The human OC SKOV-3, A2780 and OVCAR-3 cell lines, and the normal ovarian epithelial ISOE-80 cell line were purchased from the American Type Culture Collection. A2780 and OVCAR-3 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (both Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (HyClone; Cytiva). ISOE-80 cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. SKOV-3 cells were cultured in McCoys 5A medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. All cells were cultured at 37°C with 5% CO₂.

The chemically synthesized miR-134-3p mimic (miRBase, MIMAT0026481; 5′-CCUGUGGGCCACCUAGUCACCAA-3′), non-targeting mimic negative control (NC; 5′-UUCUCCGAACGUGUCACGUTT-3′), Lv-FEN1 and Lv-NC (expression vector, pcDNA3.1) were designed and obtained from Shanghai GenePharma Co., Ltd. Cell transfection was performed in SKOV-3 and OVCAR-3 cells using the cell electroporation system operator H1 (Etta Biotech Co. Ltd.) according to the manufacturers protocol. Briefly, SKOV-3 and OVCAR-3 cells were collected and re-suspended in the electroporation buffer. The cell concentration was adjusted to 6×10⁵ cells/ml and mixed with miRNA mimics (125 nM) and/or plasmid (1 mg/ml) prior to electroporation. A total of 100 µl cell suspension mixed with RNAs was then added into a 96-well plate, and used for gene transfection using the operator H1. The electroporation device was operated at DC square wave with 200 V voltage, 100 µsec duration, 2-sec interval and 6 pulses. After electroporation, the cells were diluted to appropriate concentrations and seeded into appropriate cell culture plates for use in further assays.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for the isolation of total RNA from cells. Reverse transcription was performed using the 4X Reverse Transcription Master Mix kit (EZBioscience) according to the manufacturers protocol. qPCR was performed on cDNA using FastStart Universal SYBR-Green Master Mix (Roche Diagnostics GmbH). Briefly, the reaction mixture contained 25 µl SYBR-Green Master Mix, 0.5 µl each primer (30 µmol/l), 5 µl DNA template (10 ng/µl) and molecular-grade H₂O to a final reaction volume of 45 µl. The amplification protocol consisted of one cycle of initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min. Gene expression levels were quantified using the 2^−∆∆Cq method (21). U6 and β-actin were used to normalize miR-134-3p or FEN1 expression, respectively. The PCR primer sequences for the selected genes were as follows: FEN1 forward, 5′-GTGAAGGCTGGCAAAGTCTA-3′ and reverse, 5′-GTGAAGGCTGGCAAAGTCTA-3′; β-actin forward, 5′-GCTCGTCGTCGACAACGGCTC-3′ and reverse, 5′-CAAACATGATCTGGGTCATCTTCTC-3′; miR-134-3p forward, 5′-CTGTGGGCCACCTAGTCACCAA-3′ and reverse, 5′-GCTGTCAACGATACGCTACCTA-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

The transfected SKOV-3 and OVCAR-3 cells were seeded in 96-well plates at a density of 5×10³ cells/well. The CCK-8 (Beyotime Institute of Biotechnology) colorimetric assay was performed to measure cell proliferation according to the manufacturers protocol. Briefly, the supernatant was removed after 24, 48 and 72 h of culture at 37°C and 5% CO₂. Subsequently, 100 µl of respective medium containing 10 µl CCK-8 reagent was added to each well for 3 h at 37°C. The absorbance was measured at 450 nm using a plate reader (Thermo Multiskan MK3 spectrophotometer; Thermo Fisher Scientific, Inc.). The optical density value was determined and used to construct a growth curve to assess cell proliferation.

SKOV-3 and OVCAR-3 transfected cells were seeded in 10-cm dishes (1×10³ cells/plate) and treated as follows: First, the cells were cultured in complete culture medium for ~21 days; subsequently, they were fixed in 4% paraformaldehyde for 30 min at 4°C and stained with Giemsa dye for 20 min at 4°C. Images of cells were captured using a light microscope (magnification, ×40), and the number of colonies, consisting of ≥3 cells, was calculated.

The transfected SKOV-3 and OVCAR-3 cells were cultured for ~48 h at 37°C and 5% CO₂. The apoptosis of the transfected cells was then detected by TUNEL assay as recommended in the ApopTag^® Plus Peroxidase in Situ Apoptosis kit (Sigma-Aldrich; Merck KGaA). Briefly, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C. Subsequently, the cells were incubated with 1% TdT enzyme in a humidified atmosphere at 37°C for 90 min. Subsequently, the cells were stained with DAPI (Sigma-Aldrich; Merck KGaA) at 4°C for 10 min. Finally, the cells were observed under a fluorescence microscope (magnification, ×200). An average of 10 random fields with 100–200 nuclei per field was analyzed. The number of TUNEL-positive nuclei (green fluorescence) was expressed as the percentage of total nuclei (blue fluorescence).

The effects of miR-134-3p on the cell cycle were measured by flow cytometry as previously described (10). Briefly, the transfected cells were harvested, washed and suspended in 250 µl of ice-cold PBS. Subsequently, 750 µl of 100% ice-cold ethanol were added and slowly mixed with the cell suspensions for ~8 h for fixation on ice. Subsequently, the cells were washed with PBS and incubated with RNase (50 µg/ml) and propidium iodide (50 µg/ml, Thermo Fisher Scientific, Inc.) for 30 min on ice. Finally, the cell cycle was detected using a Flow Cytometry System (BD Accuri C6; BD Biosciences) and the relative ratios of G0/G₁, S and G₂/M phases were analyzed using FlowJo VX software (v10.0.7; FlowJo LLC).

Wound healing assay was performed to examine the migration of the cells as previously described (22). Briefly, serum-starved SKOV-3 and OVCAR-3 cells were seeded in each well of a 6-well plate, and a wound was created using a 100-µl pipette tip. Following culture for a further 48 h at 37°C, the wound recovery area was evaluated using a light microscope (Nikon Corporation; magnification, ×200).

To explore the migration and invasion of SKOV-3 and OVCAR-3 cells, the Transwell chamber assay was performed as follows. The transfected SKOV-3 and OVCAR-3 cells (3×10⁴) were seeded into the upper chamber of a Matrigel pre-coated Transwell chamber and cultured in their respective serum-free medium. Complete culture medium was added to the lower chamber. Following 48 h of culture at 37°C, the cells in the lower chamber were fixed with 4% paraformaldehyde for 30 min at 4°C and stained with 5% crystal violet at 4°C for 30 min. The number of cells passing through the Matrigel matrix were counted and photographed under a light microscope (magnification, ×200).

Firstly, the starBase database (http://starbase.sysu.edu.cn/) was used for prediction of miR-134-3p target genes. R<-0.1 and P<0.05 were set as the cut-off criteria for identifying the significant miRNA/gene pairs. One of the identified target genes was FEN1. Subsequently, FEN1-wild-type (WT) or FEN1-mutant (MUT) reporter plasmids were synthesized from Shanghai GenePharma Co., Ltd. Briefly, SKOV-3 and OVCAR-3 cells were co-transfected with 0.24 µg of the FEN1-WT or FEN1-MUT reporter plasmids together with 40 nM of miR-134-3p mimic or NC mimic using the cell electroporation system operator H1, as aforementioned. Additionally, 0.05 µg of Renilla luciferase expression plasmid (Promega Corporation) was transfected into the cells as a reference control. The transfected cells were seeded into 24-well plates for 36 h of culture at 37°C. Finally, firefly and Renilla luciferase activities in the cells were measured using the Dual-Luciferase Reporter Assay System (Promega Corporation). The ratio of firefly and Renilla luciferase activities was calculated as the relative luciferase activity.

Cells were harvested using RIPA lysis buffer, Total proteins (40 µg/lane) were separated via 12% SDS-PAGE and were transferred onto PVDF membranes (EMD Millipore) using a MiniGenie blotting system (Bio-Rad Laboratories, Inc.). The membranes were then blocked with TBS-Tween (TBST; 0.1% Tween-20) containing 1% skim milk powder at room temperature for 1 h, and then incubated with rabbit monoclonal primary antibodies against human p21 (1:1,000; cat. no. 2947S), cyclooxygenase-2 (Cox-2; 1:1,000; cat. no. 12282T), matrix metalloproteinase (MMP)2 (1:1,000; cat. no. 40994S), MMP9 (1:1,000; cat. no. 13667S), cyclin D1 (1:1,000; cat. no. 55506S), CDK2 (1:1,000; cat. no. 2546S), Bax (1:1,000; cat. no. 5023S), cleaved caspase-3 (1:1,000; cat. no. 9654S), cleaved caspase-9 (1:1,000; cat. no. 20750S), Bcl-2 (1:1,000; cat. no. 4223S), β-actin (1:1,000; cat. no. 4970T) and FEN1 (1:2,000; cat. no. 82354S) (all from Cell Signaling Technology, Inc.) at 4°C overnight. After washing with TBST, the membranes were incubated with goat anti-rabbit secondary antibodies (1:10,000; cat. no. 14708S; Cell Signaling Technology, Inc.) for 1 h at room temperature, followed by visualization with an enhanced chemiluminescence system (BeyoECL Plus; Beyotime Institute of Biotechnology). The protein bands were quantified using ImageJ software (v1.48u; National Institutes of Health).

All experiments were performed in triplicate independently. Quantitative data are presented as the mean ± SD. GraphPad Prism (v6.01; GraphPad Software, Inc.) was used to perform the statistical analyses. The unpaired Students t-test was used to compare differences between two groups, while one-way ANOVA followed Bonferronis post-hoc test was used when comparing >2 groups. P<0.05 was considered to indicate a statistically significant difference.

To confirm whether miR-134-3p served a role in the progression of OC, its expression levels were compared between human ovarian cancer cell lines (SKOV-3, A2780 and OVCAR-3) and a normal ovarian epithelial cell line (ISOE-80) by RT-qPCR. The results revealed that the expression levels of miR-134-3p in the human OC cell lines were significantly lower than those in the normal ovarian cell line (P<0.01; Fig. 1A). As miR-134-3p expression was lowest in the SKOV-3 and OVCAR-3 cells, these two cell lines were selected for further analyses. Subsequently, to evaluate the effects of miR-134-3p on the progression of OC, SKOV-3 and OVCAR-3 cells were transfected with miR-134-3p mimic or NC mimic. The RT-qPCR results revealed that miR-134-3p mimic significantly upregulated miR-134-3p expression in SKOV-3 and OVCAR-3 cells (P<0.01; Fig. 1B). In addition, the CCK-8 assay revealed that miR-134-3p mimic significantly decreased the viability of SKOV-3 and OVCAR-3 cells after 72 h (P<0.05; Fig. 1C). Colony formation assay demonstrated that miR-134-3p mimic significantly decreased the proliferation of SKOV-3 and OVCAR-3 cells (P<0.01; Fig. 1D). Overall, these data demonstrated that miR-134-3p inhibited the proliferation of OC cells.

The regulation of apoptosis and cell cycle arrest serves an important role in cell proliferation (12,13). In the present study, to reveal the mechanisms of miR-134-3p in OC cell proliferation, TUNEL and flow cytometry assays were performed to examine the effects of miR-134-3p overexpression on cell apoptosis and the cell cycle. First, the results of the TUNEL assay indicated that the miR-134-3p mimic significantly facilitated SKOV-3 and OVCAR-3 cell apoptosis (P<0.01; Fig. 2A). Furthermore, the analysis of cell apoptosis-associated proteins by western blot analysis revealed that the miR-134-3p mimic significantly increased the expression levels of Bax, cleaved caspase-3 and cleaved caspase-9 (P<0.01), and significantly decreased those of Bcl-2 in SKOV-3 and OVCAR-3 cells (P<0.05; Fig. 2B). Subsequently, the data from the flow cytometry indicated that the miR-134-3p mimic significantly increased the percentage of cells at the G₀/G₁ phase, while decreasing the percentage of cells at the S and G₂/M phases in both SKOV-3 and OVCAR-3 cells (P<0.01; Fig. 2C). Finally, western blot analysis was performed to assess the expression levels of cell cycle-associated proteins. The results revealed that miR-134-3p mimic significantly decreased the protein expression levels of cyclin D1 and CDK2, while increasing the protein expression levels of p21 (P<0.05 and P<0.01, Fig. 2D). Overall, these results indicated miR-134-3p inhibited cell proliferation by facilitating cell apoptosis and inducing cell cycle arrest at the G₀/G₁ phase in OC.

To further investigate the effects of miR-134-3p on the migration and invasion of OC cells, wound healing and Transwell assays were performed. The results of the wound healing assay indicated that the miR-134-3p mimic significantly decreased the wound closure ability of SKOV-3 and OVCAR-3 cells (P<0.01; Fig. 3A), suggesting that miR-134-3p inhibited cell migration. Similarly, Transwell assays demonstrated that the miR-134-3p mimic significantly inhibited the migration and invasion of SKOV-3 and OVCAR-3 cells (P<0.01; Fig. 3B). At the molecular level, western blot analysis revealed that the miR-134-3p mimic significantly decreased the expression levels of the migration-associated proteins MMP2, MMP9 and Cox-2 (P<0.01; Fig. 3C). Collectively, these results demonstrated that miR-134-3p decreased the migratory and invasive abilities of OC cells.

To explore the detailed mechanisms responsible for the regulatory effects of miR-134-3p on OC progression, starBase was used to screen for miR-134-3p target genes, revealing that FEN1 may be one of the target genes of miR-134-3p (Fig. 4A). Subsequently, a Dual-Luciferase reporter assay was performed to verify this interaction. The results indicated that the miR-134-3p mimic significantly downregulated the luciferase activity of FEN1-WT, but not that of FEN1-MUT (P<0.01; Fig. 4B). The RT-qPCR results (Fig. 4C) and western blot analysis (Fig. 4D) demonstrated that the miR-134-3p mimic significantly downregulated the mRNA and protein expression levels of FEN1 (P<0.01). These data confirmed that miR-134-3p inhibited the expression levels of the FEN1 gene by directly binding to the 3′-UTR of FEN1 in OC cells.

To confirm the association between miR-134-3p and FEN1, miR-134-3p mimic or NC mimic were co-transfected with Lv-FEN1 or Lv-NC into SKOV-3 and OVCAR-3 cells. RT-qPCR revealed that Lv-FEN1 significantly increased the expression levels of FEN1 in SKOV-3 and OVCAR-3 cells (P<0.01; Fig. 5A). To examine the mediatory effects of FEN1 on OC cells, a CCK-8 assay was conducted, revealing that co-transfection with the miR-134-3p mimic + Lv-FEN1 or miR-134-3p mimic + Lv-NC significantly decreased the viability of SKOV-3 and OVCAR-3 cells compared with that of the cells co-transfected with NC mimic + Lv-NC (P<0.05; Fig. 5B). Additionally, the viability of SKOV-3 and OVCAR-3 cells co-transfected with miR-134-3p mimic + Lv-FEN1 was significantly higher than that of cells transfected with miR-134-3p mimic + Lv-NC (Fig. 5B). Colony formation assay revealed that, compared with Lv-NC, Lv-FEN1 significantly promoted the colony formation ability of SKOV-3 and OVCAR-3 cells transfected with miR-134-3p mimic (P<0.05; Fig. 5C). Moreover, TUNEL assay indicated that Lv-FEN1 significantly inhibited the apoptosis of miR-134-3p mimic-transfected SKOV-3 and OVCAR-3 cells compared with Lv-NC (P<0.01; Fig. 5D). Finally, Transwell assay revealed that Lv-FEN1 significantly increased the invasion of miR-134-3p mimic-transfected SKOV-3 and OVCAR-3 cells compared with Lv-NC (P<0.01; Fig. 5E). Overall, these results suggested that the overexpression of FEN1 partially reverses the effects of miR-134-3p overexpression on OC.