Patient samples and in situ hybridization

Ovarian cancer tissue microarray slides were obtained from patients recruited in the Second Affiliated Hospital of Kunming Medical University (Kunming, China), and included 3 tumor samples (epithelial ovarian cancer, Stage III/IV) and normal tissue samples (non-tumor adjacent tissue of ovarian). Histological diagnoses of the ovarian cancer samples were provided along with the tissue microarray slides. The digoxin (DIG)-labeled antisense and sense RNA probes for the full lengths of NEAT1 were used to detect NEAT1 expression in the tissue slides, as previously reported [45]. All procedures were conducted following a protocol approved by the Ethics Committee of the Second Affiliated Hospital of Kunming Medical University (RE-PJ-2021–01), with written informed consent.

Cell culture

The immortalized human ovarian surface epithelial cell line IOSE80 and human ovarian cancer cell line SKOV-3 were purchased from the American Type Culture Collection (Manassas, VA, USA). The human ovarian cancer cell line A2780 was purchased from Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). All the cell lines were tested as mycoplasma contamination free, and authenticated by STR (Short Tandem Repeat) DNA profiling analysis. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Hyclone™, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA), 100 units/mL of penicillin and 100 µg/mL of streptomycin (Gibco) at 37 °C in a humidified chamber with 5% CO2.

Gene overexpression and knockdown

The complementary DNA (cDNA) of NEAT1 was cloned into the lentivirus vector pLv201 (Invitrogen, Waltham, MA, USA), and the empty vector plasmid or NEAT1-expressing plasmid was used for transfection of human ovarian cancer cells. For cell transfection, SKOV-3 or A2780 cells were seeded into 6-well or 12-well plates and transfected with the indicated plasmids or miRNA mimics mixed with Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Stable cell lines were obtained after screening using 2 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA). The miRNA mimics were designed and synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China).

For NEAT1 knockdown, cells were infected with shRNA-expressing lentivirus particles, and the cells infected with scramble shRNA-expressing lentivirus were used as negative controls. The sequences of the shRNA are as follows: shLncRNA NEAT1, 5'-CTGTGAAATGCGGGTAAATGAATG-3'; shScramble, 5'-GAAGTGGCTAGACCTGACGCTAGG-3'. Virus packaging was conducted by co-transfection of lentiviral plasmids and the helper plasmids into 293 T cells (originally obtained from ATCC), and 48 h later, the viruses were harvested to infect the SKOV-3 or A2780 cells in logarithmic growth phase with polybrene at a multiplicity of Infection (MOI) of 20.

Cell viability

Control SKOV-3 or A2780 cells without transfection, stable SKOV-3 or A2780 cells transfected with empty vector or NEAT1-expressing vector, and SKOV-3 or A2780 cells expressing shNEAT1 RNA or scramble shRNA were seeded in 96 well-plates at the density of 5 × 103/well. The cell counting kit-8 assay (Sigma-Aldrich, St. Louis, MO, USA) was performed according to the manufacturer’s instructions. In brief, 10μL of CCK-8 solution was added to each well of different plates on Day 1, Day 2, Day 3, and Day 4, respectively, and the cells were incubated with the solution for another 1.5 h. Optical density (OD) values were measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA) to indicate the relative cell viability. The assays were carried out with triplicated samples per group, and the value of Day 1 in the control group was employed for normalization.

Cell migration and invasion

For cell migration assays, control SKOV-3 or A2780 cells without transfection, stable SKOV-3 or A2780 cells transfected with empty vector or NEAT1-expressing vector, and SKOV-3 or A2780 cells expressing shNEAT1 RNA or scramble shRNA were added into the upper chamber of the Transwell plate with 8 μm pore size (Sigma-Aldrich). For cell invasion assays, the upper chamber of the insert was pre-coated with Matrigel (ECM gel, Sigma-Aldrich). Cells were seeded in serum-free culture medium, and the medium containing 10% FBS in the lower chamber was served as chemoattractant. After incubation for 24 h, 48 h and 72 h, the cells that did not invade through the pores were carefully wiped out with a cotton swab. Then, the inserts with migrated or invaded cells were fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet for 20 min. Cells in five randomly selected visual fields per chamber were counted using an inverted microscope (Motic, Wetzlar, Germany) at 200 × magnification. Cell images were taken using the Moticam (Motic).

Flow cytometry

Apoptosis was determined using the Annexin V-phycoerythrin (PE) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s protocol. Briefly, after washing with phosphate-buffered saline (PBS) and the binding buffer for one time each, cells were stained with Annexin V-PE/7-Aminoactinomycin D (7-AAD) for 20 min at room temperature in dark. After washing with the binding buffer once, the labeled cells were detected immediately by a flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed by the FlowJo software (FlowJo, BD Biosciences).

Duo-luciferase reporter assay

The binding site of miR-214-3p and the lncRNA NEAT1 was predicted using the online tools LncBase Module (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=lncBase/index) and SPMLMI [46]. The predicted miR-214 binding site region of NEAT1 (5'- TGGCTAGCTCAGGGCTTCAG-3') and the corresponding mutated region (5'- ACCGATCGAGTCCCGAAGTC-3') were cloned into a Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP)-expressing vector pMT05-01 (Invitrogen, Waltham, MA, USA). SKOV-3 cells were co-transfected with the indicated vectors and the negative control mimic, or the miR-214-3p mimic. At 48 h after transfection, the culture supernatant and cells were harvested and subjected to luciferase activity analysis using a Dual-Luciferase Reporter Assay System (GeneCopoeia, Rockville, MD, USA) following the manufacturer’s instructions. The relative luciferase activity was quantitated according to the ratios of Gluc to SEAP.

Histology and immunohistochemistry

Tumor tissues immersed in 10% formalin solution were fixed at 4 °C for 24 h, and were then dehydrated with different grades of ethanol. The transparent tissue was embedded into paraffin, cut into 5 µm slices with a microtome, and stained with hematoxylin and eosin (H&E). The histopathological changes of the tissue sections were observed using a Zeiss Axio Lab A1 microscope (Gottingen, Germany), and images were taken with the magnification of 200 × .

For immunohistochemistry staining, the paraffin embedded tumor tissue specimens were subjected to deparaffinization in xylene and rehydration through a series of descending grades of alcohol solutions. Sections were incubated within 3% hydrogen peroxide to block endogenous peroxidase activity followed by microwaved in citrate buffer for antigen retrieval. Nonspecific staining was blocked by incubation with 10% normal goat serum in PBS for 30 min. Sections were incubated with monoclonal mouse antibodies against human SEMA4D (1:100 dilution), Plexin-B1 (1:50 dilution), Tiam1 (1:100 dilution), or RAC (1:100 dilution), CD31 (1:200 dilution) (Abcam, Cambridge, UK) at 4 °C overnight, followed by PBS washes and a further incubation with Horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulin G (IgG)(H + L) (Zsbio, Beijing, China) at room temperature for 2 h. Thereafter, the sections were stained with diami-nobenzidine (DAB) using the 3,3’-diaminobenzidine chromogenic substrate kit (Zsbio, Beijing, China). The positively stained cells in randomly selected 6 visual fields of each group were counted. The positivity percentages were determined as follows: (number of positive cells)/(total number of cells) × 100, and normalized to the group of mice inoculated with un-transfected SKOV-3 cells.

Real-time quantitative PCR (qPCR)

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Total RNA (1 μg each sample) was used to synthesize cDNA utilizing the all-in-one first-strand cDNA synthesis kit (GeneCopoeia, China). Quantitative PCR for miRNA and mRNA were performed using a standard protocol from the SYBR green master mix (GeneCopoeia) on a 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative quantification was determined by normalization to beta-actin (for mRNA or lncRNA) or U6 (for miRNA). The sequences of primers for qPCR analysis are listed in Table 1. PCR primers were synthesized by Sangon Biotech Co. Ltd (Shanghai, China). The PCR reaction protocol consisted of two steps: step one, initial denaturation for 30 s at 95 ℃; step two, denaturation for 5 s at 95 ℃, annealing and extension for 31 s at 60 ℃ and fluorescence signal acquisition. The reactions had a total of 40 cycles, and ended with a melting curve which consisted of 15 s at 95 ℃, 1 min at 60 ℃, 15 s at 95 ℃ and 15 s at 60 ℃. The experiments were repeated for 3 times and each sample was run in triplicates. PCR product specificity was confirmed by melting curve analysis. Gene expression levels were calculated with the 2−ΔΔCT method [47].

Table 1 The sequences of qPCR primers in this studyWestern blot

After washing with cold PBS, SKOV3 or A2780 cells or these cell-derived xenograft samples were lysed in radioimmunoprecipitation assay lysis buffer (RIPA; Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail on ice for 30 min. After centrifugation at 16,000 g for 10 min at 4˚C, proteins in the supernatants were quantified and equal amounts of total proteins were loaded. Samples were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and incubated with indicated primary antibodies at room temperature for 1 h. These antibodies were as follows: anti-SEMA4D antibody (1:1,000 dilution; Affinity Biosciences, USA), anti-VEGF (1:1000; Abcam, Cambridge, UK), anti-XBP1 (1:1000; Abcam), and anti-β-actin (1:5000; Abcam). The secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:10,000; TransGen Biotech, Inc., Beijing China) or anti-mouse IgG (1:10,000; TransGen Biotech, Inc.) antibodies were used. Proteins of interest were visualized using enhanced chemiluminescence kit (EMD Millipore, Burlington, MA, USA). The band intensities were quantified by densitometry using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Western blots of all the experiments were repeated at least 3 times and one representative blotting result is shown for each experiment.

Xenograft animal model

Six-eight-week-old female BALB/c nude mice were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China), and housed at the specific pathogen-free (SPF) facility at the Animal Center of Kunming Medical University (Kunming, China). Mice were maintained at room temperature (22 ± 1 ˚C) with a 12/12 h light/dark cycle and access to food and water ad libitum. Control SKOV-3 or A2780 cells without transfection, stable SKOV-3 or A2780 cells transfected with empty vector or NEAT1-expressing vector, and SKOV-3 or A2780 cells expressing shNEAT1 RNA or scramble shRNA were injected subcutaneously into the right inguen flank of each mouse (1 × 107 cells/mouse) to establish the xenograft model (n = 5/group). Tumor volume (V) was monitored every 7 days by measuring the tumor diameters starting seventeen days after tumor inoculation, and calculated with the following formula: V = ab2/2, where a is the long diameter, while b is the short diameter. Thirty days after tumor inoculation, mice were euthanized and tumor tissues were frozen at -80C for further analyses. Animal experiments were conducted in accordance with the Declaration of Helsinki and all procedures involving experimental animals were approved by the University Committee on the Use and Care of Animals (UCUCA) at the Kunming Medical University.

Statistics

Statistical analysis was performed with SPSS (Version 22.0; IBM Corporation, Armonk, NY, USA). All the parameters underwent normality tests. Data were expressed as the mean ± standard deviation. Group differences were analyzed using one-way ANOVA, followed by Bonferroni post-hoc tests for pairwise comparisons. Paired t-tests were performed for group comparisons, with P < 0.05 indicating statistical significance.