Cell lines
Human ovarian epithelial cell line (HOSEpiC) and OC cell lines (SKOV3, A2780, OVCAR3 and COV362) were obtained from American Type Culture Collection (ATCC, Manassas). All above cell lines were cultured at 37 °C in RPMI-1640 medium with a supplement of 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) in a humidified atmosphere containing 5% CO2. C646, an inhibitor of acetylation, was commercially provided by Sigma Chemical (St. Louis, MO).
Cell transfection
The miR-6744-5p mimics/inhibitor and their matched negative control (NC mimics/inhibitor), short hairpin RNAs (shRNAs) against PAXIP1-AS1 or CBP (sh-PAXIP1-AS1#1/2 or sh-CBP#1/2) and their negative control (sh-NC), and overexpressing plasmids pcDNA3.1/PCBP2 (PCBP2) and its negative control pcDNA3.1 (Vector) were synthesized by GenePharma (Shanghai, China). Using Lipofectamine 2000 (Invitrogen), Cell transfection was conducted with manufacturer’s instructions. Cultured for 48 h, RT-qPCR validated the transfection efficiency.
RT-qPCR
Firstly, Trizol reagent (Invitrogen) was used to isolate total RNAs from OC cell lines. Then, the reverse transcription of miRNA or lncRNA/mRNA was performed by miRNA First-Stand cDNA Synthesis Kit (GeneCopoeia) or cDNA Synthesis SuperMix Kit (TransGen, Beijing, China). Afterwards, RT-qPCR was performed on Applied Biosystems 7500 Real-time PCR Systems (Thermo Fisher Scientific). Gene expression was quantified by 2−ΔΔCt method normalized to U6 or GAPDH.
Colony formation assay
Cells at a density of 600 cells/well were grown in 6-well plates with RPMI-1640 medium containing 10% FBS. After culturing for two weeks, methanol was used for fixing colonies for 15 min at room temperature, and then the cells were stained with 0.1% crystal violet (Invitrogen) for 15 min. Finally, number of visible colonies were manually counted.
EdU assay
Transfected cells of SKOV3 or OVCAR3 were collected and planted into 96-well plates at the density of 1 × 104 cells each well. Later, the plates were added with EdU assay kit (Ribobio) at 37 °C for 2 h. Cell nuclei was stained by DAPI solution. Finally, fluorescence microscope (Olympus, Tokyo, Japan) was applied to observe proliferative cells.
Flow cytometry analysis
SKOV3 or OVCAR3 cells were seeded in a 6-well plate. After centrifugalization, the binding buffer, resuspended with residue, was added with 5-μL Annexin V-FITC and 5-μL propidium (PI). Then, flow cytometer (BD, Franklin Lake, NJ, USA) was used to measure cell apoptotic rate, and results were analyzed by the software WinMDI 2.9 (Invitrogen).
Western blot analysis
Cell lysis was conducted in RIPA lysis buffer, and then the lysate was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). Blocked with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C. After an incubation with secondary antibody at 37 °C for 1 h, enhanced chemiluminescence (ECL) Plus kit (Beyotime, Shanghai, China) visualized the protein bands.
Transwell assay
The migration ability was assessed by the use of transwell chambers without matrigel. In brief, cells (1 × 105) were seeded into upper chambers contained 200ul serum-free media. The lower chambers were added with 600 ul of 20% FBS media. Incubated for 48 h, cells without invasion in the upper chambers were wiped away by cotton wool. Fixed with methanol, cells were stained with 0.5% crystal violet in the lower chamber. Lastly, five random fields were photographed under an inverted microscope.
ChIP assay
ChIP assays were carried out by ChIP Assay Kit (Thermo Fisher Scientific) with SKOV3 or OVCAR3 cells. For DNA-protein cross-links, SKOV3 or OVCAR3 cells were incubated with 1% formaldehyde for 10 minutes, and then an ultrasound machine was utilized to break the cross-linked chromatin DNAs into segments sized 200 to 1000 bp. The chromatin lysate was precipitated by anti-H3K27ac (Abcam), anti-CBP (Abcam) or anti-IgG (Abcam). Finally, RT-qPCR analyzed the ChIP samples.
Subcellular fractionation assay
Firstly, the nuclear and cytoplasmic fractions of SKOV3 and OVCAR3 cells were obtained. Then, with Cytoplasmic & Nuclear RNA Purification Kit (Norgen), the fractions were separated and purified as per the manual. At last, RT-qPCR analyzed the isolated RNA (GADPH, U6, PAXIP1-AS1). GAPDH was the internal control for cytoplasm, and the distribution of PAXIP1-AS1 in nucleus was normalized to U6.
Luciferase reporter assay
To conduct luciferase reporter assay, pmirGLO vector (Promega, Madison, WI) was used. Briefly, the wild-type (WT) PAXIP1-AS1/PCBP2 or mutant (Mut) PAXIP1-AS1/PCBP2 binding element in the sequence of miR-6744-5p was cloned into pmirGLO vector for the construction of PAXIP1-AS1-WT/Mut or PCBP2-WT/Mut. Then, the reporters were separately co-transfected with miR-6744-5p mimics in SKOV3 or OVCAR3 cells. After 48 h, dual‐luciferase reporter assay system (Promega) testified the relative luciferase activity.
RNA pull down assay
NC-miRNA and miR-6744-5p-WT/Mut were labeled with biotin, and then transfected into SKOV3 and OVCAR3 cells. Streptavidin magnetic beads were used to incubate with the cell lysates for 4 h at 4 °C. Using precooled lysis buffer and salt buffer, the beads were rinsed. After that, PAXIP1-AS1 or PCBP2 level was detected following the extraction of pull-down RNAs.
RIP assay
For RNA immunoprecipitation, EZMagna RIP kit (Millipore) was applied. Cells were lysed in RIP lysis buffer after being harvested. Then, the cell lysate was incubated with magnetic beads absorbed anti-IgG (Millipore) or anti-Ago2 antibody (Millipore). Finally, RT-qPCR analyzed the purified RNA.
Statistical analysis
Three biological repeats were applied to all experimental procedures. Shown as the mean ± SD, data were statistically analyzed through GraphPad Prism 6 (GraphPad). Differences between two groups were analyzed by Student's t test or one-way ANOVA for multiple groups with P < 0.05 as cut-off value.