The long non-coding RNA CCAT2 is up-regulated in ovarian cancer and associated with poor prognosis

Background Ovarian cancer is a malignant tumor with a poor prognosis. Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) are emerging regulators in cancer biology, and can be used as potential biomarkers for cancer diagnosis, prognosis and targeted therapy. The lncRNA CCAT2 (colon cancer associated transcript 2) was recently shown to be involved in several cancers; however, its role in ovarian cancer remains unknown. Methods Expression levels of the lncRNA CCAT2 in ovarian cancer tissues, adjacent normal tissues, and cell lines were assessed by quantitative real-time PCR. Then, the associations of CCAT2 expression levels with clinicopathological features and prognosis were evaluated. In addition, CCAT2 functions in tumor progression and invasion were further determined by siRNA-induced CCAT2 silencing in vitro. Results Expression levels of the lncRNA CCAT2 in ovarian cancer tissues and cell lines were significantly higher compared with values obtained for adjacent non-tumor tissues and normal ovarian epithelial cells. Interestingly, higher CCAT2 expression levels were associated with a shorter overall survival (P = 0.006) and disease-free survival (P = 0.001) in ovarian cancer patients. In addition, CCAT2 expression was positively correlated with FIGO stage (P = 0.002), tumor grade (P = 0.006) and distant metastasis (P < 0.001). Moreover, CCAT2 knockdown in ovarian cancer cells markedly suppressed cell proliferation, migration, and invasion. Conclusions The lncRNA CCAT2 is a novel factor involved in ovarian cancer progression, and constitutes a potential prognostic biomarker and therapeutic target for patients with ovarian cancer.


Background
Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. More than 230,000 new cases are diagnosed each year, usually at a late stage; this results in the death of about 140,000 women [1]. Over the past decade, a relatively limited improvement of survival rates has been achieved in ovarian cancer. A 5-year survival rate over 90 % can be achieved with patients diagnosed and treated at early stages (I and II). Unfortunately, most ovarian cancer patients are diagnosed with advanced disease (stages III and IV), in which 5-year survival is less than 30 % [2]. Therefore, there is an urgent need to identify new biomarkers and novel therapeutic targets for therapeutic strategy improvement.
Long non-coding RNAs (lncRNAs) are larger than 200 nucleotides; they constitute an emerging class of key regulatory RNAs that do not code for proteins, and are crucial players in numerous biological processes, including chromatin remodeling, gene regulation, dosage compensation, RNA maturation (splicing, editing), and genomic imprinting [3]. Abnormal expression of lncRNAs was reported in different types of cancers [4][5][6][7][8]. In addition, recent studies demonstrated that differential expression of lncRNAs is associated with diagnostic and prognostic markers for ovarian [9], lung [10], gastric [11] and liver [12] cancers. However, the role of lncRNAs in ovarian cancer has only recently been investigated and remains largely unknown.
The lncRNA CCAT2 (colon cancer-associated transcript 2) transcript maps to the highly conserved 8q24.21 region encompassing rs6983267; CCAT2 plays a crucial role in promoting tumor metastasis, growth, and chromosomal instability in colon [13], lung [14], breast [15], and gastric [16] cancers. In colon cancer, CCAT2enhances invasion and metastasis through the MYCregulated miRNA-17-5p and miRNA-20a [13]. CCAT2 also appears to be involved in migration and chemoresistance in a SNP-independent fashion in breast cancer [15]. However, CCAT2 expression in ovarian cancer and the underlying mechanism remain unknown.
This study assessed CCAT2 expression levels in ovarian cancer tissues, normal ovarian tissues and cell lines, by quantitative real-time PCR (qRT-PCR). Then, correlation analysis was carried out to establish the associations of CCAT2 expression levels with clinicopathological features and prognosis. Finally, CCAT2 effects on ovarian cancer cell proliferation, migration and invasion were further determined via suppression of the lncRNA CCAT2 in vitro.

Patients and samples
A total of 109 ovarian cancer and 45 normal ovarian tissue samples were obtained from patients undergoing wedge biopsy of the ovaries or adnexectomy due to myoma or adenomyosis, in Department of Obstetrics and Gynecology, the First Affiliated Hospital of Nanchang University (Nanchang, China), between 2007 and 2009. No patients had received chemotherapy or radiotherapy prior to surgery. Ovarian cancer was validated by histological examination in all cases according to World Health Organization criteria [17]. Ovarian cancer and normal ovarian tissue specimens excised surgically from patients were immediately snap-frozen and stored in liquid nitrogen until use. Patient characteristics are summarized in Table 1, including age, histological subtype, distant metastasis, tumor size, tumor grade, and FIGO stage (2009). In ovarian cancer, primary cytoreductive surgery followed by platinum-based chemotherapy usually is the mainstay of treatment. All the enrolled women with ovarian cancer in this study received primary cytoreductive surgery followed by platinum-based chemotherapy as follows: paclitaxel (135 mg/m 2 ) and cisplatin (75 mg/m 2 ) or paclitaxel (175 mg/m 2 ) and carboplatin (AUC6), administered every 3 weeks for 6 cycles. Informed consent was obtained from all patients, and the study was approved by the ethics committee of the First Affiliated Hospital of Nanchang University (License No. 2013005).

RNA extraction and qRT-PCR
Total RNA was extracted from ovarian cancer tissue samples, normal ovarian tissue specimens and cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. RNA concentrations and

Wound healing assay
Migration ability of cells was measured by a wound healing assay in vitro. Briefly, 2 × 10 5 SKOV3 cells were seeded onto 6-well plates, with either si-CCAT2 or si-NC, and incubated in appropriate complete culture medium for 16 h in normoxic conditions at 37°C. The monolayer was scratched and incubated in fresh medium for 24 h. The wound width was measured after 24 h. Three different locations were visualized and photographed under a phase-contrast inverted microscope (Leica, Solms, Germany).

Transwell invasion assay
Cell invasion assay was performed using 24-well Transwell plates (8-μm pore size, Corning Life Sciences) coated with 1 mg/ml Matrigel (BD Sciences). SKOV3 cells transfected with either si-CCAT2 or si-NC were resuspended in serum-free medium at 1 × 10 5 cells/ml, respectively. Each cell suspension was seeded in the upper chamber of the Transwell plate in 200 μl FBS-free medium; the lower chamber was filled with 500 μl 20 % FBS in medium. After 48 h of incubation at 37°C with 5 % CO 2 , cells on the filter surface were fixed with methanol, stained with 0.1 % crystal violet, and photographed under a phase-contrast inverted microscope (Leica). Cells from at least five random microscopic fields (×100) were counted.

Statistical analysis
Between-group analysis was carried out by independent t-test for continuous data; Chi-square test was used for categorical data. Overall survival (OS) and disease-free survival (DFS) curves were established by the Kaplan-Meier method, and analyzed by the log-rank test. A Cox proportional hazard model was constructed to evaluate the associations of CCAT2 expression with OS and DFS, respectively. Two-sided P < 0.05 was considered statistically significant. All statistical analyses were performed with the SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA).

CCAT2 gene expression is elevated in ovarian cancer tissues and cells
Using qRT-PCR, CCAT2 gene expression was assessed in 109 ovarian cancer tissue samples, 45 normal ovarian tissue specimens, and cultured ovarian cancer-or normal cells. Starkly increased CCAT2 gene expression levels were found in ovarian cancer tissue samples compared with normal ovarian tissue specimens (Fig. 1a, P < 0.01).

Associations of CCAT2 expression with clinicopathological features in ovarian cancer patients
To further explore the role of CCAT2 in ovarian cancer, we next evaluated the associations its gene expression with several clinicopathological features. The 109 patients were divided into two groups by the median value of relative CCAT2 gene expression levels: high (n =55) and low (n =54) expression groups. As shown in Table 1, high CCAT2 level was not associated with patient age (P = 0.702), histological subtype (P = 0.918) or tumor size (P = 0.336); however it was significantly positively correlated with FIGO stage (P = 0.002), tumor grade (P = 0.006) and distant metastasis (P < 0.001). Taken together, these results supported the notion that CCAT2 up-regulation may be associated with tumor progression and development.

High CCAT2 levels are correlated with poor prognosis in ovarian cancer patients
To further explore the potential clinical value of high CCAT2 expression, the prognostic power of the lncRNA CCAT2 was determined for OS and DFS in 109 ovarian cancer patients, using the Kaplan-Meier method and log-rank test. Interestingly, high CCAT2 expression was correlated with shorter OS or DFS (P < 0.05, Fig. 2a and  2b). Univariate Cox proportional hazards regression model analysis showed that relative CCAT2 expression level, distant metastasis, tumor grade, and FIGO stage were correlated with overall survival rate in ovarian cancer patients (P < 0.05, Table 2). In addition, multivariate analysis revealed CCAT2 expression level, distant metastasis, tumor grade, and FIGO stage to be independent prognostic indicators for overall survival (P < 0.05, Table 2). These data suggested that the lncRNA CCAT2 could be a bona fide prognostic marker for ovarian cancer.

Knockdown of the lncRNA CCAT2 inhibits cell proliferation, migration and invasion
In order to explore the functional role of CCAT2 in ovarian cancer tumorigenesis, SKOV3 cells were transfected with si-CCAT2 or si-NC. 48 h after transfection, qRT-PCR was performed to assess CCAT2 gene expression. As shown in Fig. 3a, cells transfected with si-CCAT2 had significantly decreased CCAT2 mRNA levels compared with the si-NC group (P < 0.05). Next, cell viability was assessed by MTT assay. As shown in Fig. 3b, SKOV3 cell transfection with si-CCAT2 Low CCAT2 expression levels are shown. a OS of ovarian cancer patients with high CCAT2 expression was significantly poorer compared with rates found in patients with low CCAT2 levels (P < 0.05). b DFS of ovarian cancer patients with high CCAT2 expression was significantly poorer compared with rates in patients with low CCAT2 (P < 0.05) resulted in significantly decreased viability compared with the si-NC group. Furthermore, the role of the lncRNA CCAT2 in cell migration and invasion was assessed. In wound healing assay, the migration ability of SKOV3 cells transfected with si-CCAT2 was significantly decreased compared with that of the si-NC group (P < 0.05, Fig. 3c). In addition, Transwell invasion assay revealed reduced invasion capacity for SKOV3 cells transfected with si-CCAT2 in comparison with the si-NC group (P < 0.05, Fig. 3d). These findings demonstrated that CCAT2

Discussion
Ovarian cancer, a gynecological malignancy, has been a leading cause of death among women for the past decades. The dismal prognosis of ovarian cancer is due to few symptoms in early-stage; patients are generally diagnosed with advanced-stage tumors, which are less susceptible to existing treatments [19]. Therefore, it is important to explore the molecular events in ovarian cancer, which would provide insights for improved diagnosis and prognosis of this deadly disease, thus improving clinical strategies and outcomes. It was previously demonstrated that the majority of RNA transcripts in mammalian cells do not encode proteins; indeed, protein-coding genes account for only 2 % of the total genome, whereas much of the human genome, far more than expected, is transcribed into noncoding RNAs [20]. Among these, lncRNAs have been confirmed to be important biological RNAs, regulating gene expression at the transcription, epigenetic, or translation level [21]. Increasing evidence suggests that lncRNAs are emerging and important regulators of several biological processes, playing oncogenic or tumor suppressor roles in tumorigenesis [22,23]. The lncRNA CCAT2 is an imprinted and maternally expressed gene, which promotes the progression of lung [14] and gastric [16] cancers. However, CCAT2 expression and prognostic role in ovarian cancer remain unclear.
In the present study, CCAT2 gene expression levels were significantly higher in ovarian cancer tissue samples and cell lines. To assess whether CCAT2 expression can serve as a novel prognostic marker, potential associations of CCAT2 expression with clinicopathological factors were examined. Interestingly, patients with high CCAT2 gene expression levels showed poor prognostic parameters, including FIGO stage and distant metastasis.
In addition, patients with high CCAT2 expression levels had a significantly poorer prognosis compared with the low expression group. As shown above, shorter overall-and disease-free survival times were observed in the high CCAT2 expression group compared with values obtained for the low expression group. Moreover, multivariate Cox analysis indicated that high CCAT2 expression was an independent predictor of ovarian cancer. Taken together, these findings demonstrated for the first time the clinical significance of the lncRNA CCAT2 in ovarian cancer.
Zhang et al [24] demonstrated that the lncRNA CCAT2 is upregulated in esophageal squamous cell carcinoma (ESCC) tissues, with positive correlations between CCAT2 level and lymph node metastasis, TNM stage, and the number of positive lymph nodes, respectively. In addition, previous findings demonstrated that CCAT2 is upregulated in breast tumors; indeed, CCAT2 silencing decreases cell proliferation and invasion in vitro through the Wnt/βcatenin signaling pathway [25]. Thus, further experiments are needed to identify the biological processes regulated by CCAT2 in ovarian cancer. We next silenced CCAT2 in SKOV3 cells in vitro. As shown above, knockdown of the lncRNA CCAT2 inhibited cell proliferation, migration and invasion, further highlighting the role of CCAT2 in cell biology and oncogenesis of ovarian cancer cells.

Conclusion
In summary, this is the first report showing that lncRNA CCAT2 expression is significantly high in ovarian cancer tissues and cell lines. In addition, higher CCAT2 gene expression level was associated with poor prognosis in ovarian cancer patients. Moreover, CCAT2 silencing in ovarian cancer cells markedly suppressed cell proliferation, migration, and invasion. Taken together, these findings suggest a potentially important role for CCAT2, which could serve as a potential biomarker and therapeutic target in ovarian cancer.