Patients and tissue specimens
Fifty-one pairs of EOC tissues and adjacent normal tissues were obtained from Weifang People’s Hospital. All the tissues were quickly frozen in liquid nitrogen and then stored in liquid nitrogen until use. Patients who had other types of human cancer or had received chemotherapy or radiotherapy were excluded from this research. The collection and use of human tissues was approved by the Ethics Committee of Weifang People’s Hospital, and the protocols were implemented in compliance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all the patients enrolled in this research.
ES-2, an EOC cell line, was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in McCoy's 5a Medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% v/v heat-inactivated foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Three EOC cell lines, namely, OVCAR3, CAOV-3, and SK-OV-3, were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). The culture conditions for the SK-OV-3 cell line were the same as those for the ES-2 cell lines. Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS was utilized to culture the CAOV-3 cells. The OVCAR3 cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS and 0.01 mg/ml bovine insulin (Gibco; Thermo Fisher Scientific, Inc.). The human ovarian surface epithelial cell line (OSE) was purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and grown in ovarian epithelial cell medium (ScienCell Research Laboratories). All the cell lines mentioned above were cultured at 37°C in an incubator containing a humidified atmosphere with 5% CO2.
To overexpress miR-431-5p and SOX9, transfection of the miR-431-5p mimic (GenePharma; Shanghai, China) and pcDNA3.1-SOX9 (Shanghai Sangon Co., Ltd.; Shanghai, China) was performed with Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and the miRNA mimic control (miR-NC; GenePharma) and empty pcDNA3.1 plasmid were used as controls. Small interfering RNAs (siRNAs) targeting LINC01132 (si-LINC01132) and the miR-431-5p inhibitor were synthesized by GenePharma and used to knock down LINC01132 and miR-431-5p expression in EOC cells. The negative control (NC) siRNA (si-NC; GenePharma) and NC inhibitor served as the control for si-LINC01132 and the miR-431-5p inhibitor, respectively.
RNA extraction and reverse-transcription quantitative PCR (RT-qPCR)
Total RNA extraction was performed with TRIzol® reagent, and the RNA was quantified with a NanoDrop™ 2000 spectrophotometer (both from Invitrogen; Thermo Fisher Scientific, Inc.). To detect miRNA expression, first-strand cDNA was synthesized from the total RNA by performing reverse transcription using a Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Dalian, China). Next, PCR quantification was conducted on an Applied Biosystems 7500 Real-time system with a Mir-X miRNA qRT-PCR TB Green® Kit (TaKaRa). For the determination of LINC01132 and SOX9 expression, reverse transcription was carried out utilizing a PrimeScript™ RT reagent kit with gDNA Eraser (Takara), after which the cDNA was used for PCR amplification with TB Green® Premix Ex Taq™ II (Takara). U6 small nuclear RNA was used as the internal control for miRNA, and GAPDH was used as the internal control for mRNA in this assay. The 2-ΔΔCq method was used to process all the data.
Cell Counting Kit-8 (CCK-8) assay
The proliferation of EOC cells was estimated by CCK-8 assay (Dojindo Chemical Laboratory, Kumamoto, Japan). In detail, transfected cells were counted and seeded into 96-well plates. Every well was seeded with 2000 transfected cells that were suspended in 100 µl complete culture medium. At 0, 1, 2, and 3 days after adherence to the wells, the cells were further incubated with 10 µl CCK-8 solution, and the cells were cultured for an additional 2 h under the conditions described above. Finally, the absorbance at a wavelength of 450 nm was monitored via a microplate reader.
Flow cytometry analysis of cell apoptosis
Transfected cells were digested with trypsin (Gibco; Thermo Fisher Scientific, Inc.), washed with ice-cooled phosphate-buffered saline and centrifuged at 1000 xg for 5 min. The apoptosis of these cells was analysed with an Annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime Institute of Biotechnology; Shanghai, China). Briefly, the cells were resuspended in 195 μL of annexin V–FITC binding buffer and cultivated with 5 μl Annexin V-FITC and 10 μl PI at 25°C. The incubation was performed in the dark and continued for 20 mins. All the samples were analysed with a flow cytometer (FACScan, BD Biosciences, Franklin Lakes, NJ, USA).
Cell migration and invasion assays
The EOC cell migration and invasion abilities were evaluated with the Transwell method. For the migration assay, the transfected cells were washed with phosphate-buffered saline and centrifuged. The upper compartments of the Transwell chambers (8-µm pores; BD Biosciences) were loaded with 5 x 104 cells resuspended in 200 μl serum-free medium. A volume of 600 μl culture medium supplemented with 20% FBS was added to the lower compartments and served as a chemoattractant. After 1 day of incubation at 37°C, the nonmigrated cells remaining on the top surface of the membranes were removed with a cotton swab, and the migrated cells that crossed the pores were fixed with methanol and dyed with 0.1% crystal violet. The stained cells were photographed using a light microscope (Olympus, Tokyo, Japan), and the number of migrated cells in five random fields of view was counted. For the invasion assay, the experimental steps were the same, except that the Transwell chambers were precoated with Matrigel (BD Biosciences).
Tumour xenograft model
Short hairpin RNA (shRNA) targeting LINC01132 and NC shRNA (sh-NC) were prepared by GenePharma. After these molecules were inserted into a lentiviral vector, the vectors were transfected into 293T cells (National Collection of Authenticated Cell Cultures) with psPAX2 and pMD2.G. Approximately 48 h after transfection, the lentiviruses were collected and used to inject CAOV-3 cells. Three days later, fresh complete culture medium containing puromycin was used to further incubate the CAOV-3 cells, yielding cells stably expressing sh-LINC01132 or sh-NC.
All the experimental steps involving animals were conducted with approval from the Institutional Animal Care and Use Committee of Weifang People’s Hospital. Four-week-old female BALB/c nude mice were acquired from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The stably transfected cells were harvested and subcutaneously injected into the nude mice. One week after cell injection, the sizes of the subcutaneous tumours were recorded every four days, and their volumes were calculated with the formula: Volume = 1/2 × length × width2. The mice were euthanized by cervical vertebra dislocation on day 31, and the subcutaneous tumours were harvested for subsequent use.
The online software miRDB (http://mirdb.org/) was used to identify the downstream target of LINC01132. The direct target of miR-431-5p was predicted utilizing TargetScan (http://www.targetscan.org/vert_60/) and miRDB.
Subcellular fractionation assay
The lncRNA subcellular localization predictor lncLocator (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) was utilized to predict the subcellular distribution of LINC01132 in human cells. The prediction was further verified by a subcellular fractionation assay with a Cytoplasmic & Nuclear RNA Purification kit (Norgen Biotek Corp.). EOC cells were processed with cell fractionation buffer to separate the cytoplasm and nucleus. The relative expression of LINC01132 in both fractions was determined by RT-qPCR. GAPDH and U6 acted as the cytosolic and nuclear controls, respectively.
Luciferase reporter assay
Fragments of LINC01132 and the SOX9 3’-UTR containing wild-type (wt) miR-431-5p-binding sequences were amplified and inserted into the downstream region of the psiCHECK™-2 luciferase reporter vector (Promega Corporation, Madison, WI, USA). The luciferase reporter vectors were labelled psiCHECK™-2-LINC01132-wt and psiCHECK™-2-SOX9-wt. The corresponding mutant (mut) luciferase reporter vectors, namely, psiCHECK™-2-LINC01132-mut and psiCHECK™-2-SOX9-mut, were generated in the same manner. For the reporter assay, EOC cells were seeded into 24-well plates prior to being cotransfected with the miR-431-5p mimic or miR-NC and the wt or mut reporter vectors. Forty-eight hours later, the transfected cells were lysed with a Dual-Luciferase Reporter Assay System (Promega) for luciferase activity determination.
RNA immunoprecipitation (RIP)
RIP was conducted with the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). After lysing the cells in complete RIP lysis buffer, the whole-cell lysates were collected and incubated with magnetic beads coupled to an anti-Argonaute2 (Ago2) antibody or normal mouse IgG (Millipore) at 4°C overnight. In addition, 10 µL of whole-cell lysates were aliquoted for use as the input and served as the positive control. IgG acted as the negative control. Proteinase K treatment was used to detach the proteins, and the immunoprecipitated RNAs were then extracted. Eventually, the relative enrichment of LINC01132, miR-431-5p and SOX9 in the immunoprecipitated RNAs was examined via RT-qPCR.
Protein extraction and western blotting
Total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). After total protein quantification via a bicinchoninic acid kit (Beyotime Institute of Biotechnology), equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were blocked at room temperature in 5% nonfat milk for 2 h and then incubated overnight at 4°C with primary antibodies against SOX9 (cat. no. ab185966; Abcam, Cambridge, UK) or GAPDH (cat. no. ab181602; Abcam), followed by incubation with an HRP-conjugated goat anti-rabbit secondary antibody (cat. no. ab205718; Abcam) at room temperature for 1 h. Finally, a BeyoECL plus detection kit (Beyotime Institute of Biotechnology) was used to detect the protein signals.
All the experiments were conducted in triplicate, and the experimental data are expressed as the mean ± standard deviation. One-way analysis of variance with Tukey’s post hoc test was used to compare multiple groups to determine statistical significance. Comparison of the significance between two groups was conducted with Student's t test. The overall survival curves were calculated with the Kaplan-Meier method and compared with the log-rank test. Pearson's correlation analysis was used to examine the expression correlations. All the tests were two-sided, and P < 0.05 was considered statistically significant.