Bioinformatics analysis
The Cancer Genome Atlas (TCGA) datasets were used to obtain clinical and RNA sequencing information on 419 OC and 88 normal ovarian tissue samples. The docking of NRSN2-AS1 bound to PTK2 were predicted with HDOCK (http://hdock.phys.hust.edu.cn/) and visualized with PyMOL (https://pymol.org/2/) as previously described 12, 13.
Samples collection
Fresh tumor and paracancerous tissues were derived from 23 OC patients with surgical resection from January 2021 to January 2022 at the Suzhou Municipal Hospital. All participants signed informed consent, and participant information will be fully protected. Approval was received from the Research Ethics Committee of Suzhou Municipal Hospital. Collected tissues were stored in RNA Keeper Tissue Stabilizer (Vazyme, Nanjing, China) at − 80 ℃ as previously described 14.
RNA extraction and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays
Tissues fixed in RNA Keeper Tissue Stabilizer were ground to powder with liquid nitrogen. We extracted RNA with Total RNA Extraction Reagent (TRIzol, Vazyme) and reverse transcribed it into cDNA with HiScript III RT SuperMix for qPCR kit (Vazyme). The relative mRNA expression was measured using an AceQ qPCR SYBR Green Master Mix kit (Vazyme) on an Applied Biosystems 7500 RealTime PCR System. Finally, the expression level was analyzed with 2–ΔΔCT and normalized to 18sRNA. Table S1 lists the primers used in this study.
Fluorescence in situ hybridization (FISH) analysis
A tissue microarray with 48 OC and 10 normal tissue samples was purchased from Zhongke Huaguang Biotechnology Company (Shanxi, China). FISH assays were carried out with FISH kit (RiboBio Biotechnology, Guangzhou, China) following the manual. A unique probe targeting NRSN2-AS1 was synthesized by RiboBio. 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Haimen, China) was used for staining nuclei. All microscopy images were captured with a confocal laser microscope (LSM 810, Carl Zeiss, Oberkochen, Germany).
Cell culture and treatments
Human OC cell lines (OVCAR3, A2780 and SKOV3) and normal ovarian epithelial cell lines (IOSE80) were obtained from the Chinese Academy of Cell Collection (Shanghai, China), and cultured in a humidified conditions at 37 ℃ with 5% CO2. IOSE80, SKOV3 and OVCAR3 cells were cultivated in RPMI-1640 (Gibco, USA) with fetal bovine serum (FBS) (ExCell Bio, New Zealand) and 1% penicillin/streptomycin (PS) (NCM Biotech, China). Specifically, 10%FBS was needed to IOSE80 and SKOV3, and 20% to OVCAR3. Dulbecco’s modified Eagle medium (Gibco, USA) with 10% FBS and 1% PS were used to culture A2780 cells.
When the cell fusion degree reached 60–70%, small interfering RNAs (siRNAs) (GenePharma, Suzhou, China) targeting NRSN2-AS1, PTK2, and MG53 as well as overexpression plasmids (GenePharma) pcDNA3.1-NRSN2-AS1, pEX-1-MG53 and an empty vector (EV) were transfected into OC cells by Lipofectamine 2000 (Invitrogen, USA) and X-treme GENE HP DNA transfection reagent (Mannheim, Germany). The nucleotide sequences of all siRNAs are shown on Table S2.
XAV-939 and SKL2001 (Selleck, Shanghai, China) were solubilized in dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA) at 10 mM, and used in experiments at 15 µM. Following treatment, cells were harvested for further analysis 48 hours later.
Cell proliferation assays
A 96-well plate (2000 cells/well) was inoculated with the cells for the CCK-8 assay. Cell viability was measured every 24 h using a Cell Counting Kit-8 kit (Beyotime) on a microplate reader (Bio-Rad Model 680, USA) at 450 nm.
800–1000 cells were added into six-well plates for 2 weeks to evaluate cloning formation capabilities. The clones were fixed with methanol, stained with 0.1% crystal violet (Beyotime) and counted for analysis.
Cell migration assays
In the transwell assay, 4.5×104 cells in 300 µl serum-free medium were seeded into the upper chamber with 8 µm pore size (Corning, USA), whereas 700 µl complete medium was added into the lower chamber. After 48 h, the cells outside the chamber were fixed, stained and imaged for counting.
Xenograft in mice
Four-week-old female athymic BALB/c nude mice (Vital River Laboratory, China) were used to construct a subcutaneous tumor model and lung metastasis model. Mice received 12 h light/12 h dark at 22–28 ℃ and 50–70% humidity under specific-pathogen-free conditions. The approval of mice investigations obtained from the Animal Ethics and Welfare Committee of Nanjing Medical University.
For the subcutaneous tumor model 15, 1×107 cells transfected with sh-NRSN2-AS1 or sh-NC were subcutaneously injected into the left and right flanks of nude mice, respectively (n = 5). The tumor volumes were calculated every 3 days (V = 0.5 × D × d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter)). We sacrificed the mice at 11 days, follow by removing and measuring the subcutaneous tumors.
For the lung metastasis model 16, the tail veins of mice were injected with 2 × 107 treated cells (n = 5). An eight-week period ended with the mice being sacrificed, their lungs were removed and fixed in formalin.
Immunofluorescence
Immunofluorescence assays were conducted as previous descriptions 17, 18, 19, 20. Briefly, the slides containing target cells and tissues were incubated with primary and Alexa-Fluor secondary antibodies (Thermo Scientific, Waltham, USA) orderly. Confocal laser microscopes (LSM 810) were used to observe all samples. A list of the primary antibodies used can be found in Table S4.
Hematoxylin and eosin (H&E) staining
Staining of lung tissue sections with H&E was followed by ethanol dehydration, xylene hyalinization, and neutral balsam closure. A microscope (Axioskop 2 Plus, Zeiss) was used to photograph.
RNA pull-down assays and LC-MS/MS analysis
T7 RNA polymerase (Ambio Life, Shanghai, China) and Biotin RNA Labeling Mix (Ambio Life) were used to transcribe and label NRSN2-AS1 according to the manual. Next, Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific) was utilized to carry out RNA pull-down assays 21. Finally, the proteins interacting with NRSN2-AS1 were identified by LC-MS/MS analysis.
Western blot assays
We performed Western blotting according to our previous protocol 16, 22. In short, radioimmunoprecipitation assay (RIPA, Beyotime) buffer containing 1% protease inhibitor phenylmethylsulfonyl fluoride (PMSF, Beyotime) was used to lysis protein samples. Subsequently, the proteins were denaturalized at 100°C for 10 min, electrophoresed with 10% SDS-polyacrylamide gel (GenScript, China) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA). Primary antibodies and horseradish peroxidase-conjugated secondary antibodies were incubated on membranes. In the end, an Image-Pro Plus (Media Cybernetics, USA) was employed to measure the bands signal detected with the BeyoECL Plus kit (Beyotime). Tubulin antibody was used as a control. There is detailed information about the antibodies in Table S3.
RNA immunoprecipitation (RIP) assays
RIP assays were conducted with Magna RIPTM RNA-Binding Protein immunoprecipitation kit (Millipore) as previously described 21. Briefly, cell lysate, which lysed by RIP lysis buffer, was incubated with anti-PTK2 and anti-IgG antibodies at 4°C. Subsequently, the protein–RNA complexes were obtained using 0.5 mg/ml proteinase K with 0.1% SDS. RT-qPCR were done to determine the interaction between PTK2 and NRSN2-AS1.
Protein half-life assays
Cycloheximide (CHX, 100 µg/ml) was added into OC cells to interrupt protein synthesis. Western blotting was conducted to detect the expression of PTK2 at 0, 1, 2, 4 h.
Immunoprecipitation (IP) assays
IP assays were carried out according to our previously described procedure12, 23. Cell lysates were incubated with anti-PTK2 or anti-IgG antibodies overnight, followed by washed beads (Santa Cruz) for 2 h at 4 ℃. Finally, the IP product, was detected by specific antibodies via western blotting. Antibody information is provided in Table S3.
Ubiquitination assays
RIPA buffer containing 1% PMSF was used to lysed OC cells transfected with siRNAs or overexpression plasmids. Anti-PTK2 or anti-IgG antibodies were incubated overnight with cell lysates, followed by protein A/G beads for 2 h at 4°C. Finally, the products were analyzed using immunoblotting with anti-Ub and anti-Ub-K48.
Statistical analysis
The data were provided as mean ± standard deviation and analyzed using Student's t-tests for two groups, and one-way analyses of variance for three or more groups on SPSS 17.0 (SPSS, Inc., USA) and GraphPad Prism 7.0 (GraphPad Software, USA). Survival curves were profiled with Kaplan–Meier survival plots. P < 0.05 was represented as statistical significance.