EOC tissue samples were collected from patients who had undergone curative surgical treatment between March 2013 and March 2014 at the First Hospital of Jilin University (Changchun, China). All patients provided written informed consent for participation in the present study. Paired cancerous and noncancerous tissues (2 cm from the EOC tissues; n=52) were collected and stored in liquid nitrogen until further use. Two pathologists independently confirmed the EOC diagnosis. None of the enrolled patients had received any perioperative treatment, such as radio-chemotherapy. The clinicopathologic data of the patients with EOC that participated in the present study are presented (Table I). The present study was compliant with the Declaration of Helsinki and was approved by the Ethics Committee of the First Hospital of the Jilin University (Jlu20210121-1; Changchun, China).

The human EOC SKOV3 and A2780 cell lines, and human ovarian surface epithelial cells (HOSEpiCs) were purchased from the American Type Culture Collection and were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO₂ incubator at 37°C.

Lentivirus-mediated HOXA-AS2 short hairpin RNAs (sh-HOXA-AS2s) and a non-targeting control shRNA (sh-NC) were constructed and packaged by Guangzhou Geneseed Biotech. Co., Ltd. with sequences as follows: sh-HOXA-AS2#1, 5′-GCTTACCTAGAAAGATGTTTCAAGAGAACATCTTTCTAGGTAAGCG-3′; sh-HOXA-AS2#2, 5′-TTTGCGTCTACAGACCTATCTTCAAGAGAGATAGGTCTGTAGACGCAAAG-3′; sh-HOXA-AS2#3, 5′-AGTTCAGCTCAAGTTGAACATTCAAGAGATGTTCAACTTGAGCTGAACTC-3′; and sh-NC, 5′-TTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAA-3′.

SKOV3 cells were plated in 6-well plates and grown to a cell density of ~60%, and then transfected with sh-HOXA-AS2 or sh-NC using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.miR-372-3p inhibitor (anti-miR; 5′-GCUCAAAUGUCGCAGCACUUUUU-3′), inhibitor-negative control (anti-miR-NC; 5′-UUCUCCGAACGUGUCACGUTT-3′), miR-372-3p mimic (5′-AAAGUGCUGCGACAUUUGAGCGUGCUCAAAUGUCGCAGCACUUUUU-3′) and miR-NC (5′-ACGUGACACGUUCGGAGAATT-3′) were purchased from MedChemExpress. Transfection, of 100 nM miR-372 mimics, miR-NC, anti-miR-372 and anti-miR-NC into SKOV3 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was performed according to the manufacturer's protocol. Transfection efficiency was examined after 48 h using reverse transcription-quantitative PCR (RT-qPCR).

RNA was extracted from EOC samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed using the PrimeScript RT reagent kit (Takara Bio, Inc.) at 25°C for 10 min, 50°C for 45 min and 85°C for 5 min. Complimentary DNA was quantified using an ABI 7900 qPCR System with SYBR Green Real-time PCR Master Mix (Takara Bio, Inc.). The thermocycling conditions used were as follows: Denaturation at 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. All experiments were performed using previously described primers (14,18). The primers used were as follows: miR-372 forward (F), 5′-ACACTCCAGCTGGGAAAGTGCTGCGACATTT-3′ and reverse (R), 5′-GTGCAGGGTCCGAGGT-3′; HOXA-AS2 F, 5′-CCCGTAGGAAGAACCGATGA-3′ and R, 5′-TTTAGGCCTTCGCAGACAGC-3′; U6 F, 5′-CTCGCTTCGGCAGCACATATACT-3′ and R, 5′-ACGCTTCACGAATTTGCGTGTC-3′; and GAPDH F, 5′-GGGAAACTGTGGCGTGAT-3′ and R, 5′-GAGTGGGTGTCGCTGTTGA-3′. Relative gene expression levels were calculated from the data of three independent experiments using the 2^–∆∆Cq method (19). U6 was used as the internal reference for miR-372 and GAPDH was used as the internal reference for HOXA-AS2.

A PARIS Kit (Thermo Fisher Scientific, Inc.) was used to separate the nuclear and cytoplasmic fractions of SKOV3 cells according to the manufacturer's protocols. RT-qPCR was then used to assess HOXA-AS2 expression in these fractions, with U6 serving as a nuclear control and GAPDH as a cytoplasmic control, according to the aforementioned method.

Transfected EOC cells (5×10³ cells/well) were seeded and incubated in a 96-well plate for up to 72 h, with samples being isolated at 3 time points (24, 48 and 72 h) followed by the addition of CCK-8 solution (10 µl/well, Takara Bio, Inc.) for 28 h. A spectrophotometer (BioTek Instruments; Agilent Technologies, Inc.) was then used to measure the absorbance at 450 nm for each time point.

The apoptosis of HOXA-AS2-depleted SKOV3 cells was assessed using Annexin V-FITC/PI apoptosis detection kits (cat. no. A211; Vazyme Biotech Co., Ltd.) for fluorescence activated cell sorting on a BD FACSCanto™ II flow cytometer (BD Biosciences) according to the manufacturer's protocols. The apoptotic rate was calculated and analyzed using FlowJo 6.10 (FlowJo LLC).

A Transwell invasion plate with a pore size of 8 mm, which was precoated with Matrigel was used to investigate the invasion ability of SKOV3 cells. Briefly, the transfected cells (5×10⁴ cells/well) were added to the top chamber of the Transwell plate in serum-free medium, while the bottom chamber was filled with RPMI1640 medium containing 20% FBS (Gibco; Thermo Fisher Scientific, Inc.). After 24 h, the cells fixed with 4% paraformaldehyde at 25°C for 30 min and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) at 25°C for 10 min. Cells were imaged and counted in five different fields of view using an X71 inverted light microscope (Olympus Corporation).

The transfected cells (5×10⁴ cells/well) were seeded and cultured in a 6-well plate until confluent. The monolayer was then scratched with a pipette tip and cultured in a serum-free medium for 24 h. Baseline (0 h) and 24 h images were acquired using an X71 inverted light microscope (Olympus Corporation). Migration rate was calculated by dividing the change in wound width by the time spent in migration as previously described by Grada et al (20). ImageJ software (V1.8.0; National Institutes of Health) was used to measure the size of the wound.

The ENCORI database (http://starbase.sysu.edu.cn) was used to analyze the HOXA-AS2 binding interaction with miRNAs. Among the miRNAs identified, miR-372, which is known to serve tumor-suppressive roles in several cancers (18,21,22), was selected for further study. Wild-type and mutant HOXA-AS2 fragments with/without the binding sites for miR-372 were synthesized and placed into the psiCHECK2 vector (Promega Corporation) and were referred to as WT-HOXA-AS2 or MT-HOXA-AS2, respectively. The SKOV3 cells were cultured in a 12-well plate util ~80% confluent. Then, cells were co-transfected with a luciferase plasmid and either miR-372 mimics or miR-NC using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h, activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Biotinylated derivatives of wild-type, mutant or control miR-372 (Bio-miR-372-WT, Bio-miR-372-MT or Bio-NC, respectively) were purchased from Guangzhou RiboBio Co., Ltd. The biotinylated RNA was transfected into SKOV3 cells using Lipofectamine^® 2000 and cultured at 37°C with 5% CO₂ for 48 h based on the manufacturer's instructions. SKOV3 cells (1×10⁷) were lysed in the soft lysis buffer plus 80 U/ml RNasin (Promega Corporation). The cell lysate (100 µl) was then precipitated with M-280 streptavidin beads (Sigma-Aldrich; Merck KGaA) at 4°C for 12 h. The beads were harvested by centrifugation at 13,000 × g for 10 min at 4°C. The bound RNAs were purified using the RNeasy Mini kit (Qiagen GmbH). HOXA-AS2 expression was assessed in the purified RNA using RT-qPCR performed to the aforementioned method

SPSS v19.0 (IBM Corp.) was used to analyze data, which was presented as the mean ± SD. The χ² test was used to assess the relationship between HOXA-AS2 expression and the clinicopathologic features of patients with EOC. An unpaired Student's t-test (two-tailed) or ANOVA followed by Bonferroni's post hoc test were used to assess the statistical significance of differences. Kaplan-Meier analysis, coupled with the log-rank test, was performed to investigate the overall survival rate. Pearson's correlation coefficient was used to assess the correlation between HOXA-AS2 and miR-372. P<0.05 was considered to indicate a statistically significant difference.

Expression levels of HOXA-AS2 in the EOC tissues were significantly higher compared with those in the noncancerous tissues (Fig. 1A). All 52 patients with EOC were classified into two groups based on the mean expression level of HOXA-AS2, a high expression level (n=27) and a low expression level (n=25) group, to assess the relationship between levels of HOXA-AS2 and clinicopathological features. There was no significant association between the levels of HOXA-AS2 and, age or tumor size in the patients with EOC; however, a significant association was demonstrated between the expression level of HOXA-AS2 and lymph node metastasis and advanced Federation International of Gynecology and Obstetrics (FIGO) stage (Table I). Furthermore, Kaplan-Meier analysis demonstrated that elevated levels of HOXA-AS2 was associated with reduced overall survival in patients with EOC (Fig. 1B). Moreover, both EOC cell lines demonstrated significantly higher HOXA-AS2 expression levels compared with HOSEpiCs (Fig. 1C). The SKOV3 cell line showed a notably higher HOXA-AS2 expression level compared with the A2780 cell line, and was used in subsequent experiments. The localization of HOXA-AS2 in SKOV3 cells was assessed, which indicated that HOXA-AS2 was mainly located in the cytoplasm of SKOV3 cells (Fig. 1D).

To evaluate the biological role of HOXA-AS2, three lentivirus-mediated HOXA-AS2 shRNAs (sh-HOXA-AS2#1, sh-HOXA-AS2#2 and sh-HOXA-AS2#3) and a sh-NC were transfected into SKOV3 cells. Compared with sh-NC, all three shRNAs significantly reduced the expression of HOXA-AS2 in SKOV3 (Fig. 2A). sh-HOXA-AS2#3 showed the maximum reduction and was used in all subsequent experiments, where it was labeled as sh-HOXA-AS2. Furthermore, the CCK-8 assay indicated that the knockdown of HOXA-AS2 significantly decreased SKOV3 cells proliferation (Fig. 2B) and significantly induced cell apoptosis (Fig. 2C) compared with the sh-NC.

Transwell and wound healing assays were performed to assess the effect of the knockdown of HOXA-AS2 on the invasion and migration of SKOV3 cells. The results demonstrated that knockdown of HOXA-AS2 significantly decreased both cell invasion and migration in SKOV3 cells compared with the control (Fig. 3).

It has been previously reported that lncRNAs can regulate specific miRNAs involved in carcinogenesis by functioning as ceRNAs (23,24). The ENCORI database was used to predict the miR-372-HOXA-AS2 interaction. The results indicated that miR-372 contained a complementary binding sequence to HOXA-AS2 and thus, this was the potential target of HOXA-AS2 (Fig. 4A). This hypothesis was tested using a luciferase activity assay. Overexpression of miR-372 significantly reduced the luciferase activity of WT-HOXA-AS2 exclusively, with no significant difference demonstrated in MT-HOXA-AS2 (Fig. 4B). Moreover, the biotinylated RNA pull-down assay demonstrated that the bio-miR-372-WT group had significantly higher levels of HOXA-AS2 compared with both the control (bio-NC) and bio-miR-372-MT groups (Fig. 4C). HOXA-AS2 knockdown significantly increased the expression of miR-372 in SKOV3 cells compared with the negative control (Fig. 4D). However, a significant reduction in the expression of HOXA-AS2 was caused by the overexpression of miR-372 (Fig. 4E). There was a significant negative correlation between miR-372 expression and HOXA-AS2 in EOC tissues (P=0.001; r=−0.5442; Fig. 4F). This indicated that there was a direct interaction between HOXA-AS2 and miR-372, and thus that miR-372 functioned as a ceRNA.

Since HOXA-AS2 functions as a ceRNA by sponging miR-372, it was hypothesized that HOXA-AS2 might affect the progression of EOC by regulating miR-372. Rescue experiments in SKOV3 cells demonstrated that the knockdown of HOXA-AS2 significantly increased the levels of miR-372 in SKOV3 cells, while transfection with the miR-372 inhibitor significantly reversed this effect (Fig. 5A). Furthermore, downregulation of miR-372 significantly reversed the inhibitory effects of HOXA-AS2 depletion on cellular proliferation, apoptosis, invasion and migration in SKOV3 cells (Fig. 5B-E). Thus, HOXA-AS2 depletion inhibited the progression of EOC through the regulation of miR-372.