Human ovarian tumor tissues were collected from the 30 patients who had ovarian epithelial carcinoma and non-neoplastic ovarian tissues from 30 healthy individuals under 60 years of age who received surgical resection between January 2009 and December 2012 at the Department of Gynecologic Oncology at Chongqing Cancer Hospital (Chongqing Cancer Institute, Chongqing, China). The present study was approved by the Ethics Committee of the Chongqing Cancer Hospital (Chongqing Cancer Institute, Chongqing, China). Written informed consent was provided by all patients prior to enrollment in the present study.

SKOV3 and OVCAR3 human ovarian cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) in a humidified atmosphere of 5% CO₂ at 37°C.

The miR-124 mimics and the miR-124 mimics scramble were synthesized by Shanghai GeneChem Co., Ltd. (Shanghai, China). The sequences (5′-3′) were as follows: hsa-miR-124-5p mimics forward, 5′-CGUGUUCACAGCGGACCUUGAU-3′ and reverse, 5′-AUCAAGGUCCGCUGUGAACACG-3′; miR-124 mimics scramble forward, 5′-UUCUCCGAACGUGUCAGU-3′ and reverse, 5′-ACGUGACACGUUCGGAGAA-3′. The pcDNA3.1/PDCD6 and pcDNA3.1/control were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were harvested and plated in six-well plates at a density of 5×10⁵ cells/ml. All cell transfections were performed using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. In the rescue experiment, cells were co-transfected with miR-124 mimics and plasmids.

Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) from clinical tumor samples and transfected cells according to the manufacturer's instructions. RNA concentration and quality were evaluated and 0.5 µg RNA was used for RT. MMLV reverse transcriptase (Takara Bio., Inc., Otsu, Japan) was used to synthesize complementary DNA (cDNA). For synthesis of miR-124 cDNA, a miR-124 RT primer was used and U6 small nuclear RNA was used as the internal control. cDNA for other target genes was amplified using the primer oligo(dT) with β-tubulin as the internal control. Target genes and controls were analyzed by RT-qPCR using SYBR Premix Ex Taq (Promega Corporation, Madison, WI, USA). The following primers were used for qPCR: miR-124 forward, 5′-TGCGGTAAGGCACGCGGTG-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′; U6 forward, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′; PDCD6 forward, 5′-TCCAGAGGGTCGATAAAGACA-3′ and reverse, 5′-TTCTGCCAGTCCGTGATGT-3′; β-tubulin forward, 5′-TTGGCCAGATCTTTAGACCAGACAAC-3′; and reverse, 5′-CCGTACCACATCCAGGACAGAATC−3′. PCR was conducted at 94°C for 4 min, followed by 40 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec in an iQ5 Real-time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All the samples were assessed by relative quantification (2^−ΔΔCq method) (22).

The prediction of the PDCD6 3′-UTR as a miR-124 binding target was determined using TargetScan (www.targetscan.org) and PicTar (www.pictar.org).

A luciferase reporter assay was used to monitor the direct interaction between the miRNA and its target mRNA. The binding site of miR-124 in the 3′-UTR of the target mRNA was cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation), according to the manufacturer's instructions. Cells were subsequently co-transfected with miRNA and the wild type or mutant 3′-UTR for the luciferase assay. Following transfection for 48 h at room temperature, the cells were harvested for protein extraction. Luciferase intensity was examined using the Dual Luciferase Reporter Gene Assay kit (Beyotime Institute of Biotechnology, Haimen, China), according the manufacturer's protocol. Renilla luciferase intensity was used an internal control.

Cell migration was evaluated using a wound-healing assay. At 24 h after transfection, a wound was created by 20 µl pipette tips. in all culture wells. Cultures were imaged at 0, 24 and 48 h to assess cell migration. Light microscopy was undertaken at ×100 magnification at 0, 24 and 48 h to assess cell migration.

The present study used a Transwell assay with a Matrigel-coated membrane matrix (BD Biosciences, Franklin Lakes, NJ, USA) to assess cell invasion. After transfection, cells (5×10⁴) were seeded in the upper chamber of a Millicell Transwell chamber (EMD Millipore, Billerica, MA, USA) with 200 µl RPMI-1640 medium without FBS. Cells that migrated through the membrane were fixed with 4% methanol for 10 min at room temperature. and stained with 0.1% crystal violet for 15 min at room temperature. Cells were then imaged at ×100 magnification and counted using an inverted microscope.

SKOV3 and OVCAR3 cells were plated onto 96-well plates 48 h after transfection at a density of 5×10⁴ cells/ml. A total of three replicate wells were examined for each group. When cells reached 60–70% confluency, 10 µl 5 mg/ml MTT was added to each well. After 4 h, the culture medium was discarded, 100 µl dimethyl sulfoxide was added to each well and the plates were agitated at room temperature for 1 h. Absorbance (optical density) was evaluated using a microplate reader at a wavelength of 490 nm. This experiment was repeated three times.

At the indicated time, SKOV3 and OVCAR3 whole-cell lysates were extracted using radioimmunoprecipitation assay buffer and incubated for 30 min on ice. Cell lysates were prepared from cells seeded on 6-well plates at 10⁶ cells per well using radioimmunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 1% NP- 40, 1 mM MgCl2) containing protease inhibitors at 48 h post-transfection.

Proteins were quantified using the BCA method, according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.). Proteins (10 µg) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked in 5% non-fat milk/TBST for 1 h at room temperature. Thereafter, they were incubated with rabbit monoclonal anti-PDCD6 antibody (1:1,000; ab133326) and anti-β-tubulin antibody (1:1,000; ab6046) (Abcam, Cambridge, UK) overnight at 4°C. After adding peroxidase-conjugated goat anti-rabbit antibody (1:5,000; BA1055; Boster Biological Technology, Pleasanton, CA, USA) for 1 h at room temperature, the membranes were visualized by Western Lightning^®-ECL, Enhanced Chemiluminescence Substrate (PerkinElmer, Inc., Waltham, MA, USA, and X-ray film was applied to analyze the image and intensity of band.

At 48 h after transfection, 1×10⁶ cells were seeded onto 75-mm dishes. To analyze the cell cycle stage, the cells were harvested, by trypsin and washed twice with cold PBS, fixed in ice-cold 70% ethanol and stained with 100 µg/ml propidium iodide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 1 mg/ml RNase A for 30 min at room temperature in the dark. Subsequently, the cell cycle stages were analyzed using a flow cytometer and Cell Quest software version 3.3 (BD Biosciences). To analyze apoptosis, the cells were harvested from the culture dishes by trypsinization. After washing twice with cold PBS, cells were resuspended in 200 µl binding buffer and incubated with 10 µl Annexin V-R-PE and 10 µl 7-ADD (SouthernBiotech, Birmingham, AL, USA) in the dark at 4°C for 30 min. Thereafter, 380 µl binding buffer was added to each tube and the samples were analyzed by flow cytometry.

All data were presented as the mean ± standard deviation. Error bars represent standard error. One-way analysis of variance, followed by Fisher's least significance test or two-tailed Student's t-test were used to analyzed the statistical significance. P<0.05 was considered to indicate a statistically significant difference.

To analyze the expression level of miR-124 in ovarian cancer progression, RT-qPCR was performed using TaqMan probes. Ovarian cancer tissue and normal ovarian tissue have distinct pathological patterns (Fig. 1). The present study compared the expression levels of 30 clinical tumor tissue samples and 30 non-neoplastic ovarian tissues (Fig. 1C) and revealed that the miR-124 expression level was significantly decreased in the majority of ovarian cancer tissues compared with in the non-neoplastic control tissues (Fig. 1D).

To investigate the biological role of miR-124 in ovarian cancer, the present study observed its effect on the migration and invasion of the ovarian cancer SKOV3 and OCVAR3 cell lines by transient transfection with miR-124 mimics, miR-124 mimics scramble or negative control (NC). The wound-healing assay indicated that transfection with miR-124 mimics significantly inhibited migration in both cell lines relative to the scramble control or NC at 24 and 48 h (Fig. 2A and B). Furthermore, ectopic expression of miR-124 markedly decreased invasion of the cell lines compared with the scramble control or NC (Fig. 2C-F).

To investigate the potential targets of miR-124, the present study performed bioinformatics analysis using TargetScan and Pictar (Fig. 1E), which predicted that miR-124 targets the PDCD6 3′-UTR region. To determine whether the 3′-UTR of PDCD6 mRNA is a direct target of miR-124 in ovarian cancer cells, the wild-type (wt) full-length 3′-UTR of PDCD6 or a mutant (mt) sequence was cloned into a luciferase reporter vector (Fig. 1G). Cells were co-transfected with wt 3′-UTR or mt 3′-UTR vectors and miR-124 mimics. The results indicated that overexpression of miR-124 significantly decreased the luciferase activity of reporter genes with wt 3′-UTR compared with the controls. The luciferase activity for the mt 3′-UTR vector was not affected by transfection with miR-124 (Fig. 1F).

Transfection with miR-124 mimics or pcDNA3.1/vector and miR-124 mimics significantly decreased ovarian cancer cell proliferation and migration compared with the controls (Fig. 3). Conversely, co-transfection with miR-124 mimics or pcDNA3.1/PDCD6 in SKOV3 or OCVAR3 cells induced recovery of cell migration and invasion. To investigate whether miR-124 affects cancer cell migration and invasion through PDCD6, the present study performed rescue experiments by upregulating PDCD6 expression in SKOV3 and OCVAR3 cells using pcDNA3.1/PDCD6. The expression level of PDCD6 mRNA was evaluated by qPCR (Fig. 4A and B). The expression level of miR-124 significantly increased subsequent to transient transfection with miR-124 mimics; however, the expression level of PDCD6 mRNA markedly decreased, thus miR-124 downregulated PDCD6 mRNA expression level. Transient co-transfection of miR-124 mimics together and pcDNA3.1/PDCD6 rescued the expression level of PDCD6 mRNA. Similar results were obtained by western blot analysis of SKOV3 and OCVAR3 cells co-transfected with miR-124 mimics or NC and pcDNA3.1/PDCD6 or pcDNA3.1/vector (Fig. 4C). The incidence of apoptosis and cell cycle arrest following overexpression of miR-124 and overexpression of PDCD6 were confirmed by flow cytometry. The results demonstrated that transfection of miR-124 mimics markedly increased cell death (Fig. 4D) and the proportion of SKOV3 and OVCAR3 cells in the G₀/G₁ cell cycle phase, but decreased the proportion of cells in S phase (Fig. 4E). However, these effects were reversed by co-transfection of miR-124 mimics and pcDNA3.1/PDCD6.