) were collected, and the suspension was uniformly inoculated into a 6-well culture plate and cultured in an incubator with 5% CO₂ at 37°C. The control group was cultured in serum-free RMPI-1640 medium, and the experimental group was treated with drug-containing (20 µM FH535 or 20 mM LiCl) serum-free RMPI-1640 medium for 48 h at 37°C. Following scratching, images of the samples were captured at 0 and 48 h. The scratch healing was observed under an inverted light microscope at magnification, ×200. The scratch widths at 8 different sites in the groups were measured by Image-Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA) and the cell migration rate in each group was calculated.

A CCK-8 assay (Beyotime Institute of Biotechnology, Haimen, China) was utilized to assess cell viability according to the manufacturer's protocols. Briefly, transfected and un-transfected LNCaP cells were transferred to 96-well plates (3×10³ cells/well). Following incubation for 2 h at 37°C in 10 µl CCK-8, the absorbance in every well was evaluated using a microplate reader at 450 nm (Tecan Infinite M200 Micro Plate Reader; Tecan Group, Ltd., Männedorf, Switzerland).

Following transfection of the LNCaP cells for 48 h, 1×10⁶ cells were collected, and 1 ml trypsin was used to digest the cells. The mixture was then gently shaken. Following removal of the trypsin and incubation for 1 min at room temperature, the digestion was terminated by adding DMEM containing 10% FBS (Corning Inc.). The cells were centrifuged at 1,000 × g for 3 min at 4°C and the supernatant was removed. The cells were then washed twice with pre-cooled PBS and resuspended in 1X Annexin V binding buffer. According to the protocol of the manufacturer of the Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201-100; BioVision, Inc., Milpitas, CA, USA), the cells was stained with 1.25 µl Annexin V-FITC and 10 µl propidium iodide (PI) for 10 min at room temperature, and measured by flow cytometry (FACSCalibur™; BD Biosciences) using FlowJo v10.0 software (FlowJo LLC, Ashland, OR, USA)

Following transfection for 48 h, 1×10⁶ LNCaP cells/well were collected, washed twice with PBS, then fixed with 70% ethanol at 4°C overnight. Then, 500 µl PBS containing 50 mg/l PI, 100 mg/l RNase A and 0.2% Triton X-100 was added to the cells, which were incubated for 30 min at 4°C in the dark. Flow cytometry was performed, and the results were analyzed using the cell cycle fitting software ModFit LT™ software v2.0 (BD Biosciences).

Following culture of the RWPE-1, DU-145, PC-3 and LNCaP cells for 24 h in the medium, the culture solution was discarded and the cells were washed twice with PBS. Then, 500 µl TRIzol (Thermo Fisher Scientific, Inc.) was added to the wells of each 6-well plate, pipetted several times, and then the lysate was transferred to 1.5 ml RNase-free EP tubes. Then, 200 µl chloroform per 1 ml TRIzol was added into the EP tube, and incubated on ice for 10 min. Following centrifugation at 12,000 × g for 15 min at 4°C, the upper aqueous phase was carefully pipetted into a second RNase-free EP tube; the same volume of isopropanol was the added to the EP tube, and the solution was mixed by inversion several times, and incubated on ice for 10 min. Following centrifugation at 10,000 × g for 5 min at 4°C, the supernatant was discarded, an equal volume of 75% ethanol was added, and the sediment at the bottom was gently agitated, centrifuged at 7,500 × g for 5 min at 4°C, and the RNA was air-dried at the vent. Subsequently, 10 µl of DEPC water was added and the RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). A total of 1 µg RNA was used for reverse transcription into cDNA using a reverse transcription cDNA kit (Thermo Fisher Scientific, Waltham, MA, USA). The reaction conditions were as follows: 42°C for 60 min and 70°C for 5 min, followed by preservation at 4°C. SYBR-Green PCR Master Mix (Roche Diagnostics, Basel, Switzerland) was used to conduct the qPCR experiment using an Opticon RT-PCR Detection System (ABI 7500; Thermo Fisher Scientific, Inc.), The PCR thermocycler conditions were as follows: Pretreatment at 95°C for 10 min; followed by 40 cycles at 94°C for 15 sec, 60°C for 1 min and 60°C for 1 min, and then preservation at 4°C. The comparative cycle threshold (2^−ΔΔCq) method was employed to analyze the expression of mRNA (23). GAPDH expression was used for normalization. The primer sequences used are summarized in Table I.

Total proteins were collected by radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) was applied to measure the concentration of proteins, which were adjusted to a concentration of 6 µg/µl using 1X loading buffer and DEPC water. Then, 5 µl samples was separated by 10% SDS-PAGE gels and then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking of the membranes in 5% nonfat milk in PBST (0.1% Tween-20 in PBS) for 1 h at room temperature, the membranes were probed with the primary antibodies overnight at 4°C. The membranes were then washed 3 times with PBST and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse (cat. no. sc-516102; 1:2,000) and HRP-rabbit IgG (cat. no. sc-2357; 1:2,000; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA) secondary antibodies at room temperature for 2 h. Then, the membranes were washed 3 times with PBST. The EZ-ECL kit (Biological Industries, Kibbutz Beit Haemek, Israel) was used to visualize the gels, and the gray values were analyzed and counted using ImageJ software (v5.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The antibodies used were mouse anti-GAPDH (1:1,000; cat. no. LS-B1625; LifeSpan BioSciences, Inc.), rabbit anti-URG11 (1:1,000; cat. no. ab109232), mouse anti-epithelial cadherin (E-cadherin; 1:1,000; cat. no. ab1416), rabbit anti-Vimentin (1:1,000; cat. no. ab92547) and rabbit anti-α-smooth muscle actin (α-SMA; 1:1,000; cat. no. ab5694), mouse anti-cyclin D1 (1:1,000; cat. no. ab134175) and rabbit anti-MYC proto-oncogene protein (c-Myc; 1:1,000; cat. no. ab32072; all Abcam, Cambridge, MA, USA).

The results are presented as the mean ± standard deviation. Statistical software Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Comparisons between two groups were performed using un-paired Student's t-tests, and the comparisons between multiple groups were performed by one-way analysis of variance followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of URG11 were first investigated in Pca cells lines, including DU145, PC3 and LNCaP cell lines, and in the nontumor prostate epithelial RWPE-1 cell line. The URG11 mRNA levels in human prostate cells were significantly increased compared with that in the epithelial cells (Fig. 1A). Furthermore, western blot analysis was used to determine the URG11 protein level in the Pca cell lines. Similarly, the URG11 protein level exhibited the same pattern of expression as the mRNA level (Fig. 1B and C). Specifically, the LNCaP cells exhibited increased URG11 mRNA and protein expression levels in comparison with DU145 cells and PC3 cells. Therefore, LNCaP cells were used for the subsequent experiments.

To additionally explore the function of URG11 in Pca cells, overexpression plasmids and siRNA were applied to overexpress and silence URG11, respectively. Following the transfection of the URG11 plasmids and siURG11 fragments into LNCaP prostate cells, the expression level of URG11 was significantly increased in the URG11 overexpression group but suppressed in the siURG11 group compared with their corresponding control and NC groups at the mRNA (Fig. 2A) and protein levels (Fig. 2B and C). Following confirmation of the efficacy of the overexpression URG11 vector and siURG11 fragments, a CCK-8 assay was then used to detect the effects of URG11 overexpression and silencing on cell viability. As demonstrated in Fig. 2D, the cells in the URG11 overexpression group exhibited increased growth compared with the normal culture and NC groups at 24, 48 and 72 h, while siURG11 decreased cell viability compared with the normal culture and NC groups at 24, 48 and 72 h (Fig. 2E).

The specific role of URG11 in the invasion and migration of Pca cells was then determined. Cell migratory and invasive abilities were determined by wound healing and Transwell assays, respectively. The wounds generated in the cells in the URG11 overexpression group were almost healed at 48 h, at which time the wounds in normal culture and control groups were significantly wider. Treatment with siURG11 exhibited the opposite effects (Fig. 3A). Furthermore, the Transwell invasion assays revealed that the overexpression of URG11 significantly promoted the invasion of LNCaP Pca cells compared with those of in the normal culture and NC control groups, and genetic knockout of URG11 elicited the opposite effects (Fig. 3B). The quantified data from the migration and invasion assays are presented in Fig. 3C and D.

To improve understanding of the role of URG11 in Pca metastasis and the underlying mechanism of action, flow cytometry was performed with PI/Annexin V staining for apoptosis and cell cycle analyses. Using flow cytometric analysis with PI/Annexin V staining, overexpression of URG11 was observed to significantly suppress cell apoptosis, compared with that in the normal culture and NC groups (Fig. 4A). Furthermore, overexpression of URG11 significantly decreased the number of cells in G0/G1 phase and increased the number of cells in S phase, compared with that in the normal culture and NC control groups (Fig. 4B). By contrast, siURG11 elicited the opposite effects (Fig. 4A-D).

Due to the importance of EMT in the development of LNCaP cells, the effect of URG11/siURG11 on EMT markers in LNCaP cells was assessed. The mRNA and protein levels of EMT markers were examined by RT-qPCR and western blot analysis, respectively. As indicated in Fig. 5A, overexpression URG11 treatment significantly decreased E-cadherin mRNA levels and increased vimentin and α-SMA mRNA levels compared with the controls (Fig. 5A). Furthermore, western blot analysis was used to determine the protein levels of those EMT markers, and the results demonstrated that the effects of URG11 on protein levels of E-cadherin, vimentin and α-SMA were in accordance with mRNA levels (Fig. 5B and C). However, siURG11 elicited the opposite effects (Fig. 5D-F).

As the overexpression of URG11 and siURG11 significantly affected proliferation and cell cycle arrest in human LNCaP cells, western blot analysis was performed to examine the protein expression levels of cyclin D1 and c-Myc, which are involved in the regulation of cell proliferation and the cell cycle. It was identified that the transfection of LNCaP cells with URG11 overexpression plasmid vectors significantly promoted the expression of cyclin D1 and c-Myc at the mRNA (Fig. 6A) and protein levels (Fig. 6B and C). However, gene knockdown of URG11 markedly inhibited the levels of cyclin D1 and c-Myc at the mRNA (Fig. 6D) and protein levels (Fig. 6E and F).

Due to the importance of Wnt/β-catenin signal pathway in URG11-induced cell viability, migration and invasion in LNCaP cells, the cells were treated with URG11 overexpression plasmids and the Wnt/β-catenin inhibitor FH535 (20 µM), together and individually. Then, the cell viability, migration and invasion were respectively determined by CCK-8, wound healing and Transwell assays. With these functional experiments, it was identified that FH535 treatment significantly inhibited the plasmid vector URG11 transfection-induced effects on cell viability (Fig. 7A), migration (Fig. 7B and D) and invasion (Fig. 7C and E). The images of migration and invasion were presented in Fig. 7D and E, respectively.

In order to additionally validate the role of the Wnt/β-catenin signaling pathway in LNCaP cells proliferation, the cells were treated with siURG11 and the Wnt/β-catenin agonist LiCl (20 µM), together and individually. CCK-8, wound healing and Transwell assays were also conducted to determine the effects on cell viability, migration and invasion, respectively. Notably, it was identified that that the cell viability (Fig. 8A), migration (Fig. 8B) and invasion (Fig. 8C) were increased in the siURG11+ LiCl group compared with those in the siURG11 group. The images of migration and invasion were presented in Fig. 8D and E.