Patients and tissue specimens
Fifty-six EOC tissues were collected from patients with EOC in Qilu Hospital of Shandong University. A total of 21 ovarian surface epithelial tissues were obtained from patients who underwent oophorectomy or hysterectomy for nonmalignant reasons. Patients that had received preoperative radiotherapy or chemotherapy were excluded from the study. All fresh tissues were stored in liquid nitrogen until further use. The study was approved by the Ethics Committee of Qilu Hospital of Shandong University and carried out in accordance with the World Medical Association Declaration of Helsinki. Written informed consent was obtained from all participants.
Cell culture and transfection
Human ovarian surface epithelial cells (OSE) were obtained from ScienCell Research Laboratories (cat. no. 7310), and cultured in ovarian epithelial cell medium (cat. no. 7311; ScienCell Research Laboratories). Four EOC cell lines, ES-2, OVCAR3, CAOV-3, and SK-OV-3, were purchased from the Cell Bank of Type Culture Collection, Chinese Academy of Science (Shanghai, China). McCoy's 5A (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.,) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.,) was used to culture ES-2 and SK-OV-3 cells. CAOV-3 cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.,) containing 10% FBS, 1% penicillin/streptomycin mixture and 1% sodium pyruvate 100 mM solution (Gibco; Thermo Fisher Scientific, Inc.,). RPMI-1640 medium was used to the culture OVCAR3 cells and supplemented with 0.01 mg/ml bovine insulin (Gibco; Thermo Fisher Scientific, Inc.,) and 20% FBS. All cells were kept at 37°C in a humidified atmosphere with 5% CO2.
To silence PTPRG-AS1 expression, EOC cells were transfected with small interfering RNAs (siRNA) targeting PTPRG-AS1 (si-PTPRG-AS1). The negative control siRNA (si-NC) was used as the control for si-PTPRG-AS1. The miR-545-3p mimic, negative control (NC) mimic, miR-545-3p inhibitor, and NC inhibitor were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The histone deacetylase 4 (HDAC4) overexpressing plasmid pcDNA3.1-HDAC4 (pc-HDAC4) was chemically synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and used to induce HDAC4 overexpression. EOC cells were collected and seeded into 6-well plates. After growing to 70%–80% confluency, cells were transfected with the molecular products described above using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.,). The total RNA was reverse-transcribed to cDNA using an miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany). Quantitative PCR was done to measure miR-545-3p expression using an miScript SYBR Green PCR kit (Qiagen GmbH). miR-545-3p expression was normalized to that of U6 small nuclear RNA.
For mRNA detection, reverse transcription was performed with the PrimeScript™ RT reagent Kit (Takara, Dalian, China). PTPRG-AS1 and HDAC4 mRNA expression was quantified by quantitative PCR using the TB Green Premix Ex Taq (Takara). GAPDH used as an internal inference for PTPRG-AS1 and HDAC4. Relative expression was calculated using the 2−ΔΔCq method.
Cell Counting Kit-8 (CCK-8) assay
Transfected cells were collected, counted and seeded into 96-well plates at a density of 2,000 cells per well. After culturing for 0 h, 24 h, 48 h or 72 h, 10 μl of CCK-8 solution (KeyGen BioTECH; Nanjing, China) was added into each well followed by a 2 h incubation period at 37°C in a humidified atmosphere with 5% CO2. The absorbance was read at a 450 nm wavelength using a microplate reader (BioTek, Winooski, VT, USA).
Cell apoptosis analysis by flow cytometry
An Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (Biolegend, San Diego, CA, USA) was used for measuring the relative number of apoptotic cells. Briefly, transfected cells were harvested after 48 h of culture, washed with phosphate buffer saline (PBS), and centrifuged. The collected cells were resuspended in 1 × binding buffer and stained with 5 μl annexin V-FITC and 10 μl propidium Iodide. The apoptotic cells were quantified by flow cytometry (FACScan; BD Biosciences, San Jose, CA, USA).
Transwell cell migration and invasion assays
After a 48-hour incubation, transfected cells were trypsinized using 0.25% trypsin, washed with PBS, and resuspended in serum-free culture medium. The concentration of cell suspension was adjusted to 5 × 105 cells/ml. Transwell chambers (8 μM pore size; BD Biosciences) were used for the cell migration assays, whereas cell invasion assays were done with Matrigel-coated (BD Biosciences) chambers. The apical chambers were loaded with 200 μL cell suspension, while 500 µl of complete culture medium containing 10% FBS was added into the basolateral chambers. Twenty-four hours later, the cells that had migrated or invaded through the pores were collected with a cotton swab. The migrated and invaded cells were fixed with 5% glutaraldehyde, stained with 0.1% crystal violet, and washed three times with PBS. After drying, the cells were imaged using an optical microscope (Olympus, Tokyo, Japan). The number of migrated and invaded cells was counted in five randomly selected fields and was considered to be a reflection of the migratory and invasive capacities.
In vivo tumor xenograft study
The Institutional Animal Care and Use Committee of Qilu Hospital of Shandong University approved the experiments and procedures involving animals. The in vivo tumor xenograft study was performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. The lentivirus plasmids overexpressing PTPRG-AS1 short hairpin RNA (sh-PTPRG-AS1) or negative control shRNA (sh-NC) were obtained from Shanghai GenePharma Co., Ltd., and transduced into HEK293T cells in the presence of lentivirus packaging plasmids. The supernatants were collected after 72-hour incubation and used to infect CAOV-3 cells. Puromycin (0.5 μg/ml) was used to select CAOV-3 cells stably expressing sh-PTPRG-AS1 or sh-NC. In total, 1 × 107 CAOV-3 cells stably transfected with sh-PTPRG-AS1 or sh-NC were subcutaneously injected into BALB/c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.; Beijing, China). Tumor width and length was recorded at a 4 day interval for totally 4 weeks, and the data were used for calculating tumor volumes by the following equation: volume = 0.5 × (length × width2). All mice were euthanized and the tumor xenografts were excised and weighed.
Two bioinformatics tools, miRDB (http://mirdb.org/) and StarBase 3.0 (http://starbase.sysu.edu.cn/), were used to identify miRNAs that potentially target PTPRG-AS1. The molecular targets of miR-545-3p were predicted by means of miRDB, StarBase 3.0 and TargetScan (http://www.targetscan.org/).
EOC cells were washed with ice-cold PBS and centrifuged. Subcellular fractionation was conducted to isolate cytoplasmic and nuclear fractions of EOC cells using the Nuclear/Cytosol Fractionation Kit (Biovision, San Francisco, CA, USA). The localization of PTPRG-AS1 expression in EOC cells was determined by RT-qPCR analysis.
Luciferase reporter assay
The wild-type (WT) fragments of PTPRG-AS1 and HDAC4 were amplified and subcloned into the pmirGLO reporter vector (Promega Corporation, Madison, WI, USA). The resulting luciferase reporter plasmids were termed as PTPRG-AS1-WT and HDAC4-WT. Mutation sequences were generated using a Site-Directed Mutagenesis Kit (Agilent, Santa Clara, USA), and the mutant (MUT) fragments were inserted into pmirGLO reporter vectors to obtain PTPRG-AS1-MUT and HDAC4-MUT.
For reporter assays, EOC cells were cotransfected with WT or corresponding MUT reporter plasmids and miR-545-3p mimic or NC mimic using Lipofectamine® 2000. Transfected cells were lysed 48 h after incubation and the luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation).
RNA immunoprecipitation (RIP) assay
RIP assay was performed to examine the interaction between miR-545-3p and PTPRG-AS1 in EOC cells following the instructions of Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Bedford, MA, USA). A complete RIP lysis buffer was used to lyse the EOC cells and the cell lysates were incubated at 4°C with magnetic beads, which were combined with human anti-Argonaute2 (Ago2) antibody or normal mouse IgG (Millipore). After 24 h, the magnetic beads were rinsed and treated with Proteinase K to digest protein. Finally, the immunoprecipitated RNA was analyzed by RT-qPCR to determine the expression of miR-545-3p and PTPRG-AS1.
RIPA buffer (Beyotime Institute of Biotechnology; Shanghai, China) supplemented with a protease inhibitor cocktail (Beyotime Institute of Biotechnology) was used for total protein extraction. Equal amounts of protein were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membranes. Tris-buffered saline containing 0.1% Tween-20 (TBST) supplemented with 5% nonfat dried milk was employed for blocking the membranes for 2 h at room temperature. Next, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies against HDAC4 (cat. no. ab235583) and GAPDH (cat. no. ab128915) were purchased from Abcam (Cambridge, MA, USA) and used at a dilution of 1:1000. The membranes were washed three times with TBST and incubated at room temperature for 2 h with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. ab205718; Abcam). Protein bands were visualized using the Immobilon Western Chemilum HRP substrate (Millipore).
All measured data were expressed as the mean ± standard deviation based on at least three independent experiments. Comparisons between two groups were done with Student's t-test, whereas one-way ANOVA and Tukey’s post-hoc test was used for determining differences among multiple groups. The expression correlation analysis for PTPRG-AS1, miR-545-3p and HDAC4 was calculated by Pearson's correlation analysis. The overall survival of EOC patients was determined by Kaplan-Meier survival analysis and the survival curves were compared using the log-rank test. P values less than 0.05 were considered to be statistically significant.