A total of 25 pairs of ovarian cancer tissue and adjacent non-tumor tissue were collected at Nanjing Medical University Affiliated Wuxi Second Hospital (Wuxi, China) from patients during resection. Paired non-tumor tissues (adjacent to the tumor) were obtained from the same patient that the tumor tissue samples were collected, during the same procedure. The tissue samples were obtained during surgery and immediately frozen in liquid nitrogen until RNA extraction. The mean age of the patients was 51.5 years (range, 34.0–69.0 years). None of the patients had received radiation or chemotherapy prior to surgery. Written informed consent was obtained from all patients. The present study was approved by the Ethical Committee of Nanjing Medical University Affiliated Wuxi Second Hospital (Wuxi, China).

Ovarian cancer cell lines (ES2, HO8910, OVCAR3, A2780, SKOV3 and HeLa) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (GE Healthcare Life Sciences, Logan, UT, USA), at 37°C in a humidified atmosphere with 5% CO₂. miR-494 mimic and negative control oligonucleotide sequences were obtained from Shanghai GenePharma Co., Ltd., (Shanghai, China). The sequences of miR-494 mimic and negative control were as follows: miR-494 mimic forward, 5′-UGAAACAUACACGGGAAACCUC-3′ and reverse, 5′-GGUUUCCCGUGUAUGUUUCAUU-3′; the negative control forward, 5′-UUCUCCGAACGUGUCACGUTT−3′ and reverse, ACGUGACACGUUCGGAGAATT−3′. A total of 2×10⁵ cells were seeded into a six-well plate (Corning, Inc., Corning, NY, USA) and 100 nM miR-494 mimic and negative control were transiently transfected into cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) once the cells had reached 70–90% confluence, respectively.

Total RNA from the tissue samples and cell lines was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Reverse transcription was performed to obtain complementary DNA using a reverse transcription kit (PrimeScript™ RT reagent Kit with gDNA Eraser; Takara Bio, Inc., Otsu, Japan), which also contained DNase. Briefly, 1 µg RNA, 2 µl of 5X gDNA Eraser buffer and 1 µl of gDNA Eraser and RNase Free dH₂O were mixed and incubated at 42°C for 2 min to remove the DNA. The RT reaction system contained 10 µl of the aformentioned reaction solution, 1 µl of PrimeScript RT Enzyme Mix I, 1 µl of RT Primer Mix, 4 µl of 5X PrimeScript Buffer 2 and 4 µl of RNase Free dH₂O. The incubation conditions were 37°C for 15 min, followed by 85°C for 5 sec. A sample with no DNA was used as the negative control. All reactions were performed with an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression of miR-494 was analyzed by PCR using the SYBR^® Premix Ex Taq™ II kit (Takara Bio, Inc.). U6 was used for normalization. The primers were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China), and the sequences used were as follows: miR-494 forward, 5′-TGACCTGAAACATACACGGGA−3′ and reverse, 5′-TATCGTTGTACTCCACTCCTTGAC−3′; U6 forward, 5′-AAAGACCTGTACGCCAACAC-3′ and reverse, 5′-GTCATACTCCTGCTTGCTGAT−3′. The PCR cycling conditions were 95°C for 3 min, followed by 40 cycles at 95°C for 30 sec, 62°C for 30 sec and 72°C for 30 sec.

The expression of FGFR2 was examined using a SYBR^® Premix Ex Taq™ II kit (Takara Bio, Inc.). β-actin was used as the internal control. Experiments were repeated three times. The primer sequences were as follows (Genscript, Nanjing, China): FGFR2 forward, 5′-TGACATTAACCGTGTTCCTGAG-3′ and reverse, 5′-TGGCGAGTCCAAAGTCTGCTAT-3′; β-actin forward, 5′-GACCTCTATGCCAACACAGT-3′ and reverse, 5′-AGTACTTGCGCTCAGGAGGA-3′. The PCR cycling conditions were 94°C for 5 min, followed by 32 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec. The relative expression levels were evaluated by the 2^−ΔΔCq method (16).

Cell proliferation was assessed by CCK-8 assay. Cells were seeded into 96-well plates (Corning, Inc.) and transfected with miR-494 mimic or negative control as described previously. A total of 24, 48, 72, 96 and 120 h subsequent to transfection, 10 µl CCK-8 (Beyotime Institute of Biotechnology, Haimen, China) was added into 100 µl medium. Following 2 h incubation with the diluted CCK-8 in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), the cells were lysed in radioimmunoprecipitation assay buffer and centrifuged at 13,000 × g for 10 min at 4°C. The absorbance of the supernatants was measured at 450 nm on an imark microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell apoptosis was analyzed using a propidium iodide (PI)/Annexin V-fluorescein isothiocyanate (FITC) double staining cell apoptosis detection kit (KeyGEN BioTECH, Nanjing, China), according to the manufacturer's protocol, on a FACSCalibur™ (BD Biosciences, Franklin Lakes, NJ, USA). A total of 3×10⁵ cells were seeded into a 6-well plate (Corning, Inc.) and transfected with miR-494 mimic or negative control. A total of 48 h later, cells were collected and stained with Annexin V-FITC/PI, and the percentage of apoptotic and viable cells was determined by flow cytometry.

The wild-type and mutant 3′-UTRs of FGFR2 were synthesized and cloned into the luciferase reporter vector pGL3 (catalog no., E1761; Promega Corp., Madison, WI, USA). HeLa cells were seeded into 24-well plates (Corning, Inc.) and cultured until they reached 70% confluence. Subsequently, 100 ng wild-type or mutant pGL3-FGFR2-3′-UTR and 10 ng pRL-TK plasmid (E2241, Promega Corp.), together with 30 nM miR-494 mimic or negative control were cotransfected into cells using Lipofectamine 2000. The Renilla luciferase activity was analyzed at 48 h post transfection using the Dual-Luciferase^® Reporter Assay System (Promega Corp.) on a Lumat³ LB 9508 Single Tube Luminometer (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Renilla luciferase activity was normalized to firefly luciferase activity to control for transfection efficiency.

Following transfection, cells were washed twice with ice-cold phosphate-buffered saline, harvested using trypsin (Gibco; Thermo Fisher Scientific, Inc.) and lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Following 30 min of incubation, the lysed cells were centrifuged at 13,000 × g for 10 min at 4°C, and the supernatant was removed and frozen at −80°C. Total protein extracted was quantified with a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (80 µg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The following primary antibodies were used: FGFR2 (dilution, 1:500; rabbit monoclonal; catalog no., #11835; Cell Signaling Technology, Inc., Beverly, MA, USA) and β-actin (dilution, 1:1,000; rabbit polyclonal; catalog no., BA0410; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Following incubation with the primary antibodies overnight at 4°C, the membranes were washed with TBST and incubated with secondary antibody (dilution, 1:2,000; goat anti-rabbit; catalog no., BA1054; Wuhan Boster Biological Technology, Ltd.) at room temperature for 2 h. Protein bands were detected using an enhanced chemiluminescence detection system (Beyotime Institute of Biotechnology).

The independent Student's t-test was used to compare two preselected groups, which were expressed as the mean ± standard deviation. All statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the potential role of miR-494 in the development of ovarian cancer, the present study detected the expression level of miR-494 in 25 ovarian cancer tissues and 5 cell lines (ES2, HO8910, OVCAR3, A2780, SKOV3) by RT-qPCR. The level of miR-494 expression in tumor tissues was reduced compared with that in normal ovary tissues (P=0.012; Fig. 1A). Similarly, compared with the three normal ovarian tissues that were pooled and used as the normal control, the expression of miR-494 in the 5 ovarian cancer cell lines was reduced at different extent (A2780 vs. Non-tumor, P=0.017; HO8910 vs. Non-tumor, P=0.089; OVCAR3 vs. Non-tumor, P=0.28; ES2 vs. Non-tumor, P=0.057; SKOV3 vs. Non-tumor, P=0.015; Fig. 1B). Taken together, these results suggest that miR-494 may be involved in the development of human ovarian cancer.

To investigate the role of miR-494 on cell growth and apoptosis, the present study performed a gain-of-function analysis using A2780 and SKOV3 cell lines. miR-494 mimic or negative control was transiently transfected into the two cell lines, and RT-qPCR was performed to confirm the effect 48 h later. As demonstrated in Fig. 2A and B, the expression of miR-494 in A2780 [miR-494 vs. negative control (NC), P=0.019] and SKOV3 (miR-494 vs. NC, P=0.021) cells was significantly increased following transfection. CCK-8 was performed to detect the growth ability of cells. The results of this assay indicated that overexpression of miR-494 resulted in a significant decrease in cell proliferation in 120 h (Fig. 3A, miR-494 mimic vs. NC, P=0.011; Fig. 3B, miR-494 mimic vs. NC, P=0.038). The present study additionally examined the apoptotic changes following miR-494 transfection with miR-949 mimic. The results revealed that the apoptotic rate in miR-494-transfected cells was increased compared with the control (Fig. 3C, miR-494 vs. NC, P=0.017; Fig. 3D, miR-494 vs. NC, P=0.024). Taken together, these data suggest that miR-494 may inhibit ovarian cancer cell growth by inducing apoptosis.

According to the predictions made by the online software TargetScan (www.targetscan.org), Pictar (pictar.mdc-berlin.de/) and miRanda (www.microrna.org), a large number of genes were identified as direct targets of miR-494. The present study selected FGFR2 as a likely regulator of cell apoptosis. A luciferase reporter assay was performed in order to investigate whether FGFR2 was a target for miR-494. The wild-type (pGL3-FGFR2-WT) and mutant (pGL3-FGFR2-Mut) binding site sequences of miR-494 are listed in Fig. 4A. The results of this assay demonstrated that the luciferase activity of pGL3-FGFR2-WT in the two cell lines was markedly reduced compared with the mutant pGL3-FGFR2-Mut (miR-494 mimic vs. NC, P=0.043; Fig. 4B), which suggested that miR-494 may directly target FGFR2.

In order to confirm the endogenous regulatory role of miR-494 in relation to FGFR2 in ovarian cancer cells, the present study measured the level of FGFR2 in A2780 and SKOV3 cells following transfection with miR-494 mimic or negative control. The results of this investigation indicated that the FGFR2 mRNA and protein level were significantly downregulated in A2780 (miR-494 mimic vs. NC, P=0.031) and SKOV3 (miR-494 mimic vs. NC, P=0.037) cells that had been transfected with miR-494 mimic (Fig. 4C and D). Taken together, these results indicate that FGFR2 may be a direct target of miR-494.