The ovarian cancer cell line SKOV3 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). SKOV3 cells were routinely maintained in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and maintained at 37°C with 5% CO₂. For the E2 induction experiments, cells (plated at 20–30% confluence) were grown for 3 days in phenol red-free RPMI-1640 (Gibco) containing 5% activated, charcoal-treated foetal bovine serum (Serana, Bunbury, Australia). Next, the cells were treated for 24 h with 10⁻⁸ M E2 or vehicle alone (DMSO, 0.01% of final volume) as a control.

TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from SKOV3 cells with or without 24-h treatment with 10⁻⁸ M E2. There were three replicates of each sample; they were purified using an RNeasy Micro kit (cat. #74004; Qiagen GmbH, Hilden, Germany). An Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) was used to quantify the RNA and evaluate its integrity; the 28S:18S ratio was determined, and an RNA integrity number (RIN) was assigned to each sample. RNA with no evidence of degradation and no signs of DNA contamination (as indicated by an RIN ≥7.0 and a 28S:18S ratio ≥0.7) was processed for further analysis.

The lncRNA and mRNA expression profiles were obtained using the Glue Grant Human Transcriptome (GG-H) Array, which was manufactured by Affymetrix and Stanford University. This array contains 5,869 probes covering 730 non-coding, functional RNAs and 3,292,929 probes covering 27,670 coding genes collected from RefSeq, Ensembl and UCSC Known Genes, based on human genome assembly hg18 (22). The array experiments and computational analysis were performed according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA). Briefly, 0.2 μg of total RNA was amplified and labelled, and 20 μg of labelled cDNA was loaded onto the array. The array was hybridized and washed using the GeneChip^® Hybridization, Wash and Stain kit (cat. #900720), Hybridization Oven 645 (cat. #00-0331-220V) and Fluidics Station 450 (cat. #00-0079). The slides were scanned in a GeneChip^® Scanner 3000 (cat. #00-00212). The raw data were obtained using Command Console Software 3.1 with the default settings and were processed using Affymetrix Power Tools with Robust Multiarray Analysis (RMA) for background correction, normalization and summarization. Differentially expressed genes [defined as a fold-change ≥1.5 and a P-value <0.05 (t-test)] were selected for further study.

For the GO and pathway analyses, the putative targets were initially inputted into the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/), which searched the GO terms to identify the molecular function represented in the gene profile (25), and then into the database of the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.ad.jp/kegg/) to analyze the roles of the targets in molecular pathways (26).

The study included 95 patients who underwent surgery for primary ovarian cancer in the Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University between January 2006 and December 2008. Patients were included based on the availability of tissue and follow-up data. Patients with borderline ovarian tumors or with two or more different malignancies were excluded from the study. None of the patients had received preoperative radiotherapy, chemotherapy or hormonal therapy. All EOC tissue samples were frozen immediately after surgery and stored in liquid nitrogen until use.

Clinical and pathological data of EOC patients were evaluated by reviewing medical charts and the original pathology reports. Staging and grading were evaluated in accordance with the criteria of the International Federation of Gynecologists and Obstetricians (FIGO) and the World Health Organization (WHO). Follow-up data were obtained by reviewing the out patient charts, contacting patients or correspondence. Overall survival (OS) was calculated from the date of surgery until the date of mortality or end of follow-up (January 2013). The present study was approved by the Research Ethics Committee of Fudan University, China. Informed consent was obtained from all the patients.

The immunohistochemical study of ERα was performed using a standard streptavidin-peroxidase method. The endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min. For the antigen retrieval, slides were immersed in 10 mM citrate buffer (pH 6.0) and boiled for 15 min in a microwave oven. Non-specific binding was blocked by 5% normal goat serum for 10 min. The slides were incubated with a 1:50 dilution of monoclonal antibody against ERα (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight in a moist chamber. The slides were sequentially incubated with biotinylated goat anti-mouse IgG (1:100 dilution; Santa Cruz Biotechnology) and then streptavidin-peroxidase conjugate, each for 30 min at room temperature. Isotope-matched human IgG was used in each case as a negative control. Finally, the 3,5-diaminobenzidine (DAB) Substrate kit (Dako) was used for color development followed by Mayer’s hematoxylin counterstaining. ERα+ cases were defined as tumors with >10% stained nuclei (27).

qRT-PCR analysis of lncRNA expression was performed using FastStart Universal SYBR-Green Master (Rox; Roche) and an ABI Prism 7900 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Briefly, total RNA was extracted from cells and tissues and converted to cDNA using an iScript cDNA Synthesis kit (Bio-Rad Laboratories) according to the manufacturer’s protocol. The PCR amplifications were performed in a 10-μl (total volume) reaction that included 1 μl of cDNA template (~5 ng), 5 μl of FastStart Universal SYBR-Green Master (Rox), 3.6 μl of double-distilled water and 0.2 μl of each pair of forward and reverse primers (Table I; Sangon Biotech Co., Ltd., Shanghai, China). The PCR conditions included an initial denaturation step at 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 30 sec. All the experiments were performed in triplicate, and all the samples were normalized to GAPDH expression. The expression fold-changes were calculated using the 2^−ΔΔCt method.

The data were processed using SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). Comparison of continuous data was analyzed using the Student’s t-test, whereas categorical data was analyzed using the Chi-square test and Fisher’s exact test where appropriate (when the expected frequency was <5). OS curves were plotted according to the Kaplan-Meier method, with the log-rank test applied for comparison. Variables were used in multivariate analysis on the basis of the Cox proportional hazards model. A P-value <0.05 was considered to indicate a statistically significant difference (P<0.05).

Based on the overlap between the targets predicted by bioinformatics and the differentially expressed mRNAs detected in the microarray, we constructed 55 E2-regulated lncRNA-target mRNA pairs (Table III). The GO (Fig. 2) and pathway (Table IV) analyses showed that a set of E2-regulated lncRNAs that mapped to the target mRNAs were correlated with several cellular processes and pathways known to be related to cancer progression, such as cell cycle and proliferation, developmental processes, cell adhesion, cell death, MAPK signaling, Hedgehog signaling, Jak-STAT signaling and cancer pathways, suggesting their great potential to contribute to cancer progression.

In order to confirm the potential of some E2-regulated lncRNAs to contribute to cancer progression, we initially selected the four lncRNAs (TC0100223, TC0101686, TC1500845 and TC0101441) as candidates and tested their expression levels in EOC tissues. Considering the fact that ERα is the main form expressed in malignant ovarian tumors and as ERα has been reported to promote poor prognosis in EOC patients (28,29), we determined whether the expression of in vitro E2-regulated lncRNAs, detected in ERα+ ovarian cancer cells, could discriminate between ERα+ and ERα− EOC tissues. Based on the qRT-PCR assay, we found that ERα+ tissues had lower expression of TC0100223 and TC0101686 and higher expression of TC0101441 (Fig. 3, ERα+, n=64 vs. ERα−, n=31, P<0.01; Fig. 3A shows the representative immunohistochemistry results of ERα expression in EOC tissues). In contrast, TC1500845 was not differentially expressed between ERα+ and ERα− EOC tissues. These results may be suggestive of the potential clinical significance of TC0100223, TC0101686 and TC0101441 in ERα+ EOC.

According to the median value which was used as the cut-off (30), specific lncRNA expression in ERα+ EOC tissues, equal or more than median value was defined as high lncRNA group, and less than median value was defined as low lncRNA group. As shown in Table V, low-expression of TC0100223 and TC0101686 and high-expression of TC0101441 were closely related to ERα+ EOC tissues with advanced FIGO stage and/or high histological grade (P<0.05), suggesting that aberrant expression of the three candidate lncRNAs is associated with a more malignant ovarian cancer phenotype.

We investigated whether the expression of TC0100223, TC0101686 and TC0101441 correlated with the postoperative survival of ERα+ EOC patients. Among the 64 ERα+ EOC patients, 38 died during follow-up. In univariate analysis, OS was associated with the FIGO stage, histological grade and expression of TC0100223 and TC0101441 (P<0.05; Table VI). Multivariate analysis further confirmed that high TC0101441 expression, advanced FIGO stage and high histological grade were independent factors for evaluation of OS in ERα+ EOC patients (P<0.05, Table VI; Fig. 4 shows the OS curves according to TC0101441 expression). Thus, it was concluded that of the three candidate lncRNAs, TC0101441 could be used as an independent prognostic factor for OS of ERα+ EOC patients.