Human EOC cells SK-OV-3, OVCAR-3, CAOV-3 and ES-2 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and A2780 was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). Human non-tumorous ovarian surface epithelial cells (HOSEpiC) were obtained from Guangzhou Jennio Biotech Co., Ltd. (Guangzhou, China). SK-OV-3 and CAOV-3 cells were cultured in DMEM (Corning Life Sciences, Manassas, VA, USA). A2780, OVCAR-3 and HOSEpiC cells were respectively cultured in RPMI-1640 media (Corning Life Sciences). ES-2 cells were cultured in McCoy's 5A medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). All media were supplemented with 10% fetal bovine serum (FBS; Gibco; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and were replaced with fresh medium every three days.

All tissue samples (n=32) were derived from hospitalized patients between June 2012 to October 2016 at Jinshan Hospital, Fudan University. None of the patients with OC had received chemotherapy or radiotherapy prior to surgery. Control ovarian tissues were obtained from 12 patients with non-tumorous ovaries. The tumor and normal tissue specimens were frozen in liquid nitrogen after collection and stored at −80°C until use. The present study was approved by the Ethics Committee of Jinshan Hospital and informed consent was obtained from each patient.

The Kaplan-Meier Plotter database, an online bioinformatics tool (www.kmplot.com), is available to evaluate the effect of genes on survival information in OC (27). Before starting the use of the tool, the samples from patients were filtered by stage, histology, grade, and treatment elements containing debulking status and applied chemotherapy. To assess the clinical value of MALAT1, patients with OC were selected for the calculation of OS and PFS and were divided into two groups using the median, a group with low expression of MALAT1 and a group with high expression of MALAT1. The Kaplan-Meier survival curve was plotted. The hazard ratio (HR) with 95% confidence intervals (CIs) and the log-rank P-value were calculated. Online software LncBase Predicted v.2 was used to predict a candidate of miRNA that interacted with MALAT1 (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex). Online software miRWalk 2.0 database was used to find a potential target of hsa-miR-143-3p (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/index.html) (28).

The MALAT1-siRNA (siMALAT1) and negative control-siRNA (siNC) were obtained from RiboBio Co., Ltd. (Guangzhou, China). The sequence of siMALAT1 was 5′-GCAAATGAAAGCTACCAAT-3′. Briefly, cells were seeded in a 6-well plate at a density of 2×10⁵ (OVCAR-3) or 1.5×10⁵ (SK-OV-3) cells/well. After culture for 24 h, the cells were transfected with siRNA using an X-treme GENE Transfection Reagent (Roche Applied Science, Indianapolis, IN, USA) according to the protocol recommended by the manufacturer. The cells were then collected for subsequent experiments. The knockdown efficiency of siMALAT1 was confirmed by qRT-PCR analysis.

Total RNA from tissues and cells was extracted using an Axygen Bioscience kit (Suzhou, China) according to the manufacturer's protocol. The cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science). The reaction conditions of reverse transcription were: 25°C for 10 min, 50°C for 60 min, 85°C for 5 min, and 4°C for 70 min. The qPCR experiments were conducted using a SYBR-Green Master kit (Roche Applied Science). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S and U6 served as a control for cells, tissues, and miRNAs, respectively. The primers were: MALAT1 forward, 5′-GTGTGCCAATGTTTCGTTTG-3′ and reverse, 5′-AGGAGAAAGTGCCATGGTTG-3′; hsa-miR-143-3p forward, 5′-CTGAGATGAAGCACTGTAGCTC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′; 18S forward, 5′-GACTCTGGCATGCTAACTAG-3′ and reverse, 5′-GACATCTAAGGGCATCACAG-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The expression of the target gene was analyzed by threshold cycle (Ct) 2^−ΔΔCt method obtained from Sequence Detection Software v1.4 (7300 Real-Time PCR System; Applied Biosystems; Thermo Fisher Scientific, Inc.). The assay was performed at least three times.

In brief, cells were cultured in 96-well plates at a density of 6×10³ cells/100 µl medium/well overnight. After transfection and culture for 0, 24 and 48 h, the viability of cells was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. The optical density (OD) values at 450 nm were detected using a plate reader (Epoch; BioTek Instruments, Inc., Winooski, VT, USA). At least three independent experiments were conducted.

The cell migration and invasion capacities were evaluated by Transwell assays. Briefly, a Transwell chamber with a membrane of polycarbonate (6.5 mm in diameter with 8-µm pores; Corning Life Sciences) was placed into a well of a 24-well plate. In the lower chamber, 700 µl suitable medium containing 10% FBS was added. For the migration assay, cells were seeded in the upper chamber at a concentration of ~6×10⁴/100 µl with serum-free culture medium. For the invasion assay, cells were seeded in the upper chamber with a Matrigel-coated membrane (BD Biosciences, Bedford, MA, USA) at a concentration of ~8×10⁴ cells in a 100 µl volume of serum-free culture medium. After incubation for 48 h, the non-migrated or non-invaded cells on the upper chamber were carefully removed with a cotton swab and washed with phosphate-buffered saline (PBS). Migrated or invaded cells on the reversed membrane were fixed with 4% paraformaldehyde for 15 min, stained with crystal violet (Sigma-Aldrich; Merck KGaA) for 30 min, photographed, and finally counted in three random fields under a light microscope (BX43; Olympus, Tokyo, Japan) at an ×200 magnification. Experiments were conducted three times.

293T cells were cultured in a 24-well plate and 70–80% confluency was reached prior to the experiment. Cells were co-transfected with 0.4 µg luciferase reporter vector (pmirGLO-MALAT1-wt or pmirGLO-MALAT1-mut) and 50 nM miRNA (miR-143-3p mimics or miR-negative control (miR-Ctrl; RiboBio Co., Ltd.) using the X-tremeGENE Transfection Reagent (Roche Applied Science) and cultured for 24 h. Firefly and Renilla luciferase activities were detected using Luc-Pair™ Duo-Luciferase Assay Kit 2.0 (GeneCopoeia, Inc., Rockville, MD, USA) following co-transfection according to the instructions recommended by the manufacturer. The relative firefly luciferase activity was corrected in accordance with the Renilla luciferase activity.

SK-OV-3 and OVCAR-3 cells were lysed in SDS buffer with a phosphatase inhibitor (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). After separation on SDS-PAGE, total proteins were transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA) and incubated with either mouse anti-CMPK (cytidine monophosphate kinase; Cell Signaling Technology, Inc., Danvers, MA, USA) or rabbit anti-GAPDH (Abcam, Cambridge, UK) primary antibody at 4°C overnight. After incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG (Cell Signaling Technology) for 1 h at room temperature, the signals were detected using Tanon-4500 Gel Imaging System (Tanon Science and Technology Co., Ltd., Shanghai, China) with an Immobilon™ Western Chemiluminescent HRP Substrate (EMD Millipore).

SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used to analyze the collected data. For comparison between two groups, a Student's t-test was used. The survival curve was evaluated with a log-rank test. All data are displayed as the mean ± the standard error of the mean (SEM) from three independent experiments. A P-value <0.05 was considered to indicate a statistically significant difference.

In order to identify functional MALAT1 relevant to the progression of ovarian tumors, we performed qRT-PCR to evaluate the expression of MALAT1 in human ovarian normal tissues (n=12), benign tumors (n=8), and malignant tumors (n=12, serous adenocarcinoma). The results revealed that the expression level of MALAT1 was significantly higher in ovarian malignant tumors than normal ovarian tissues and ovarian benign tumors (P<0.01) (Fig. 1A). In addition, the expression of MALAT1 between diverse OC cell lines and normal ovarian HOSEpiC cells was detected by qRT-PCR. The expression level of MALAT1 was high in EOC cell lines SK-OV-3, OVCAR-3, CAOV-3 and A2780 cells compared to normal ovarian HOSEpiC cells and ovarian clear cell carcinoma ES-2 cells (P<0.05) (Fig. 1B).

Based on the Kaplan-Meier Plotter online database, we further analyzed the effect of MALAT1 on the OS and PFS of patients with OC. The survival plots revealed that the expression levels of MALAT1 were correlated with OS and PFS. The patients with high MALAT1 expression had low OS as shown in Fig. 2A (Affymetrix ID: 226675_s_at) and Fig. 2B (Affymetrix ID: 224567_x_at) and PFS as shown in Fig. 2C (Affymetrix ID: 226675_s_at) and Fig. 2D (Affymetrix ID: 224567_x_at) (all P<0.05).

In order to investigate the potential function of MALAT1 on the biological behaviors of OC cells, we conducted a loss-of-function assay. Knockdown of MALAT1 by MALAT1-siRNA (siMALAT1) was confirmed by qRT-PCT in OVCAR-3 and SK-OV-3 cells (Fig. 3A and B). Cells transfected with siMALAT1 had a low expression of MALAT1 compared with cells transfected with negative control-siRNA (siNC) and blank control (Blank). Next, we assessed cell viability using a CCK-8 assay. We found that the knockdown of MALAT1 significantly inhibited OC cell viability after 48 h of transfection with siMALAT1 (Fig. 3C and D).

Next, we investigated the effect of MALAT1 knockdown on OC cell migration and invasion. Using Transwell migration assays, we determined that the number of migrated cells of OVCAR-3 (Fig. 4A and B) and SK-OV-3 (Fig. 4C and D) was significantly decreased after MALAT1-siRNA transfection (siMALAT1) for 48 h compared with non-transfected cells (Blank) and negative control-siRNA (siNC) transfected cells. Furthermore, the knockdown of MALAT1 also significantly decreased the number of invaded cells of OVCAR-3 (Fig. 5A and B) and SK-OV-3 (Fig. 5C and D).

One of the functions of lncRNAs is to regulate RNA expression. The regulatory mechanism of lncRNAs on miRNAs is that lncRNAs can act as sponges to influence the function of miRNAs (29). Using online software LncBase Predicted v.2, we predicted a candidate of miRNA hsa-miR-143-3p that is a potential target of MALAT1. Using the loss-of-function approach, we demonstrated for the first time that MALAT1 expression was correlated with miR-143-3p expression. The expression of miR-143-3p was significantly increased in OVACR-3 (Fig. 6A) and SK-OV-3 (Fig. 6B) cells after MALAT1 knockdown (siMALAT1) for 48 h compared with the controls (Blank and siNC) (P<0.05) as detected by qRT-PCR. Next, we used a dual-luciferase reporter assay to confirm the interaction between MALAT1 and miR-143-3p. Three sequences are shown in Fig. 6C: hsa-miR-143-3p, a wild-type MALAT1 containing a miR-143-3p binding site (position at 3990–3997 of MALAT1), and a mutated MALAT1 in which the binding site was changed. The Dual-Luciferase reporter assay confirmed that wild-type MALT1, not mutated MALAT1, bound to miR-143-3p in 293T cells after 72 h of co-transfection (Fig. 6D). These data revealed that MALAT1 could directly bind to miR-143-3p. Using miRWalk 2.0 database, we found that miR-143-3p potentially targets CMPK, a molecule previously demonstrated to play a role in EOC (30). Western blotting revealed that treating OVCAR-3 and SK-OV-3 cells with miR-143-3p mimics significantly decreased CMPK protein expression (Fig. 6E and F).