Between 2014 and 2015, 26 ovarian cancer tissues and para-carcinoma tissues were obtained from patients through surgery at Hepatobiliary and Enteric Surgery Research Center, Xiangya Hospital, and were diagnosed with ovarian cancer based on histopathological evaluation according to the International Federation of Obstetrics and Gynecology (FIGO) criteria. All patients were operated without any local or systemic treatment. All samples were collected and frozen in liquid nitrogen immediately, and then stored at −80°C for RNA or protein extraction. All participants provided consent for the use of their specimens in this research, and the study was approved by the Institute Research Ethics Committee of Xiangya Hospital. All procedures performed in studies involving human participants were in accordance with the Ethical Standards of the Institutional and/or National Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Human ovarian surface immortalized epithelial cells (IOSE25) were cultured as described (16). Human ovarian cancer cell line SKOV3 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The ovarian cancer cell SKOV3 was cultured according to the vendor's instructions. In brief, cells were cultured in McCoy's 5A medium modified (ATCC) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), and the medium contained 1% penicillin-streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin). All cells were cultured and maintained under standard conditions.

PAX2 3′UTR containing the putative binding sites for miR-497 was cloned from the genome DNA and constructed into downstream of psi-CHECK2 vector (Promega), named PAX2-3′UTR-WT. PAX2 mutant 3′UTR recombinant plasmid was produced by QuikChange Site-Directed Mutagenesis kit (Stratagene, USA), which generated a mutation of 7 bps from UGCUGCU to ACGACGG in the predicted miR-497 target binding sites, named as PAX2-3′UTR-MUT. All plasmids were confirmed by DNA sequencing.

The miR-497 mimics and mock were purchased from a commercial manufacturer (Ribobio, China). PAX2-siRNA (5′-GAAGUCAAGUCGAGUCUAU-3′) and negative control siRNA (NC-siRNA) (5′-AGGUAGUGUAAU CGCCUUGTT-3′) were obtained from Genechem Biotech (Shanghai, China). Cells (1×10⁴) were seeded in a 24-well plate and incubated for 24 h, then ovarian cancer cells were transfected with mimics (100 mM), siRNA (100 mM) or vectors (2 µg) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in serum-free medium in accordance with the manufacturer's instructions.

Total RNA from tissues or cultured cells was extracted using TRIzol reagent (Invitrogen Inc.). Two micrograms of total RNA was used for cDNA synthesis by using M-MLV Reverse Transcriptase (Promega, USA). The relative expression of miRNA-497 was analyzed using a SYBR PrimeScript miRNA RT-PCR kit (Takara, China) and results were normalized to U6. SYBR Green Real-Time Master Mix (Toyobo, Japan) was used to perform quantitative real-time PCR to detect the relative expression of PAX2, and GAPDH was used as an internal control. The gene-specific primers were synthesized by Sangon (Shanghai, China) (Table I). qRT-PCR and data collection were performed on Applied Biosystems 7500 Sequence detection system (ABI, USA). The miR-497 and PAX2 expression levels were calculated using the 2^−ΔΔCt method.

Total proteins from tissues or cultured cells were extracted with SDS lysis buffer (Beyotime, China) and BCA protein assay kit (Pierce, USA) was used to detect the protein concentration. Equal amounts of total proteins were boiled using loading buffer for 10 min and separated in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF) (Millipore, USA). The membranes were blocked in Tris-buffered saline (TBS) containing 5% nonfat-dried milk at 37°C for 2 h and incubated with PAX2 antibodies (1:800; Abcam) for 1 h at room temperature, then membranes were incubated with goat anti-rabbit IgG-HRP (1:2,000; Santa Cruz Biotechnology, USA) at 37°C for 1 h. Proteins were detected by enhanced chemiluminescence (ECL; Beyotime). GAPDH (1:2,000; Abcam), a constitutive expression gene, was used as an internal control.

Cells (100 µl) (1×10⁶/ml) were cultured in 24-well plates and incubated for 24 h. miR-497 mimics or Mock (100 nM) were co-transfected with 20 ng PAX2-3′UTR-WT or -MUT psi-CKECK2 vectors into cells using Lipofectamine 2000 reagent. Dual-luciferase reporter assay system (Promega) was used to detect luciferase activities after 48 h of transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.

The cell proliferation was analyzed using Cell Counting Kit-8 (Dojindo, Japan). Ovarian cancer cells (1×10⁴) were seeded in 96-well plate. After transfected with mimics or siRNA, cells were cultured with 10% FBS for 24, 48 and 72 h. Then, the cells were treated with 10 µl CCK-8 solution at 37°C for 2 h, The absorbency at 450 nm was measured using a microplate reader Thermo Plate (Rayto Life and Analytical Science Co., Ltd., Germany).

Transwell insert (24-well) with 8-µm pore size (Corning Costar Corporation) was used to perform migration assay and invasion assay. For migration assay, 1×10⁴ SKOV3 cells were plated in 100 µl medium without FBS into upper chamber of Transwell insert with non-coated membrane. For invasion assay, 1×10⁵ SKOV3 cells suspended in 100 µl serum-free medium were loaded in the upper Matrigel-coated chamber instead. In both assays, 500 µl of culture medium containing 15% FBS was added to the lower chamber for culture. Then, cells were allowed to migrate or invade for 24 h at 37°C. The migrated or invaded SKOV3 cells were fixed with 100% methanol for 30 min at room temperature and stained with 0.5% crystal violet (Sigma, USA) for 15 min, and the permeating cells were counted under a phase-contrast microscope (Olympus, Tokyo, Japan).

Annexin V-FITC/PI apoptosis detection kit (Sigma) was used to detect cell apoptosis by flow cytometry. In brief, after transfected with mimics or siRNA for 48 h, 1×10⁶ SKOV3 cells were harvested and stained using the Annexin V-FITC/PI apoptosis detection kit according to the manufacturer's instructions. Cells were analyzed using Becton-Dickinson flow cytometer. Annexin V-FITC(+)/PI(−) and Annexin V-FITC(+)/PI(+) represent the early cell apoptosis and late apoptosis/necrosis, respectively.

All results are expressed as the mean ± SD from three independent experiments. Statistical analysis was performed using SPSS 20.0 software (SPSS Inc., USA). Statistical significance between groups was determined using a Student's t-test or one-way analysis of variance (ANOVA) test. P-value of <0.05 was considered to be statistically significant.

The expression levels of miR-497 in human ovarian cancer tissues and cells were investigated using qRT-PCR. miRNA-497 was significantly decreased in ovarian cancer tissues compared with matched adjacent normal tissues (Fig. 1A). Furthermore, the expression of miR-497 was significantly downregulated in SKOV3 cells compared to IOSE25 cells (Fig. 1B).

To determine the role of miR-497 in cell proliferation, migration and invasion, SKOV3 cells were transfected with miR-497 mimics (Fig. 2A). The SKOV3 cell proliferation was significantly inhibited after transfection of miR-497 mimics (Fig. 2B). Migration and invasion abilities of SKOV3 cells after overexpression of miR-497 were analyzed using Transwell assay. The SKOV3 cell migration and invasion were significantly suppressed after transfection of miR-497 mimics (Fig. 2C and D).

To evaluate the effect of miR-497 on SKOV3 cell apoptosis, cell apoptosis assay was performed using the Annexin V-FITC/PI staining method by flow cytometry. The results showed that overexpression of miR-497 induced SKOV3 cell apoptosis (Fig. 3).

Firstly, the protein expression of PAX2 in human ovarian cancer tissues and cells was detected using western blot analysis, the result showed PAX2 was increased in tumor tissues (Fig. 4A) and SKOV3 cells (Fig. 4B). In addition, to determine that miR-497 regulates PAX2 expression directly in ovarian cancer, the expression of PAX2 in miR-497 mimic-transfected SKOV3 cells was analyzed using western blot analysis (Fig. 4C). Furthermore, miRNA target predication databases (TargetScan: www.targetscan.org and MICRORNA.ORG: www.microrna.org) were used for computational analyses. miR-497 has one predictive target site in the human PAX2 3′UTR (Fig. 4D). Moreover, dual-luciferase assay was performed to verify whether PAX2 is a direct target gene of miR-497. The results showed that miR-497 mimics suppressed the luciferase activity of the reporter (Fig. 4E). Our results indicated that this site of PAX2 3′UTR was the exact regulation site for miR-497.

To determine the role of PAX2 in cell proliferation, migration and invasion, PAX2 was knocked down using small interference RNA (Fig. 5A and B). CCK-8 assay results showed SKOV3 cell proliferation was significantly suppressed after PAX2-siRNA transfection (Fig. 5C). Transwell assay was performed to analyzed cell migration and invasion, the results showed knockdown of PAX2 inhibited SKOV3 cell migration and invasion (Fig. 5D and E).

To investigate the effect of PAX2 on ovarian cancer cell apoptosis, the Annexin V-FITC/PI staining method was used to perform apoptosis assays by flow cytometry. The results showed that knockdown of PAX2 promoted SKOV3 cell apoptosis (Fig. 6).