All of the specimens used in the present study were collected from the Affiliated Zhongda Hospital of Southeast University (Nanjing, China) between March 2012 and April 2014. Ethical approval was obtained from the relevant ethics committee at the Affiliated Zhongda Hospital of Southeast University. All of the samples were collected upon receipt of written informed consent from patients. Benign prostate hyperplasia (BPH) tissues were obtained from 10 patients who had undergone transurethral prostatic resection (TURP). Pca tissues were obtained from 30 androgen-dependent Pca (ADPC) patients who had undergone radical prostatectomy. All patients were divided into three groups based on a Gleason score of <7, 7 or >7 (13). Nine patients were diagnosed with CRPC, as their serum prostate-specific antigen levels continued to increase despite maximum androgen deprivation therapy. All patients with CRPC were in stage T4 (distant metastasis) and presented with a Gleason score of >7 (13). These patients had undergone TURP due to urinary retention. Histological diagnosis was conducted on freshly frozen sections following hematoxylin and eosin staining (Beyotime Institute of Biotechnology, Shanghai, China). Ten ADPC samples with Gleason score >7 and >60% tumor content and nine patients with CRPC were included in the present study for quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The specimens used for miR qRT-PCR were snap-frozen in liquid nitrogen (Nanjing University Physics Refrigeration Laboratory, Nanjing, China).

All surgical samples were fixed in 10% buffered formaldehyde solution (BioSharp, Hefei, China) and embedded in paraffin (Sigma-Aldrich, St. Louis, MO, USA). Paraffin sections (4-µm thick) were reacted with polyclonal antibodies against PAK6 (1:50; cat. no. 13539-1-AP; ProteinTech Group, Inc., Chicago, IL, USA). Phosphate-buffered saline (PBS; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) served as a negative control (NC).

Total RNA was extracted from specimens using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA synthesis was performed using PrimeScript^® 1st Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China). qRT-PCR reactions were performed using the SYBR Green PCR Master mix from the Hairpin-it™ miRs RT-PCR Quantitation kit (GenePharma Co., Ltd., Shanghai, China) according to the manufacturer's instructions. PCR conditions were used to detect miRs as follows: 95°C for 3 min, 40 cycles at 95°C for 12 sec and 62°C for 40 sec. The miR-328 expression relative to U6 was calculated using the 2^−ΔΔCt method.

Specimens and cells were lysed with radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined by the bicinchoninic acid method (Beyotime Institute of Biotechnology). Equal amounts of proteins (30 µg) were separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) at 80 V for 30 min then 100 V for 1.5 h. Electrophoresed proteins were transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA) and subsequently blocked with 5% skimmed milk (BioSharp) at room temperature for 1 h. The membranes were incubated with rabbit anti-human PAK6 polyclonal antibody (1:400; cat. no 13539-1-AP; ProteinTech Group, Inc.), rabbit anti-human cleaved caspase-3 antibody (1:500; cat. no. 9654; Cell Signaling Technology, Danvers, MA, USA), mouse anti-human caspase-9 monoclonal antibody (1:500; cat. no. 9492; Cell Signaling Technology), rabbit anti-human bcl-2 polyclonal antibody (1:500; cat. no. 12789-1-AP; ProteinTech Group, Inc.), or rabbit anti-human GAPDH polyclonal antibody (1:1,000; cat. no. sc-25778; Santa Cruz Biotechnology Inc., Dallas, TX, USA) in 5% skimmed milk overnight at 4°C. The blots were washed with Tris-buffered saline with Tween 20, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:3,000; cat. no. ZB-2301; Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 37°C for 1 h, and visualized using Immobilon Western Chemilum HRP Substrate (EMD Millipore). Protein levels were determined by normalization against GAPDH.

A PAK6 3′-UTR-luciferase reporter was created by ligating the PAK6 3′-UTR PCR product into the XhoI and NotI restriction sites of the psiCHECK-2™ Vector (Promega Corp., Madison, WI, USA). Deletion of the binding site for miR-328 generated the mutant reporter. Following a 48-h cotransfection, luciferase activity was evaluated using a dual-luciferase reporter assay system (Promega Corp.).

The PC-3 and DU-145 human Pca cell lines, and HEK-293 cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). HEK-293 and DU-145 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA), and PC-3 was cultured in DMEM F12 (GE Healthcare Life Sciences) supplemented with 100 U/ml penicillin (GE Healthcare Life Sciences), 100 mg/ml streptomycin (GE Healthcare Life Sciences), and 10% fetal bovine serum (GE Healthcare Life Sciences). All cell cultures were incubated at 37°C in an atmosphere of 5% CO₂.

miR mimic oligonucleotide duplexes were chemically synthesized by GenePharma, Co., Ltd. based on the following sequences: Sense, 5′-CUGGCCCUCUCUGCCCUUCCGU-3′ and antisense, 5′-GGAAGGGCAGAGAGGGCCAGUU-3′ for hsa-miR-328 mimic; and sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and anti-sense, 5′-ACGUGACACGUUCGGAGAATT-3′ for the NC. For cell transfection, DU-145 and PC-3 cells were seeded in 6-well plates and transfected at 60–70% confluence using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions.

DU-145 and PC-3 cells were seeded in 6-well plates, cultured at 37°C in an atmosphere containing 5% CO₂ overnight, transfected with oligonucleotides, and cultured for a further 48 h. Subsequently, the cells were trypsinized (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) and seeded at a density of 3,000 cells/well (200 ml/well) in 96-well plates. Following overnight incubation at 37°C in an atmosphere containing 5% CO₂, the cells were treated with various docetaxel concentrations. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Absorbance was detected at 450 nm using an automatic multi-well MK3 spectrophotometer (Thermo Fisher Scientific, Inc.). Five wells were analyzed for cell viability in each treatment group.

Approximately 48 h post-transfection, cells were harvested and stained with propidium iodide [PI; MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China] for cell cycle assay and by Annexin V-fluorescein isothiocyanate and PI (Ubio Biological Technology PVT Ltd., Jinan, China) for the apoptosis assay, according to the manufacturer's instructions. Treated cells were analyzed by flow cytometry (FACS101; BD Biosciences, Franklin Lakes, NJ, USA).

Six 4-week-old immunodeficient BALB/c-nu/nu male mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) for injection of PC-3 cells. The mice were maintained under specific pathogen-free conditions under a 12 h light/12 h dark cycle at 26–28°C and 50–65% humidity. Cell suspensions (100 µl; 5×10⁶ cells) were subcutaneously injected into the dorsal scapular region of each mouse. Tumor volume was measured using a caliper every three days and the following formula was used: Volume (mm³) = (length × width²)/2. When the tumor volume reached 40–50 mm³, the mice were randomly divided into two groups with three mice per group. The mice were treated with 200 pmol NC or hsa-miR-328 mimics in 10 µl RNA free water by local injection of the xenograft tumor at multiple sites. This treatment was performed once every five days for 15 days, and tumors were harvested one week later. Animal experiments were undertaken in accordance with National Institute of Health Guide for the Care and Use of Laboratory Animals guidelines and were approved by the ethics committee of the Affiliated Zhongda Hospital of Southeast University.

Data from at least three independent experiments are presented as means ± standard error of the mean. Differences between groups were calculated by Student's t-test or one-way analysis of variance using the SPSS 16.0 software package (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

PAK6 expression levels were detected using immunohistochemistry. Cytoplasmic staining was observed, although no nuclear expression was demonstrated in the Pca tissue samples (Fig. 1A). In the BPH tissue samples, PAK6 was predominantly expressed in the epithelium; however, the staining was weak in the majority of BPH tissues (Fig. 1B). In the Pca samples, the staining intensity appears to be greater in groups with Gleason scores of 7 and >7, when compared with the group with Gleason score of <7 (Fig. 1C–E). The staining intensity was notably increased in the CRPC tissue samples (Fig. 1F). The results of immunostaining are summarized in Table I. PAK6 expression levels were detected by western blot analysis in three pairs of ADPC and CRPC tissues, and overexpression of PAK6 was observed in the CRPC tissue samples (Fig. 1G). These results are consistent with those of a previous study (6).

To determine which miR affects PAK6 expression, bioinformatics analysis was performed using TargetScan Human 6.2 (http://www.targetscan.org/) and miRanda (http://www.microrna.org/microrna/home.do). Of the miRs identified, miR-328 was identified to be bound to the 3′-UTR of PAK6 mRNA with high scores (Fig. 2A). miR-328 expression was subsequently detected in 10 ADPC and nine CRPC tissue samples by qRT-PCR, and weak expression was observed in CRPC tissues (Fig. 2B). In addition, PAK6 protein expression was inhibited in cells transfected with miR-328 mimics (Fig. 2C). These results indicate that PAK6 is the potential target of miR-328. To determine direct miR-target interactions, a luciferase reporter assay was conducted by constructing wild- and mutant-type cells with the luciferase vector. A significant decrease was observed in the luciferase activities of HEK-293 and PC-3 cells when compared with those of the control and mutant-type cells (Fig. 2D). These characteristics indicate that miR-328 directly targets PAK6.

To investigate the functional roles of miR-328 in Pca progression, miR-328 or NC mimics were transfected into PC-3 and DU-145 CRPC cell lines. NC mimic-incorporated green fluorescent protein demonstrated high transfection efficiency (Fig. 3G). CCK-8 assay and nude mouse transplantation tumor experiments demonstrated that miR-328 overexpression inhibits PC-3 cell proliferation in vivo and in vitro (Fig. 3A–3C). In addition, reduced staining was observed in miR-328-treated xenografts (Fig. 3D). The proapoptotic effect of miR-328 was observed in PC-3 and DU-145 cells by flow cytometry assay (Fig. 3E and F). The same assay, however, indicated that miR-328 does not affect cell cycle progression (data not shown). The viability of caspase-3, -9 and bcl-2 was examined in cells following transfection. Compared with cells transfected with NC mimics, increased cleavage of caspase-3 and -9 and decreased bcl-2 were observed in cells transfected with miR-328 (Fig. 3H). These results indicate that miR-328 may inhibit cell growth and promote cell apoptosis.

A previous study demonstrated that PAK6 knockdown in prostate cell lines increases docetaxel sensitivity (8). To determine if miR-328 exerts a similar effect, assessment of docetaxel drug sensitivity following transfection with miR-328 was conducted. Cells were transfected with miR-328 or NC, and treated with docetaxel at concentrations of 5, 10, 20, 50 or 100 nM for 24 h. Subsequent to miR-328 transfection, the docetaxel drug concentration that inhibited 50% of cell proliferation significantly decreased from 25.45 to 8.30 nM (P<0.05) in PC-3 cells and from 36.63 to 16.78 nM (P<0.05) in DU-145 cells (Fig. 4).