The present study was conducted in accordance with the Declaration of Helsinki 1991 and was approved by the Institutional Review Board of Tianjin Central Hospital of Gynecology and Obstetrics (Tianjin, China). Written informed consent was obtained from all patients. A total of 70 paired OC tissues and corresponding normal tissues were collected from patients (aged 28–72 years) at Tianjin Central Hospital of Gynecology and Obstetrics between February 2007 and August 2015. The following inclusion criteria were required: i) Good indication of suitability for surgical intervention; ii) must not have received radiotherapy or chemotherapy prior to surgery; and iii) complete clinical history. The following exclusion criteria were required: i) Previous surgical intervention; ii) severe liver and/or kidney dysfunction; iii) abnormal coagulation; and iv) incomplete clinical history. All clinical and pathological information was obtained from each patient's history record. An experienced gynecological pathologist at the hospital assessed all tissue specimens. After surgery, each patient was followed up every 3 months for a period of 60 months. The OS time was defined as the time between the date of surgery and the date of death or last follow-up. All clinical specimens were immediately snap-frozen in liquid nitrogen and subsequently stored at −80°C until required for RNA extraction.

The two human OC cell lines, SK-OV-3 (cat. no. HTB-77) and OVCAR-3 (cat. no. HTB-161), were purchased from the American Type Culture Collection. The human ovarian surface epithelial cell line HOSEpiC (cat. no. 7310) was obtained from ScienCell Research Laboratories, Inc. All cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences), supplemented with 10% FBS (HyClone; GE Healthcare Life Sciences) and 100 µg/ml penicillin/streptomycin. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

Two short hairpin (sh)RNAs targeting CCEPR, shCCERP1 (cat. no. H-4861, 5′-ATGTTATAGCTAAATGGATGTGACTAG-3′) and shCCEPR2 (cat. no. H-4862, 5′-CATTTTATGTCTTGACAATGCCTCGATTTG-3′), and the corresponding scrambled shRNAs [negative control (NC)], shNC1 (cat. no. H-4861-1, 5′-ACTGGCTCATTACCTGAGGAAATGTGT-3′) and shNC2 (cat. no. H-4862-1, 5′-AATGTTGGACCCTTTACAGTTGGAAATC-3′), were designed and cloned into psi-H1 vector from GeneCopoeia, Inc (Guangzhou, China). Sequences of all oligonucleotides were confirmed by Sanger sequencing (Thermo Fisher Scientific, Inc.). Upon reaching 80% confluence, cells in 6-well plates at 8×10⁶ cells/well were transfected with 2 µg/well shRNA or NC using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h at 37°C, according to the manufacturer's protocol. Cells were incubated with RPMI-1640 at 37°C supplemented with 10% FBS for 48 h prior to subsequent experimentation. The efficiency of cell transfection was analyzed using reverse transcription-quantitative PCR (RT-qPCR) analysis.

To measure the cell proliferation rate, a total of 3×10³ transfected OC cells/well were plated into 96-well plates and cultured overnight. Subsequently, 10 µl Cell Counting Kit-8 (CCK-8) solution (Dojindo Molecular Technologies, Inc.) was added to each well at the at 0, 24, 48 and 72 h. Following incubation for 3 h at 37°C, the absorbance was measured at 450 nm using an automatic microplate reader (Bio-Rad Laboratories, Inc.).

Total protein was extracted from cells with ice-cold mammalian cell total protein lysis buffer (Sangon Biotech Co., Ltd.) and centrifuged at 13,000 × g for 30 min to obtain the protein samples at 4°C. Total protein was quantified using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.) and 25 µg protein/lane was separated via SDS-PAGE on a 12% gel. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked with 5% non-fat milk in TBS-0.1% Tween 20 for 1 h at 37°C. The membranes were incubated with the following primary antibodies overnight at 4°C: Anti-Myc (1:1,000; cat. no. D199941; Sangon Biotech Co., Ltd.), anti-MMP-7 (1:2,000; cat. no. MAB9071-100; R&D Systems, Inc.), anti-cyclin D1 (1:1,000; cat. no. D198702; Sangon Biotech Co., Ltd.), anti-β-catenin (1:500; cat. no. D199519; Sangon Biotech Co., Ltd.) and anti-GAPDH (1:2,500; cat. no. D190090; Sangon Biotech Co., Ltd.). Following the primary antibody incubation, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,000; cat. no. D110098; Sangon Biotech Co., Ltd.) for 1 h at 37°C. Protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) and protein expression was semi-quantified using a v4.6.6 Quantity One software (Bio-Rad Laboratories, Inc.), with GAPDH as the loading control.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway online database (https://www.genome.jp/kegg/pathway.html) was used to predict the downstream signaling pathways of CCEPR.

Human Bcl-2 (cat. no. CSB-E08853h), Bax (cat. no. CSB-E09344h) and caspase-3 (cat. no. CSB-E08856h) ELISA kits (Cusabio Technology LLC) were used to analyze the expression levels of Bcl-2, Bax and caspase-3 in transfected cells. Briefly, a total of 1×10⁶ transfected OC cells were harvested and lysed with lysis buffer (Cusabio Technology LLC) provided by the kits on ice for 30 min at 4°C and centrifuged at 13,000 × g for 30 min to obtain the supernatants at 4°C. A total of 100 µl/well supernatant was added to the 96-well ELISA plates and incubated at 37°C for 30 min. Subsequently, 100 µl 1X HRP-conjugate solution was added to each well and incubated for 30 min at 37°C. Then, 90 µl tetramethylbenzidine substrate solution/well was added to the plates and incubated for 20 min at 37°C in the dark. Finally, 50 µl stop solution was added to each well to terminate the reaction and the optical density value at 570 nm was measured using an automatic microplate reader (Bio-Rad Laboratories, Inc.).

Total RNA was extracted from OC cell lines and cancer/normal tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The quality was assessed using a SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, Inc.) and 1 µg RNA was reverse transcribed into cDNA using a cDNA Synthesis kit (Toyobo Life Science), according to the manufacturer's protocol. qPCR was subsequently performed using the SYBR^® qPCR mix (Toyobo Life Science) and a 7900HT Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The following primer pairs (Sangon Biotech Co., Ltd.) were used for qPCR: CCEPR, forward, 5′-AAGGTCCCAGGATACTCGC-3′, reverse, 5′-GTGTCGTGGACTGGCAAAAT-3′; and GAPDH, forward, 5′-CGCTCTCTGCTCCTCCTGTTC-3′ and reverse, 5′-ATCCGTTGACTCCGACCTTCAC-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 60 sec; followed by 40 cycles at 95°C for 10 sec, 60°C for 30 sec and 72°C for 45 sec, finally at 4°C for 30 min. Expression levels were quantified using the 2^−ΔΔCq method (19) and normalized to the internal reference gene GAPDH.

A total of 7×10⁴ transfected cells/well were plated into 6-well plates and cultured for 48 h in 5% CO₂, prior to being harvested. Subsequently, cells were centrifuged at 1,000 × g for 10 min at 37°C and 1×10⁶ cells were fixed overnight with 70% ice-cold ethanol at 4°C. The cells were resuspended in PBS containing 50 mg/ml RNase A (cat. no. ST576; Beyotime Institute of Biotechnology) for 30 min at 37°C, and blocked with 10% goat serum (Solarbio Science & Technology Co., Ltd., Beijing, China) for 2 h at 37°C. Next, the cells were stained with 3 µl propidium iodide and 5 µl Annexin V-FITC (Thermo Fisher Scientific, Inc.) in the dark for 1 h at 37°C. The apoptotic cells were visualized using a BD FACSCalibur^™ flow cytometer (BD Biosciences) and subsequently analyzed using BD CellQuest^™ Pro version 5.1 software (BD Biosciences).

The invasion assay was performed using a 24-well Boyden chamber (pore size, 8 µm; Corning, Inc.). A total of 5×10⁴ transfected cells/well were resuspended in 200 µl serum-free RPMI-1640 medium and were plated in the upper chambers, which were precoated with Matrigel. A total of 500 µl RPMI-1640 medium supplemented with 10% FBS was plated in the lower chambers as a chemoattractant. After 48 h, the invasive cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min and subsequently stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) at 37°C for 15 min. Stained cells were counted in five randomly selected fields using an Olympus light microscope at ×200 magnification (Olympus Corporation).

Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.). Data are presented as the mean ± SD and are representative of ≥3 experimental repeats. The association between CCEPR expression and clinicopathological variables was analyzed using a χ² test; Kaplan-Meier curves and the log-rank test were used to analyze the association between CCEPR expression levels and OS in patients with OC; Student's t-tests were used to identify statistical differences between two groups; and differences among >2 groups were analyzed using one-way ANOVA followed by a Bonferroni's correction post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the potential role of CCEPR in the progression of OC, RT-qPCR was performed to analyze the expression levels of CCEPR in 70 OC and matched normal tissues. The expression levels of CCEPR were significantly increased in OC tissues compared with the matched normal tissues (Fig. 1A; P<0.05). Furthermore, CCEPR expression was also examined in two OC cell lines, SK-OV-3 and OVCAR-3. Compared with the HOSEpiC cell line, CCEPR expression levels were significantly increased in the two OC cell lines (Fig. 1B and C; P<0.05).

To determine the clinical value of CCEPR in OC, the relationship between the expression levels of CCEPR and clinicopathological variables was analyzed. Based on the median expression levels of CCEPR in OC tissues, patients with OC were divided into two groups: High expression group (n=35) and low expression group (n=35). Increased CCEPR expression levels were associated with an increased invasive ability and a higher International Federation of Gynecology and Obstetrics (FIGO) stage (Table I; P<0.05), but not with patients' age, pathological type, differentiation, tumor size, CA125 levels or lymph node metastasis. Kaplan-Meier curves revealed that the 5-year OS rate was significantly lower in patients within the high expression group (11.43%) compared with patients in the low expression group (25.71%; Fig. 2A; P<0.05). The median survival time of patients with OC in the low expression group was 45.0 months, whereas the median survival time of patients with OC in the high expression group was 19.6 months. These data suggested that increased CCEPR expression levels may be related to aggressive clinicopathological behaviors and may predict an unfavorable prognosis in patients with OC.

Given the association of CCEPR expression with invasion and FIGO stage in OC, the biological effects of CCEPR on the proliferation rate and invasive ability of OC cells were further investigated by loss-off function assays. CCEPR expression levels were significantly decreased in SK-OV-3 and OVCAR-3 cells following transfection with shCCEPR1 compared with shNC1-transfected cells (Fig. 2B; P<0.05); however, transfection with shCCEPR2 only significantly decreased expression levels in OVCAR-3 cells compared with shNC2-transfected cells (Fig. 2C). Therefore, the shCCEPR1 shRNA was chosen for subsequent experiments. The CCK-8 assay revealed that the proliferative ability of cells was markedly suppressed in shCCEPR-1-transfected SK-OV-3 and OVCAR-3 cells compared with the shNC1-transfected group (Fig. 2D and E; P<0.05). In addition, a Transwell invasion assay was performed to investigate the effect of CCEPR expression on tumor cell invasion. CCEPR knockdown significantly decreased the invasive abilities of SK-OV-3 and OVCAR-3 cells (Fig. 3A-D; P<0.05) compared with the cells transfected with the shNC1. These findings suggested that the knockdown of CCEPR may impede cell growth and invasion in vitro.

To further confirm whether CCEPR knockdown suppressed OC cell proliferation through regulating cell apoptosis, flow cytometric analysis was performed. The results revealed that the apoptotic rate in the shCCEPR1-transfected cells was significantly increased compared with the shNC1-transfected cells (Fig. 4A-D; P<0.05). Furthermore, the expression levels of cell apoptosis-related proteins, including Bcl-2, Bax and caspase-3 were analyzed using ELISA following CCEPR knockdown. The protein expression levels of Bcl-2 were significantly decreased in OC cells post-transfection with shCCEPR1 compared with the shNC1-transfected cells (Fig. 4E; P<0.05), whereas the protein expression levels of Bax and caspase-3 were significantly increased in shCCEPR1-transfected cells compared with shNC1-transfected cells (Fig. 4F and G; P<0.05). These results suggested that the knockdown of CCEPR may induce apoptosis of OC cells in vitro.

The potential mechanism through which CCEPR regulates OC progression was subsequently investigated. KEGG analysis was used to predict the downstream signaling pathways of CCEPR and the Wnt/β-catenin signaling pathway was highly enriched; thus, western blotting was used to verify this. Significantly decreased expression levels of proteins involved in the Wnt/β-catenin signaling pathway, including cyclin D1, β-catenin, Myc and MMP-7, were observed in shCCEPR1-transfected OC cells compared with the shNC1-transfected cells (Fig. 5A-D; P<0.05). These results suggested that CCEPR may be involved in the progression of OC through regulating the Wnt/β-catenin signaling pathway.