Between 2008 and 2010, 63 ovarian cancer tissues and matched adjacent normal tissues were obtained from patients who underwent surgery at the First Affiliated Hospital of Sun Yat-sen University, and were diagnosed with ovarian cancer (stages II, III and IV) based on histopathological evaluation according to the International Federation of Obstetrics and Gynecology (FIGO) criteria. Clinical pathology information was available for all samples (Table I). No local or systemic treatment was conducted in these patients before the operation. All tissues were collected and immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. The present study was approved by the Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from every participant.

Human ovarian surface epithelial cells (IOSE25) were immortalized and cultured as previously described (22). Five human ovarian cancer cell lines, A2780, OVCAR3, SKOV3 and 3AO, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), respectively. The ovarian cancer cell lines were maintained according to the vendor's instructions. In brief, A2780 and 3AO cell lines were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA). SKOV3 cells were cultured in McCoy's 5A Medium Modified (ATCC) with 10% FBS (Gibco). OVCAR3 cells were cultured in RPMI-1640 medium (ATCC) with 20% FBS (Gibco). All the media contained 1% penicillin-streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin). All ovarian cancer cells were cultured and maintained in a humidified incubator at 37°C and supplemented with 5% CO₂.

Total RNA was extracted from tissue samples or cultured cells using TRIzol reagent (Invitrogen Inc., Carlsbad, CA, USA). For qRT-PCR, 2 µg of total RNA was used for reverse transcription reaction and cDNA synthesis using a Reverse Transcription kit (Takara, Dalian, China). Quantitative real-time PCR analyses were performed using SYBR-Green Real-Time Master Mix (Toyobo, Tokyo, Japan). Results were normalized to a constitutive expression gene, GAPDH. The PCR primers for GAS5 were: 5′-CTTCTGGGCTCAAGTGATCCT-3′ (forward) and 5′-TTGTGCCATGAGACTCCATCAG-3′ (reverse); for GAPDH, 5′-ACACCCACTCCTCCACCTTT-3′ (forward) and 5′-TTACTCCTTGGAGGCCATGT-3′ (reverse). qRT-PCR and data collection were performed on Applied Biosystems 7500 Sequence Detection system (ABI, USA). The relative expression of GAS5 was calculated and normalized using the 2^−ΔΔCt method relative to GAPDH.

Total proteins were extracted from tissue samples or cultured cells with SDS lysis buffer (Beyotime, Shanghai, China) on ice for 20 min and the protein concentration was determined using BCA Protein Assay kit (Pierce, Rockford, IL, USA). Equal amounts of total proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 µm polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and incubated with specific antibodies. Proteins were detected by enhanced chemiluminescence (ECL) as described by the manufacturer (Beyotime) and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). GAPDH antibody (1:2,000; Abcam) was used as control, anti-cyclin D1 (1:800), P21 (1:1,500) and apoptosis protease activating factor 1 (APAF1) (1:1,000) were purchased from Abcam.

To generate a GAS5 expression vector, the entire sequence of human GAS5 (2,651 bp) was synthesized and subcloned into the pCDNA4.1 vector. In addition, the plasmids were confirmed by DNA sequencing (Sangon, Shanghai, China).

Ovarian cancer cells were seeded in 24-well plates (1×10⁵ cells/well) and incubated for 24 h, and then the cells were transfected with pCDNA3.1-GAS5 (2 µg) or the empty vector (2 µg) using Lipofectamine 2000 (Invitrogen) in serum-free medium in accordance with the manufacturer's instructions.

The cell proliferation assay was performed with Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. In brief, 2×10⁴ cells/well were seeded in a 96-well plate. Cell viability was evaluated with CCK-8 at daily intervals from the next 24, 48 and 72 h after seeding. After treatment with CCK-8 at 37°C for 1 h, ovarian cancer cells were used to measure the absorbance at 450 nm using a microplate reader Thermo Plate (Rayto Life and Analytical Sciences Co., Ltd., Germany).

Twenty-four-well Transwell inserts with 8-µm pore size (Corning Costar, Inc., Corning, NY, USA) were used for Transwell migration and Matrigel invasion assays, separately. For the Transwell migration assay, 2×10⁴ ovarian cancer cells suspended in 100 µl serum-free corresponding culture medium were loaded into the upper chamber of the Transwell insert with a non-coated membrane. For the Transwell invasion assay, 4×10⁴ ovarian cancer cells were plated in 100 µl corresponding culture medium without FBS in the upper Matrigel-coated chamber instead. In both assays, 500 µl of culture medium containing 20% FBS was added to the lower chamber for culture. Cells were then allowed to migrate or invade for 24 h at 37°C. The cells that migrated or invaded into the bottom chamber were fixed with 100% methanol for 30 min and stained using 0.5% crystal violet (Sigma, St. Louis, MO, USA) for 20 min, and the permeating cells were counted under a phase-contrast microscope (Olympus, Tokyo, Japan). All experiments were independently repeated at least three times.

Cell apoptosis was evaluated by flow cytometry using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BestBio, Shanghai, China) in accordance with the manufacturer's instructions. Briefly, cells were harvested and washed with cold phosphate-buffered saline (PBS). Cells (1×10⁶) were resuspended and stained using the Annexin V-FITC/PI apoptosis detection kit according to the manufacturer's instructions. Then, the samples were analyzed using Becton-Dickinson flow cytometer. Annexin V(+)/PI(−) and Annexin V(+)/PI(+) represent the ovarian cancer cells in early and late apoptosis/necrosis, respectively. Moreover, cell apoptosis was also detected using Hoechst staining. Ovarian cancer cells were cultured in 6-well cell culture plates, and Hoechst 33342 (Sigma) was added to the culture medium. Fluorescence microscopy with a filter for Hoechst 33342 (365 nm) was used to detect nuclear morphology.

Four-week-old female BALB/c athymic nude mice (Vital River Laboratory Animals, Beijing, China) were maintained under specific pathogen-free conditions and manipulated according to protocols approved by the Ethics Committee of Animal Experiments of the First Affiliated Hospital of Sun Yat-sen University. pCDNA3.1-GAS5 or pcDNA3.1 vector stably transfected SKOV3 cells were harvested and washed with PBS, then resuspended at a concentration of 5×10⁷ cells/ml. A volume of 100 µl of the suspended cells was subcutaneously injected into the right side of the posterior flank of each mouse. The subcutaneous tumor growth was examined every seven days, and tumor volumes were calculated using the equation: V = 0.5 × D × d² (V, volume; D, longitudinal diameter; d, latitudinal diameter). At five weeks post injection, the mice were sacrificed and tumor weights were measured. Each neoplasm was collected and stored at −80°C for further analysis. All procedures performed in these studies involving animals were in accordance with the ethical standards of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

For quantitative data, all results are expressed as the mean ± SD. Statistical analysis was performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance between groups was determined using a Student's t-test or a Chi-square test. Survival analysis was performed using the Kaplan-Meier method, and the log-rank test was used to compare the differences between patient groups.

To investigate the expression level of GAS5 in human ovarian cancer tissues, qRT-PCR was used to examine GAS5 expression in 63 paired ovarian cancer samples and matched adjacent normal tissues. As shown in Fig. 1A, the expression level of GAS5 was significantly downregulated in the ovarian cancer tissues compared with that in the corresponding adjacent histological normal tissues. In tumor specimens, the expression level of GAS5 was lower than that of the normal tissues, with the median ratio of 0.69 compared with the normal counterparts.

The clinical pathological findings of 63 ovarian cancer patients are listed in Table I. The 63 ovarian cancer patients were classified into two groups based on the median ratio of relative GAS5 expression (0.69) in the tumor tissues: group 1, high GAS5 expression group (n=32, GAS5 expression ratio ≥ median ratio); group 2, low GAS5 expression group (n=31, GAS5 expression ratio ≤ median ratio) (Fig. 1B). The clinicopathological factors compared between the two groups are shown in Table II. The low-GAS5 group (group 2) was correlated with larger tumor size (p=0.0024), deeper depth of invasion (P=0.0009) and higher TNM stage (P=0.0379) than the high-GAS5 group (group 1). However, the GAS5 expression level was not associated with other parameters such as age (P=0.4527), histologic differentiation (P=0.8347) and lymphatic metastasis (P=0.5321) (Table II).

In order to confirm whether the GAS5 expression level is correlated with the outcome of ovarian cancer patients after ovariectomy, disease-free survival (DFS) and overall survival (OS) curves were plotted according to GAS5 expression level by the Kaplan-Meier analysis and log-rank test, respectively, and the results are presented in Fig. 1C and D. Markedly, patients with a low GAS5 expression level had poorer disease-free survival (P<0.0001) and overall survival (P=0.0016). For OS, the 3-year overall accumulative survival rates of ovarian cancer patients with a high level and a low level of GAS5 expression were 47.68 and 22.36%, respectively. Low GAS5 expression indicated a shorter overall survival time for the ovarian cancer patients (median OS, 13 months) compared with high GAS5 expression patients (median OS, 29 months). Moreover, the 3-year disease-free survival rates for high GAS5 expression and low GAS5 expression were 36.59 and 19.76%, respectively. The median survival time of patients with high GAS5 expression was 23 months, while the value was 6 months for patients with low GAS5 expression.

To evaluate the impact of GAS5 on ovarian cancer cell proliferation, the expression level of GAS5 in a variety of cell lines, including ovarian cancer cell lines SKOV3, OVCAR-3, A2780, 3AO and human ovarian surface epithelium cells were detected using qRT-PCR. The results showed that GAS5 was obviously downregulated in the ovarian cancer cell lines (Fig. 2A). In addition, SKOV3 and A2780 cells were chosen for the present study. GAS5 was overexpressed in the SKOV3 and A2780 cells by pCDNA3.1-GAS5 vector transfection. The expression of GAS5 was increased 22.37- and 25.70-fold in the pCDNA3.1-GAS5-transfected SKOV3 and A2780 cells compared with the vector controls, respectively (Fig. 2B). CCK-8 assay revealed that cell proliferation was significantly impaired in the pCDNA3.1-GAS5-transfected SKOV3 (Fig. 2C) and A2780 (Fig. 2D) cells.

To evaluate the effect of GAS5 on tumor cell migration and invasion, Transwell assay was used to detect the migration and invasion abilities of the SKOV3 and A2780 cells after pcDNA3.1-GAS5 vector transfection. Overexpression of GAS5 inhibited the migration and invasion of the SKOV3 (Fig. 3A and B) and A2780 (Fig. 3C and D) cell lines.

To investigate the effect of GAS5 on cell apoptosis, the Annexin V-FITC/PI staining method was used to perform apoptosis assays. The results showed increased apoptosis rates of SKOV3 and A2780 cells following pcDNA3.1-GAS5 vector transfection compared with pcDNA3.1 vector transfection (Fig. 4). The apoptosis rates of SKOV3 cells transfected with pCDNA3.1 and pCDNA3.1-GAS5 were 10.7±1.9 and 36.7±3.9%, respectively (Fig. 4A and B). In addition, the apoptosis rates of the A2780 cells transfected with pCDNA3.1 and pCDNA3.1-GAS5 were 10.0±1.8 and 35.9±3.5%, respectively (Fig. 4C and D). Moreover, Hoechst staining results showed that the number of SKOV3 and A2780 cells with condensed and fragmented nuclei following pcDNA3.1-GAS5 transfection were significantly different than the empty vector-transfected cells (Fig. 4E and F).

In order to explore the underlying mechanisms involved in GAS5 overexpression-mediated suppression of cell growth, western blotting was carried out to examine the expression of potential targets after transfection with pcDNA3.1-GAS5 or pcDNA3.1 (Fig. 5). The results showed that the expression of cyclin D1 was significantly decreased in the SKOV3 (Fig. 5A and B) and A2780 (Fig. 5C and D) cells transfected with pCDNA3.1-GAS5 compared to those with pcDNA3.1. Moreover, p21 and APAF1 protein were increased in the SKOV3 (Fig. 5A and B) and A2780 (Fig. 5C and D) cells transfected with pcDNA3.1-GAS5 compared to those with pcDNA3.1. The results suggest that GAS5 may act as an oncogene by regulating cyclin D1, p21 and APAF1, and the underlying mechanism should be elucidated in further experiments.

In order to confirm the above finding that overexpression of GAS5 affects tumorigenesis, pCDNA3.1-GAS5 or pcDNA3.1 vector stably transfected SKOV3 cells were inoculated into female nude mice for 28 days following injection. The group injected with pCDNA3.1-GAS5 formed substantially smaller tumors than the control group (Fig. 6A and B). Moreover, the mean tumor weight of the pCDNA3.1-GAS5 group (0.54±0.06 g) was markedly lower than that in the pcDNA3.1 group (1.07±0.14 g) at the end of the experiment (Fig. 6C). Then, the expression level of GAS5 in each neoplasm was assessed using qRT-PCR. The results showed that the expression of GAS5 in tumor tissues formed from the pCDNA3.1-GAS5 cells was significantly higher than those of tumors formed in the pcDNA3.1 group (Fig. 6D). Furthermore, western blotting was used to detect the protein expression levels of cyclin D1, p21 and APAF1 in the neoplasms. The results showed that the expression level of p21 and APAF1 protein in the neoplasms formed from the pCDNA3.1-GAS5 cells were significantly higher than those of the neoplasms in the pcDNA3.1 group, and the expression of cyclin D1 was significantly lower in neoplasms formed from the pCDNA3.1-GAS5 cells than the control group (Fig. 6E and F). Taken together, our results indicate that overexpression of GAS5 inhibited tumor growth and may regulate cyclin D1, p21 and APAF1 in vivo.