The SKOV3 ovarian cancer cell line was from the Molecular Medicine and Cancer Center, Chongqing Medical University, and the cells were maintained in medium containing 10% fetal bovine serum, 2 mmol/l l-glutamine, penicillin (100 units/ml) and streptomycin (100 μg/ml). A 375-bp NFATc1 primer (5′-TCTGGGAGATG GAAGCAAAGACTG-3′; 5′-AGGGCTATCACGTGGTG TGAAGAG-3′) and a 180-bp primer (5′-GCCCTGACAGCTCCCAAGCCT-3′; 5′-ATGCGTCATGCCGCTTCCCAG-3′) were designed for PCR. Three DNA oligos (siRNA-880, anti-sense, 5′-UUCCGGCACAGUCAAUGACGGCUCG-3′ and sense, 5′-CGAGCCGUCAUUGACUGUGCCGGAA-3′; siRNA-1169, antisense, 5′-AGAGAAUUCGGCUUGCACAGGUCCC-3′ and sense 5′-GGGACCUGUGCAAGCCGAAUUCUCU-3′; and siRNA-1307, antisense 5′-AGACGUAGA AACUGACGUGAACGGG-3′ and sense 5′-CCCGUUCACGUCAGUUUCUACGUCU-3′) were designed as green fluorescent small interfering RNA (siRNA) against NFATc1 mRNA to target the open reading frame of NFATc1 cDNA. These DNA oligos were contained in Lipofectamine™ 2000 and were used to infect SKOV3 cells; the highest transfection efficiency oligo was selected by RT-PCR to use for interference. The subjects can be divided into three groups as follows according to the intervention measures: A, Blank; B, Control and C, siRNA.

To evaluate the ability of cells to form tumors, 4- to 8-week-old BALB/c athymic female nude mice from the Animal Experimental Center, Chongqing Medical University were given bilateral injections of SKOV3 tumor cells, with a total of 18 mice used. All mouse experiments were performed in accordance with the institutional guidelines approved by the Institutional Animal Care and Use Committee. Each subcutaneous injection consisted of 5×10⁶ cells (0.2 ml) for SKOV3. Control mice were injected with SKOV3 cell lines that expressed NFATc1 siRNA. The mice were kept in a specific pathogen-free environment and were checked every 2 days for 30 days. After 30 days, the mice were sacrificed by exposure to 5% carbon monoxide. The tumor inhibition rate was calculated with the use of the tumor weight: Tumor inhibition rate (%) = (control weight - experimental weight)/control weight × 100%. The tumor volume was calculated with the use of the following formula: Tumor volume (in mm³) = (L × W²)/2, where L is the length and W is the weight). All tumors for each group were excised, fixed in 10% formalin overnight, and subjected to routine histologic examination and immunostaining of NFATc1 (antibody cat. no. ab25916, Abcam, 1:200), CK (antibody cat. no. BM0030, BOSTER, 1:500), CD34 (antibody cat. no. SC-9095, Santa Cruz, 1:500) and CXCR2 (antibody cat. no. bs-1629R, Bioss, 1:800) by investigators who were blinded to the tumor status. The assay was repeated twice.

Immunohistochemical staining for NFATc1 (ab25916, 1:200) was performed using avidin-biotin-peroxidase methods. Briefly, tissue slides were deparaffinized in xylene and rehydrated in a graded series of ethanol, and the sections were subjected to antigen retrieval by boiling in 0.01 mol/l sodium citrate buffer (pH 6.0) in a microwave oven for 10 min. After blocking endogenous peroxidase activity with 0.3% hydrogen peroxide and blocking non-specific protein binding with 1.5% normal goat serum, the sections were incubated overnight with an antibody at 4°C in a humidified chamber. Then, the sections were incubated with biotinylated goat anti-mouse IgG for 30 min and detected with the LSAB system (Dako). Sections were lightly counterstained with hematoxylin. The primary antibody was replaced with 1X PBS as a negative control. Differences in proportions between NFATc1 expression and FIGO stage, age at diagnosis, family history, relapse, level of debulking surgery, clinical response, the presence of ascites, and chemoresponse were calculated by χ² analysis or Pearson's correlation as appropriate. Entire tissue sections were evaluated by H-score values, which are objective measurements of staining intensity, and the percentage of tumor cells that stained positive. Five fields of each slice were randomly selected with a magnification of ×400, and the numbers of positive cells were counted per 100 cells per field. Staining intensity was scored as negative (<5% of positive tumor cells), 1+ (mild intensity), 2+ (moderate intensity), or 3+ (intensity greater than that of the positive control). The sections were evaluated by two pathologists using a double-blind method to determine the immunohistochemistry results. The formula was H-score = ∑(i + 1) × p_i, where i indicates the intensity of staining, p_i was the percentage of cells with positive staining/the total number of tested cells, and 1 was the correction factor.

Total protein extracts from the SKOV3 cells and transplanted tumor tissue were obtained using lysis buffer, and equal amounts (30 μg per load) were analyzed by immunoblotting. An antibody against β-actin was obtained from Sigma-Aldrich (A5441, 1:20,000). Antibodies used were against NFATc1 (ab25916; 1:1,000), and against CXCR2 (antibody cat. no. bs-1629R, 1:800). The secondary antibodies were anti-rabbit immunoglobulin horseradish peroxidase-linked F(ab)2 fragment from donkey (Amersham Biosciences). Western blot reagents were from an electrochemiluminescence kit (Amersham Biosciences).

To detect the inhibition rate of cell proliferation, each group of cells in the logarithmic growth phase was plated at 5×10³ cells/well, and each condition was repeated thrice. The experimental cells were transfected by NFATc1 siRNA. After incubation for 6 to 24 h, 200 μl of RPMI-1640 was added per well. The 1640 reagent was removed after 24 h, and after 48 h, RPMI-1640 and 20 μl of MTT was added. The cells were cultured for 4 h at 37°C; the reagent was then removed, and 150 μl of dimethylsulfoxide was added per well. The samples were shaken slowly for 10 min, and the absorbance in each well, including the blanks, was measured at 490 nm using a microtiter plate reader. The cell growth inhibition rate (%) = (1− experimental group A₄₉₀ mean value/control group A₄₉₀ mean value) × 100%.

The number of apoptotic cells was detected by flow cytometry and was analyzed by CellQuest software. To detect apoptosis, 1×10⁵ cells were stained with Annexin V and propidium iodide, according to the Annexin V-Fluorescence Apoptosis Detection Kit I (BD Biosciences) and were subjected to analysis with a FACStation equipped with CellQuest software. The percentage of apoptotic cells was calculated in terms of peaks (M2) in the histogram, representing the early apoptotic population (Annexin V⁺/propidium iodide⁻). The experiment was performed in duplicate and repeated three times.

SKOV3 cells were treated with siRNA as described above. After incubation for 24 h, the cells were removed by trypsinization, counted and plated at 5×10⁵/ml in 6-well dishes. The cells were incubated overnight, yielding confluent monolayers for wounding. Wounds were created using a pipette tip, and images were captured immediately (time zero) and at 24 and 48 h after wounding. The cell monolayer that migrated from the wounded edge during this time period was counted. The number of migrated cells after siRNA treatment (control and targeted) was compared. Experiments were performed in triplicate and repeated at least five times.

The number of mice (sample size) required to reach significance was determined in preliminary pilot studies that used the following formula: n = 16 × (SD/difference in mean tumor volume)² + 1. The results of that pilot study indicated that six mice would be required to detect differences in tumor size with 80% power at a P-value of <0.05. Each mouse received two bilateral flank injections, from which, the mean volume of tumor in each mouse generated from 5×10⁶ cells (for SKOV3) was computed to determine the growth curve (the mean tumor volume in each group = total mean volume from each mouse divided by the number of mice). Statistical analysis was performed using Fisher's exact test at different time points for the mean tumor sizes of each group. Differences in proportions were evaluated by the Fisher's exact test as appropriate. The correlation between NFATc1 expression in tissue arrays (based on the scores of CXCR2 immunostaining intensity) and patient survival was analyzed by the Kaplan-Meier method using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The clinical correlation in terms of NFATc1 expression and patient survival was evaluated by excluding missing data. The association between the expression of NFATc1 and clinical pathological parameters was analyzed with contingency tables and Pearson W2 test. P<0.05 was considered significant. All statistical tests were two-sided.

We first examined NFATc1 expression in the ovarian cancer tissues by immunohistochemical staining. As shown in Fig. 1A and Table I, epithelial ovarian cancer samples expressed high levels of NFATc1 compared with two types of benign ovarian tumors. Then, using Lipofectamine™ 2000 infection, we delivered siRNA against NFATc1 into the ovarian cancer cell line SKOV3. As shown by immunofluorescence and RT-PCR (Fig. 1B and C; Tables II and III), NFATc1 was notably silenced in cells treated with the specific NFATc1 siRNA compared with the control group, which led to a marked decrease in the secreted expression of NFATc1 as detected by western blotting (Fig. 1D). To investigate whether NFATc1 downregulation is associated with in vitro and in vivo tumor growth in ovarian cancer cells, we investigated cell proliferation and mouse xenograft tumor growth after inoculation. As indicated in Fig. 2 and Table IV, NFATc1 expression (Fig. 2A) and the subcutaneous tumor size in animals (Fig. 2B and Table V) were both distinctly reduced in cells treated with NFATc1 siRNA compared with the controls. These data showed that NFATc1 was upregulated in ovarian cancer cells and that silencing of NFATc1 diminished tumorigenicity and aggressiveness of the ovarian cancer cells both in vitro and in vivo.

We first tested the overall level of cell proliferation by MTT staining. Knockdown of NFATc1 decreased the number of proliferating cells by at least 2-fold in the SKOV3 cells (Fig. 3A; Table VI). To explore the underlying mechanism by which NFATc1 induces ovarian tumor growth, we first examined the effect of NFATc1 on cell apoptosis by flow cytometry. As shown in Fig. 3B and Table VII, knockdown of NFATc1 promoted cell apoptosis compared with the control cells. These results strongly suggest that NFATc1 promotes ovarian development at least in part through regulation of cell proliferation and apoptosis.

The cells that migrated through the filter pores to the underside of the filter in the experimental group were significantly increased compared with the cells in the control and blank groups (P<0.05; Fig. 4A and Table VIII). In the wound-healing assays, the distance migrated by the cells after wounding a cell monolayer on plastic after treatment with NFATc1 siRNA and control was obtained. The results (Fig. 4B, Table IX) are reported as migration indices, which represent the distance migrated by the control or NFATc1 siRNA-treated cells to close the wound, expressed relative to the distance migrated by the control-treated cells. In the SKOV3 cells, NFATc1 siRNA significantly decreased migration compared with the non-targeted controls (P<0.05). This result was consistent with results obtained for this cell line in the Transwell assay, as described above.

It is well known that VEGFA (angiogenic factor A) promotes tumor angiogenesis. As previously mentioned, research has shown that VEGFA activates calcineurin and induces NFATc1 transcriptional activity. To confirm whether NFATc1 is associated with angiogenesis, we first examined the xenograft mouse tumor tissues generated from animals injected with SKOV3/NFATc1 siRNA-1169. The overall blood vessel density was decreased in tissues expressing NFATc1 siRNA, as indicated by the reduced number of tissue microvessels stained with CD34 (Fig. 5A and Table X). In addition, we then detected the expression of CXCR2 in mouse tumor tissues treated with or without NFATc1 siRNA. We found that CXCR2 was significantly decreased in the xenograft tumor tissues treated with NFATc1 siRNA (Fig. 5B and Tables XI and XII). These results suggest that NFATc1 promotes ovarian cancer angiogenesis by activating CXCR2, which may be mediated through the upstream signaling of VEGFA.

Immunohistochemical staining of NFATc1 in 93 specimens of high-grade ovarian carcinoma showed that both the cell membrane and cytoplasm of epithelial cancer tissues were specifically stained with NFATc1, whereas little NFATc1 expression was detected in benign ovarian tumors. Representative images are shown in Fig. 1A. Statistically, patients with low NFATc1 expression lived longer (mean, 49 months) than patients with high NFATc1 expression (mean, 36 months; P<0.01, Fig. 6). In terms of disease-free survival, patients with high NFATc1 expression also relapsed earlier in the course of the disease (mean, 22 months) than did patients with low expression (mean, 35 months; P<0.01; Fig. 6). Univariate and multivariate analyses of FIGO stage, age at diagnosis, and NFATc1 expression showed all of these factors to be independent prognostic factors for overall survival, whereas FIGO stage and age at diagnosis were independent prognostic factors for disease-free survival (Table XIII). These data suggest that NFATc1 overexpression is significantly associated with EOC prognosis.

The association between patient clinical characteristics and NFATc1 expression is summarized in Table XIII. Low levels of NFATc1 expression were noted in a higher proportion of patients with early-stage carcinoma compared with patients who had late-stage disease with high levels of NFATc1 expression (P=0.005). A higher proportion of patients with a positive family history of cancer had low NFATc1 expression compared with patients with no family history of cancer (P=0.03). Patients who did not experience a relapse had low NFATc1 expression compared with patients whose disease progressed (P=0.005).