The IF11 and IA8 human prostate cancer cell lines (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (Shanghai Genebase Gen-Tech Co., Ltd., Shanghai, China) at 37°C in a 5% CO₂ incubator.

Normal prostate epithelial tissues were obtained from patients at the Department of Pathology of Tangdu Hospital (Xi’an, China). All patients provided written informed consent for the use of the excess pathological specimens for research purposes. The use of human tissues in the present study was approved by the Institutional Review Board of The Fourth Military Medical University (Xi’an, China) and was conducted in accordance with the International Guidelines for the Use of Human Tissues.

The hsa-miR-429 mimics, hsa-miR-429 inhibitor, negative control miRNA mimics (Mock/mimics NC) and negative control miRNA inhibitor (inhibitor NC) were chemically synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Prior to transfection, the logarithmically growing cells were harvested and seeded in 6-well plates (4×10⁵ cells per well), 24-well plates (1×10⁵ cells per well) or 96-well plates (1×10⁴ cells per well). Following overnight proliferation, RNA oligonucleotides (mimics NC/inhibitor NC or hsa-miR-429 mimics/inhibitor) were transfected into the adherent cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

A luciferase reporter assay was conducted using pMIR-REPORT™ vectors (Guangzhou RiboBio Co., Ltd., Guangzhou, China). For recombinant vector construction, a full-length 3′UTR of the p27^Kip1 gene was cloned and inserted downstream of the firefly luciferase gene in the pMIR-REPORT plasmid as follows: cDNA from the IA8 cells was amplified by polymerase chain reaction (PCR) using p27^Kip1-3′UTR-wild-type (wt) primer for p27^Kip1-3′UTR-wt cloning. The PCR products were then digested with MluI and SacI (Takara Bio, Inc., Shiga, Japan), and inserted into the multiple cloning site of the pMIR-REPORT Luciferase vector (Ambion^®; Thermo Fisher Scientific, Waltham, MA, USA). The recombinant vector was designated as pMIR-p27^Kip1-3′UTR-wt. Using this as a template, the pMIR-p27^Kip1-3′UTR-mutant (mut) plasmid, which carried the mutated p27^Kip1 3′UTR sequence in the complementary site for the seed region of miRNA-429, was generated by overlap PCR using p27^Kip1-3′UTR-mut-1 and p27^Kip1-3′UTR-mut-2 primers. The primers used in the PCR are shown in Table I. The cells were transiently cotransfected with miR-429 mimics or miR-NC mimics and the pMIR-p27^Kip1-3′UTR-wt/pMIR-p27^Kip1-3′UTR-mut vector. Luciferase activity was measured 48 h after transfection with a Dual-Luciferase assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA (2 μg) from each sample was used for cDNA synthesis using RT primers for miR-429 and U6 small nuclear RNA (Guangzhou Ribobio Co., Ltd). qPCR was performed using the SYBRII Premix Ex Taq™ (Takara Bio, Inc., Shiga, Japan). The amplification was performed under the following thermal program: Initial denaturation (95°C, 20 sec), 40 cycles of 95°C for 10 sec, 60°C for 20 secs and 70°C for 10 sec. The qPCR primer sets for miRNA-429 and U6 small nuclear were purchased from Guangzhou Ribobio Co., Ltd. miR-429 and U6 small nuclear RNA were quantified according to a standard curve and this was performed in triplicate. U6 small nuclear RNA was used for normalization. The relative expression levels of miRNA-429 were calculated using the following equation: Copies miR-429/copies U6. The quantitative analysis of the change in expression levels was calculated by qPCR analysis software (Bio-Rad CFX manager 2.0; Bio-Rad, Hercules, CA, USA).

Total proteins were isolated 24 h after transfection. The protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad). Equal quantities of total protein (20 μg) were separated on a 12% SDS-polyacrylamide gel, then transferred to a polyvinylidene membrane (Millipore, Billerica, MA, USA). Subsequent to blocking, the membrane was incubated with mouse monoclonal anti-p27^Kip1 antibody (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or rabbit polyclonal anti-β-actin antibody (1:1,000; Bioss Biotechnology, China) followed by incubation with polyclonal goat anti-mouse or polyclonal goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.). Signals were determined with a chemiluminescence detection kit (NEN™ Life Science Products, Inc., Boston, MA, USA).

The cells were seeded in 96-well plates at ~1×10⁴ cells/well and cultured in growth medium. At 0, 24, 48 and 72 h after miR-429 mimic/inhibitor transfection, the effect of miR-429 overexpression/knockdown on cell viability was determined by an MTT assay. Each experiment was performed in triplicate. The absorbance value of each well was measured with a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) at 570 nm. All proliferation assays were repeated as independent experiments at least three times. For the colony formation assay, the transfected cells and control cells were plated on 10-cm plates (500 cells/plate), respectively, cultured for another 14 days, fixed with 10% formaldehyde for 5 min, stained with 1.0% crystal violet for 30 sec and counted without microscopy.

The cells were harvested by trypsinization, washed with ice-cold phosphate-buffered saline, fixed in 75% ice-cold ethanol and stained with propidium iodide (10 mg/ml; 15 min; Invitrogen). A total of 2×10⁴ cells were analyzed with a FACSCalibur Flow Cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The experiments were performed in triplicate.

All experiments were repeated at least three times and data are expressed as the mean ± standard deviation. The statistical significance between groups was determined using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-429 were examined in two prostate cancer cell lines and six normal prostate epithelial tissues by qPCR. The results revealed that miR-429 expression was upregulated in the IF11 and IA8 prostate cancer cell lines, compared with the normal prostate epithelial tissue (Fig. 1), indicating a potential role for miR-429 in the tumorigenesis of prostate cancer.

In order to investigate the effect of miR-429 on prostate cancer cell proliferation, the has-miR-429 inhibitor was transfected into the IF11 and IA8 prostate cancer cells. Colony formation assays and MTT were employed to analyze cell proliferation. As shown in Fig. 2A and B, downregulation of miR-429 in IF11 and IA8 cells significantly inhibited cell proliferation (P<0.05), compared with the NC transfection. To further demonstrate the effect of miR-429 on cell proliferation, has-miR-429 mimics was transfected into IF11 and IA8 prostate cancer cells; compared with the mock group, the proliferation of tumor cells in the miR-429 mimics transfected group was significantly increased (P<0.05; Fig. 2C and D).

An important characteristic of tumor cells is the increased proliferative capability, which commonly results from impaired regulation of the cell cycle. Thus, the effect of miR-429 on the prostate cancer cell cycle was investigated using flow cytometry. As shown in Fig. 3, miR-429 mimic-transfected IF11 and IA8 cells exhibited lower percentages of cells in the G1 phase (IF11, 51.39%; IA8, 54.37%) and increased percentages of cells in the S (IF11, 42.47%; IA8, 40.04%) and G2/M phases (IF11, 6.14%; IA8, 5.59%), compared with the mock control group cells (G1: IF11, 62.57%; IA8, 63.55%; S: IF11, 28.69%; IA8, 27.25%; and G2/M: IF11, 8.74%; IA8, 9.21%). However, the miR-429 inhibitor-transfected IF11 and IA8 cells exhibited higher percentages of cells in the G1 phase (IF11, 72.39%; IA8, 74.63%) and reduced percentages of cells in the S (IF11, 21.72%; IA8, 18.95%) and G2/M phases (IF11, 5.89%; IA8, 6.42%), compared with the mock control groups. These results indicated that downregulation of miR-429 mainly arrested IF11 and IA8 cells in the G1 phase, which may result in cell proliferation inhibition.

miRNAs usually exert effects by binding to the 3′UTR of target genes; thus, the targets of miR-429 mimics were investigated to elucidate the underlying mechanism of this effect in IF11 and IA8 prostate cancer cells. Bioinformatic analysis using the miRanda algorithm (http://www.microrna.org) and target scan (http://www.targetscan.org/) indicated that the 3′UTR of p27^Kip1 contains a predicted binding site for miR-429 (Fig. 4A). To verify whether p27^Kip1 is a direct target of miR-429, a Dual-luciferase reporter system using pMIR-REPORT™ luciferase vectors containing wt or mutant p27^Kip1 3′UTR was employed (Fig. 4A). Cotransfection with miR-429 significantly suppressed the luciferase activity of the reporter containing wt 3′UTR but did not suppress the mutant reporter (P<0.05; Fig. 4B). Consistent with these results, transfection with miR-429 mimics significantly reduced the endogenous p27^Kip1 mRNA and protein expression levels in IF11 and IA8 cells (P<0.05; Fig. 4C and D). In conclusion, these data suggest that p27^Kip1 is a direct target gene of miR-429.

To further confirm whether miR-429 mediates tumorigenic effects through p27^Kip1 in prostate cancer cells, IA8 cells were cotransfected with miR-429 mimics and the p27^kip1 expression plasmid; the increased p27^Kip1 expression levels were confirmed by PCR and western blotting, as shown in Fig. 5A. MTT analysis revealed that cotransfection of the p27^Kip1 expression plasmid partially reversed the effect of miR-429 on cell proliferation (Fig. 5B). Furthermore, cotransfection of the p27^Kip1 expression plasmid also partially abrogated the effect of the miR-429 mimics on G1 phase arrest (Fig. 5C). These findings demonstrate that the cell cycle arrest effect of miR-429 is achieved, at least in part, by the direct downregulation of p27^Kip1 expression. Consequently, cell cycle arrest results in proliferation inhibition in prostate cancer cells.