PC-3 prostatic carcinoma cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured at 37°C in a humidified environment containing 5% CO₂. The cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1:100 penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.).

Anti-mouse ERK5 antibody (dilution 1:2,000; cat. no. sc-398015) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-rabbit RERG antibody (1:2,000 for western blotting; 1:200 for immunohistochemistry; cat. no. 10687-1-AP) was purchased from ProteinTech Group, Inc. (Rosemone, IL, USA). Anti-MMP-2 antibody (dilution 1:2,000; cat. no. ab92536), anti-MMP-9 antibody (dilution 1:2,000; cat. no. ab137867), anti-B cell lymphoma (Bcl)-2 antibody (dilution 1:2,000; cat. no. ab196495), and anti-c-Myc antibody (dilution 1:2,000; cat. no. ab39688) were purchased from Abcam (Cambridge, UK; dilution 1:2,000). Anti-GAPDH antibody (dilution 1:4,000; cat. no. 100242-MM05) was purchased from Sino Biological, Inc. (Beijing, China). All of the secondary antibodies, including goat anti-mouse IgG antibody-(cat. no. 715-035-003) and goat anti-rabbit IgG antibody (cat. no. 111-035-045)-conjugated horseradish peroxidase (HRP), were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA; dilution 1:4,000). XMD8-92, an ERK5 inhibitor that specifically inhibits the phosphorylation of ERK5, but not the phosphorylation of ERK1/2, was purchased from Selleck Chemicals (Houston, TX, USA). EGF was obtained from Sino Biological, Inc. The final concentration of the XMD8-92 and EGF treatment was 5 µM and 0.5 ng/ml, respectively. The cells were serum starved overnight followed by treatment with XMD8-92 as indicated for 1 h, and/or stimulated with EGF for 20 min, as previously described (21).

The human RERG open-reading frame was amplified by RT-polymerase chain reaction (PCR) from the total RNA of PC-3 cells. The primer sequences of RERG are presented in Table I. The RERG cDNA were amplified by PCR and inserted into the EcoRI/XbaI sites of the pCDNA3.1(+) eukaryotic expression vector (Invitrogen; Thermo Fisher Scientific, Inc.) with T4 DNA ligase (New England Biolabs, Ipswich, MA, USA). To construct pSpCas9-RERGtarget-2A-GFP, the RERG sgRNA oligo was designed in http://crispr.mit.edu, phosphorylated, ligated, and inserted in the pSpCas9-2A-GFP vector (Addgene, Cambridge, MA, USA) after BbsI digestion (Thermo Scientific, Inc.).

To establish PC-3 cells with stable RERG protein overexpression or inhibition, PC-3 cells were transfected with pCDNA3.1(+)-RERG, pSpCas9-RERGtarget-2A-GFP recombinant plasmid, or pCDNA3.1(+) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Briefly, for transfection, 1×10⁶ cells were seeded in a 6-well plate. A total of 5 µl Lipofectamine^® 3000, and 5 µg pCDNA3.1(+)-RERG or pSpCas9-RERGtarget-2A-GFP were diluted in 200 µl Opti-MEM medium (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated for 30 min, respectively. The mixture was then added and incubated in the cell culture medium for 48 h. For the selection of RERG-overexpressing PC-3 cells, cells were transfected with pCDNA3.1(+)-RERG and selected by G-418 treatment for three weeks. For the selection of cells with silenced RERG expression, pSpCas9-RERGtarget-2A-GFP-transfected PC-3 cells were sorted by flow cytometry on a FACSAria II system (BD Biosciences, San Jose, CA, USA), and the selected fluorescently labelled cells were then cultured in 6-well plates. PC-3 cells were also transfected with pCDNA3.1(+) and selected using G-418, as vector control cells.

To prepare protein extracts, the cells were washed with PBS three times. Following centrifugation, the harvested cells were resuspended and protein extracted using lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) for 30 min on ice. The lysates were centrifuged at 16,000 × g for 10 min at 4°C. The supernatants of the lysates were mixed with SDS sample buffer and boiled for 10 min. The samples (~100 µg of each lane) were separated on a 10% SDS polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% (w/v) dry skim milk for 1 h at room temperature, and then blotted with the corresponding antibody in PBST buffer (0.1% Tween-20 in PBS) under gentle shaking at room temperature for 2 h. After being washed with PBST three times, the membranes were incubated with the indicated secondary antibody for 2 h at room temperature. The signals were detected using a SuperSignal West Pico Substrate kit (Pierce; Thermo Fisher Scientific, Inc.). The signals were measured by fluorescence intensity with ImageJ software (version no. 1.8.0; National Institutes of Health, Bethesda, MD, USA).

Total RNA was extracted from the cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using a SYBR PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan) on a Rotor-Gene 6000 Real-Time Genetic Analyser (Qiagen, Valencia, CA, USA), and quantified according to the previously described method (22). The primer sequences of RERG, MMP-2, MMP-9, and GAPDH are presented in Table I. The PCR protocol included a denaturation programme (95°C for 2 min), followed by 40 cycles of an amplification and quantification programme (95°C for 5 sec and 55–57°C for 30 sec) and a melting curve programme (55–95°C, with a 0.5°C increment each cycle). Each sample was replicated three times.

A paraffin-embedded tissue microarray (Alenabio, Xi'an, China), which contains 58 prostatic carcinoma specimens and 6 normal prostate tissues, was dewaxed with xylene and rehydrated in descending concentrations of ethanol. The slide was blocked with Immuol Staining blocking buffer (cat. no. P0102; Beyotime Institute of Biotechnology, Shanghai, China) for 60 min at room temperature, and then was incubated with anti-RERG antibody (dilution 1:200; Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing three times in PBST buffer for 10 min, the slide was processed according to the instructions of the Super Sensitive Polymer HRP Detection System/DAB kit (Thermo Fisher Scientific, Inc.) and counterstained with haematoxylin for 15 min at room temperature. The intensity of the immunostaining (1, weak; 2, moderate, and 3, intense) and the percentage of positive cells (0, <0.5%; 1, 5–35%; 2, 26–50%; 3, 51–75%; 4, >75%) were assessed in at least 5 high-power fields under a light microscope (Nanjing Jiangnan Inc., Nanjing, China). All values for each sample were multiplied to obtain a final score, and the tissues with scores <4 and ≥4 were determined to have low and high expression, respectively.

Cell counting kit (CCK)-8 assay was performed to examine PC-3 cell proliferation. The cells were seeded at 2×10³ cells/well in phenol red-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS in a 96-well plate. The cell growth rate was determined by CCK-8 assay (Beyotime Institute of Biotechnology). A total of 10 µl of CCK-8 working solution was added to each well on days 1–5, followed by incubation for 2 h at 37°C, and the absorbance at a wavelength of 450 nm was measured using a model 3550 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A total of 5×10² cells were added to a 24-well plate. All of the cells were then cultured for 2 weeks with fresh medium every three days. Cells were washed with PBS for three times, and stained with 0.1% crystal violet for 10 min at room temperature, and destained with PBS three times. Colonies were counted using ImageJ software for each well, and each condition was tested in triplicate.

A 24-well micropore polycarbonate membrane filter with 8-µm pores (Corning Inc., Corning, NY, USA) was coated with 10 µl of Matrigel (BD Biosciences). Then, 200 µl of cell suspension (1×10⁴/ml) was seeded into the top chamber in serum-free DMEM medium. The bottom chamber was filled with DMEM supplemented with 10% FBS. After 24 h of incubation, the membranes were fixed and stained by 0.1% crystal violet for 15 min at room temperature, and the cells on the upper surface were then removed with a cotton swab. The invasive cells on the lower surface were observed and counted under a phase-contrast microscope. Each condition was tested with five replicates.

Cell monolayers in all groups were wounded using a 200-µl pipette tip in 6-well plates and then incubated in DMEM supplemented with 10% FBS. The wound width was imaged at 0, 12, 24, or 36 h post-scratching under a phase-contrast microscope.

Hoechst 33342 staining (Beyotime Institute of Biotechnology) was performed to examine apoptosis. In brief, cells were seeded on coverslips in 24-well plates and cultured for 24 h; then, the DMEM was removed, and the cells were washed three times. Cells in all of the groups were incubated in Hoechst labelling solution (2 µg/ml) for 20 min at room temperature before being washed with PBS three times. The cells were then observed under a ZEISS Observer A1 fluorescence microscope at 350 nm.

In total, 5×10⁴ cells were seeded into 24-well plates and cultured for 24 h. Cells in the different groups were transfected with 0.3 µg of pNF-κB-Luc plasmid (Agilent Technologies, Inc., Santa Clara, CA, USA) with Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 48 h after transfection, the cells were lysed in lysis buffer. The luciferase activity was determined using a luciferase assay kit (Promega Corp., Madison, WI, USA). Each experiment was repeated at least three times.

Data are presented as the mean ± standard error of the mean. The data were analysed using SPSS software, version 14.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5.0. (GraphPad Software, Inc., La Jolla, CA, USA). Statistical analyses were performed by analysis of variance using Dunnett's multiple comparison test, or Students t-test. P<0.05 was considered to indicate a statistically significant difference. Each experiment was replicated at least three times.

Our previous study indicated that activation of the ERK5 pathway induces prostatic carcinoma cell proliferation (13). To further investigate the function of ERK5 in the progression of prostatic carcinoma, the present study activated or inhibited the ERK5 pathway with treatments with EGF or XMD8-92, respectively. Fig. 1 revealed that phosphorylated ERK5 was markedly upregulated in EGF-treated PC-3 cells, indicating that the ERK5 pathway can be activated by EGF treatment (21). The results indicated that EGF treatment can activate the ERK5 pathway, which induces more phosphorylated ERK5 in total ERK5 protein. Thereafter, EGF- and XMD8-92-treated PC-3 cells were harvested and examined by microarray analysis (Sangon Biotech, Shanghai, China). Fig. 1B revealed the regulation of various genes in EGF- and XMD8-92-treated PC-3 cells. Notably, it was noted that RERG expression was significantly upregulated in XMD8-92-treated PC-3 cells, but inhibited in EGF-treated PC-3 cells, compared with control, which was also confirmed by western blot analysis (Fig. 1C). Similarly, RT-PCR also demonstrated that RERG was regulated by ERK5 signalling at the transcriptional level (Fig. 1D). Therefore, the results suggested that RERG may be associated with ERK5-mediated cell proliferation in prostate tumour cells.

To investigate the role of RERG expression in prostatic carcinoma, the present study then analysed the expression level of RERG in the specimens of tissue microarray, which contained 58 prostatic carcinoma specimens and 6 normal prostate tissues. The characteristics of the patients with prostatic carcinoma are presented in Table II. Immunohistochemistry revealed that RERG expression was lower in advanced prostatic carcinoma tissues, whereas RERG protein expression was higher in normal prostate tissues and less malignant tumour tissues (Fig. 2A), suggesting that the loss of RERG expression may result in cancer progression. In addition, the results indicated that the expression level of RERG was negatively associated with malignancy in prostatic carcinoma (Fig. 2B), which is consistent with previous reports regarding other tumours (16–18).

The aforementioned results indicated that RERG expression may be involved in the malignancy of prostatic carcinoma. It was expected that RERG expression may play a role in tumourigenesis in prostatic carcinoma. To confirm our hypothesis, the present study stably overexpressed and knocked down RERG expression in PC-3 cells. RT-qPCR and western blot analysis demonstrated the overexpression and knockdown of RERG mRNA and protein in PC-3 cells (data not shown and Fig. 3A, respectively). Additionally, cell proliferation assays demonstrated that the inhibition of RERG expression promoted cell proliferation compared with RERG overexpression and the control (empty vector; Fig. 3B). Moreover, the colony formation assay indicated that the colony number was much greater in RERG-inhibited PC-3 cells compared with RERG-overexpressing or empty-vector-transfected PC-3 cells (Fig. 3C and D), suggesting that RERG expression is associated with prostate cancer cell proliferation.

As RERG has been reported to be associated with tumour metastasis in breast cancer, the present study then examined whether the regulation of RERG expression was associated with tumour invasion and metastasis in prostatic carcinoma. The invasion and migration of RERG-overexpressing, RERG-inhibited, and empty-vector-transfected PC-3 cells was examined. The in vitro wound healing assay demonstrated that RERG-inhibited PC-3 cells migrated into the scratched area more rapidly than RERG-overexpressing or empty-vector-transfected PC-3 cells at 12, 24, and 36 h (Fig. 4A and B). Further transwell studies demonstrated that RERG-inhibited PC-3 cells displayed an increased invasive capacity compared with RERG-overexpressing and empty-vector-transfected PC-3 cells (Fig. 4C and D).

MMP-2 and MMP-9 have been reported to be associated with the invasion and migration of many malignant tumours (23). Therefore, the present study examined the function of MMP-2 and MMP-9 in RERG-associated invasion and migration in prostatic carcinoma. The expression levels of MMP-2 and MMP-9 in RERG-overexpressing, RERG-inhibited, and empty-vector-transfected PC-3 cells were determined. Notably, western blot analysis revealed that the expression levels of MMP-2 and MMP-9 were inhibited in RERG-overexpressing PC-3 cells, but upregulated in RERG-inhibited PC-3 cells (Fig. 4E). Therefore, the results indicated that MMP-2 and MMP-9 play important roles in RERG-associated invasion and migration in prostatic carcinoma.

As apoptosis plays important roles in the regulation of cell proliferation in various malignant tumours, it was expected that RERG may promote cell proliferation by inhibiting apoptosis in prostatic carcinoma cells. To clarify the mechanism of RERG function in prostatic carcinoma cell proliferation, Hoechst staining was performed to confirm whether the regulation of RERG expression was associated with the induction of cell apoptosis in RERG-overexpressing or RERG-inhibited PC-3 cells. The results indicated that cell apoptosis was increased in RERG-overexpressing PC-3 cells, suggesting that the regulation of RERG expression was involved in the apoptosis of prostatic carcinoma cells (Fig. 5A and B).

To further illustrate the possible mechanism of RERG-induced apoptosis in prostatic carcinoma cells, the present study then determined the protein expression levels of c-Myc and Bcl-2, which are key factors in apoptosis (24,25), in RERG-overexpressing, RERG-inhibited, and empty-vector-transfected PC-3 cells. Notably, it was revealed that the expression level of Bcl-2 was significantly inhibited, whereas that of c-Myc was upregulated in RERG-overexpressing PC-3 cells compared with empty-vector-transfected PC-3 cells (Fig. 5C). Bcl-2 is an anti-apoptotic factor that is also a regulator of the NF-κB signalling pathway (26). Inactivation of the NF-κB pathway can also induce apoptosis in malignant tumours (27,28). Thus, NF-κB activation in RERG-overexpressing, RERG-inhibited, and empty-vector-transfected PC-3 cells was investigated. As expected, the results indicated that RERG overexpression blocked NF-κB signalling pathway activation (Fig. 5D), which suggested that NF-κB-mediated apoptosis may be involved in RERG-regulated cell proliferation in prostatic carcinoma.