Human prostate cancer cell lines (PC-3, DU-145 and LNCaP) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cell lines were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) (both from Hyclone, Salt Lake City, UT, USA) and 1% penicillin/streptomycin (Gibco, Rockville, MD, USA). Additionally, the human non-neoplastic prostatic epithelial RWPE-1 obtained from the American Type Culture Collection (ATCC; Rockwell, MD, USA) were maintained in keratinocyte serum-free medium (Gibco) containing 50 μg/ml of bovine pituitary extract, 0.5% penicillin-streptomycin mix, and 5 ng/ml epidermal growth factor. The cell lines were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.

Tumor tissues and matched adjacent normal tissues were obtained from Fourth Military Medical University and tissues were cryopreserved immediately following laparoscopic radical prostatectomy. The set of prostate tissues included 10 tumor tissues and 10 matched adjacent normal tissues. The study was approved by the hospital ethics board.

Total RNA was extracted from cell lines using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA). The method of total RNA extraction from tissues was performed according to the literature (36). Subsequently, the total RNA was synthesized into cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific) as per the protocol. All of the RT-qPCR reactions used 20 μl, with 10 μl of SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) and primers as follows: FOXQ1 forward, 5′-ATTTCTTGCTATTGACCGATGC-3′ and reverse, 5′-CCCAAGGAGACCACAGTTAGAG-3′; GAPDH forward, 5′-GGAAGATGGTGATGGGATT-3′ and reverse, 5′-GGATTTGGTCGTATTGGG-3′. E-cadherin forward, 5′-GGCTGCGGCTGCAGAGACTGG-3′ and reverse, 5′-TACACTGCCCAGGAGCCAGA-3′; and vimentin forward, 5′-TGAGTACCGGAGACAGGTGCAG-3′ and reverse, 5′-GGAGCCACTGCCTTCATAGTCAA-3′. The relative levels of gene expression were estimated by the 2^−ΔΔCt method.

Total proteins were extracted from the cell lines using lysis buffer (Beyotime, Nantong, China) and phenylmethanesulfonyl fluoride (100:1). The concentrations of proteins were measured using a BCA kit (Beyotime), and the equivalent volume containing 25 μg of protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to a nitrocellulose membrane using a semi-dry blotting apparatus (both from Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% skim milk diluted in Tris-buffered saline at room temperature for 2 h and then incubated with primary antibodies against anti-FOXQ1 (1:500), anti-BCL11A (1:800), anti-MDM2 (1:500), anti-E-cadherin (1:500), anti-vimentin (1:500) and anti-GAPDH (1:1,000) purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) overnight at 4°C. The membrane was blotted with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA; 1:1,000) for 2 h followed by washing with Tris-buffered saline with Tween (TBST). Finally, the target proteins were analyzed using a Bio-Rad ChemiDoc apparatus, and the signal intensity was measured by Image-Pro Plus 6.0 software.

Caspase-3 activity assay in the cells was assessed on the basis of the standard instructions of a Caspase-3 activity assay kit (Beyotime).

Apoptosis was assessed according to the manufacturer's instructions in the Annexin V-FITC Apoptosis detection kit (Bender MedSystems, Vienna, Austria). The cells (5×10⁵ cells/ml) were resuspended in the binding buffer diluted in distilled deionized water (1:4) and mixed with 5 μl of Annexin V-FITC in 195 μl of the cell suspension. The mixture was then incubated at room temperature for 10 min, then washed in phosphate-buffered saline. Thereafter, the cells were suspended in 190 μl of diluted binding buffer and 10 μl of PI was added. Apoptotic cells were counted using a FACS analyzer (Beckman Coulter, Kraemer Boulevard Brea, CA, USA).

Cell growth and viability were tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were plated in 96-well plates and cultured in an incubator with 5% CO₂ at 37°C. A total of 25 μl MTT (5 g/l) was added after removing the cell culture medium and allowed to react for 5 h at 37°C. Afterwards, the crystals were dissolved in 150 μl of dimethyl sulfoxide. The results were measured using an ELISA reader (Titertek Plus MS 212; ICN, Eschwege, Germany) at a wavelength of 490 nm. All experiments were performed in triplicate.

Cell proliferation was assessed by a BrdU cell proliferation assay kit (Millipore, Billerica, MA, USA). The protocols were performed as per the instructions. Cells were plated in 96-well plates and 10 μl of BrdU solution was added per well and allowed to react for 1 h. The culture medium was replaced by denaturing solution (100 μl/well) and incubated for 20 min. Thereafter, the detection antibody solution was added and incubated for 1.5 h at room temperature. The secondary antibody solution (100 μl/well) was added following three washes with wash buffer. Next, 100 μl tetramethylbenzidine (TMB) substrate was added and allowed to incubate for 25 min. The DNA incorporation of BrdU was detected at 450 nm on a SpectroFluor Plus Multiwell plate reader (Tecan, Research Triangle Park, NC, USA). All experiments were performed in triplicate.

Prostate cancer cell invasion ability was detected using Bio-Coat cell migration chambers (BD Biosciences, San Jose, CA, USA). Filters (Corning Incorporated, Toledo, NY, USA) were coated with Matrigel (Becton Dickinson, Bedford, MA, USA). The cells were resuspended in 300 μl of serum-free medium (2×10⁵ cells) and added to the insert (top chamber). A total of 500 μl of culture medium with 10% FBS was then added to the lower chamber and cultured in an incubator with 5% CO₂ at 37°C for 48 h. Non-invading cells were gently removed by a cotton swab, and invasive cells were fixed, stained and observed using a microscope. The number of invaded cells was counted in six random fields per membrane and averaged. Each assay was repeated three times.

Full-length human BCL11A (NM_022893.3) was amplified using the reverse transcription polymerase chain reaction and then subcloned into the plasmid pcDNA.3.1/myc-His(−)Avector (Invitrogen, Carlsbad, CA, USA). The restriction sites of BCL11A cDNA and pcDNA.3.1/myc-His(−)Avector were EcoRI and BamHI. Next, the transformation of recombined plasmids into E. coli DH5α (Tiangen, Beijing, China) was conducted, and the recombined plasmids were amplified overnight at 37°C. Finally, the amplified plasmids were extracted and sequenced, and designated as pcDNA.3.1/-BCL11A.

The PC-3 (3×10⁴ cells/well) and DU-145 (3×10⁴ cells/well) cells were plated in 24-well plates, separately. Transfection of FOXQ1 small-interfering RNA (siRNA, 5′-CGCGGACTTTGCACTTTGA-3′), non-specific siRNA (5′-TTCTCCGAACGTGTCACGT-3′) or co-transfection of pcDNA.3.1/-BCL11A and FOXQ1 siRNA was performed using Turbofect (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. The transfected cells were maintained in an incubator (Thermo Fisher Scientific) with 5% CO₂ at 37°C for 48 h, and the transfection efficiency was measured by RT-qPCR and western blot analyses.

The statistical analyses were performed to test for statistically significant differences between two groups using Student's t-test and between multiple groups using one-way ANOVA. Data are expressed as the mean ± standard deviation (SD). A p-value of <0.05 was considered statistically significant.

To investigate the expression of FOXQ1 in prostate cancer, we tested the relative mRNA expression using RT-qPCR in tumor tissues and matched adjacent normal tissues. The data show that the mRNA expression of FOXQ1 was significantly higher in tumor tissue than in the matched adjacent normal tissues (Fig. 1). Furthermore, FOXQ1 mRNA and protein expression in prostate cancer cell lines, including PC-3, DU-145 and LNCaP, were measured by RT-qPCR and western blot analysis, respectively. The results show that the mRNA (Fig. 2A) and protein (Fig. 2B) expression of FOXQ1 in prostate cancer lines (PC-3, DU-145 and LNCaP) was obviously upregulated compared with the non-neoplastic cell line RWPE-1. These results indicate that FOXQ1 may play an important role in prostate cancer.

To explore the influence of FOXQ1 on BCL11A and MDM2, we performed a FOXQ1 loss-of-function experiment in PC-3 and DU-145 cells by transfection with siRNA targeted to FOXQ1. The results show that the mRNA (Fig. 3A) and protein (Fig. 3B) expression of FOXQ1 were both markedly decreased by FOXQ1 siRNA, as determined by RT-qPCR and western blot analysis, indicating that FOXQ1 inhibition was successful. Moreover, we found that FOXQ1 inhibition significantly decreased the protein expression of BCL11A and MDM2 (Fig. 3C) in prostate cancer cells.

Next, we tested the effect of FOXQ1 inhibition on apoptosis in PC-3 and DU-145 cells using the Annexin V-FITC/PI assay and by assessing caspase-3 activity. In the Annexin V-FITC/PI assay, apoptosis was markedly increased by FOXQ1 siRNA (Fig. 4A). Furthermore, the results show that caspase-3 activity was greatly increased by FOXQ1 inhibition (Fig. 4B). These results demonstrate that suppression of FOXQ1 promotes apoptosis in prostate cancer cells.

To further investigate the biological effect of FOXQ1 inhibition in prostate cancer cells, we measured its effect on prostate cancer cell proliferation and invasion. The results show that prostate cancer cell proliferation was significantly restrained by FOXQ1 inhibition, as detected by the MTT (Fig. 5A) and BrdU (Fig. 5B) assays. Moreover, the Transwell invasion assay showed that FOXQ1 suppression significantly restrained the invasion ability of prostate cancer cells (Fig. 6A). Western blot analysis indicated that FOXQ1 suppression markedly increased E-cadherin expression and inhibited vimentin expression in prostate cancer cells (Fig. 6B). Above all, these results suggest that FOXQ1 inhibition suppresses the proliferation and invasion of prostate cancer cells.

To investigate the potential molecular mechanism of FOXQ1 in prostate cancer, we performed co-transfection of FOXQ1 siRNA with the recombinant plasmid pcDNA.3.1/-BCL11A. The western blot analysis showed that upregulation of BCL11A significantly reversed the inhibitory effect of FOXQ1 siRNA on BCL11A expression (Fig. 7A and B). Furthermore, the decreased expression of MDM2 induced by FOXQ1 siRNA was also significantly reversed by BCL11A overexpression (Fig. 7A and B). Additionally, the induction of apoptosis (Fig. 8A) and inhibition of proliferation (Fig. 8B) and invasion (Fig. 8C) caused by FOXQ1 suppression were all obviously reversed by BCL11A overexpression.