Human ovarian epithelial cell line (ISOE80) and human ovarian cancer cell lines (SKOV-3, A2780, OVCAR-3 and HO8910) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). After resuscitation, ISOE80 was cultured in a DMEM medium, while other cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Beyotime, Beijing, China) in a humidified environment. When cells attained 70% to 80% confluence, transfection experiments could be conducted.
The LINC00665-targeting shRNA sequences were designed and inserted into pENTR/U6 plasmids to construct LINC00665 shRNA. The NC mimic and miR-181a-5p mimic were purchased from Ribobio corporation (Guangzhou, China). Cell transfection was performed by using cell electroporation system operator H1 (Suzhou Etta Biotech Co. Ltd., Suzhou, China.) according to the manufacturer’s manual. Cell transfection efficiency was measured by qRT-PCR at 48 hours post-transfection.
After cell transfection, SKOV-3 and OVCAR-3 were seeded into 96-well plates at 5 × 103 per well. After incubated for 0 hours, 24 hours, 48 hours and 72 hours, 10 μL CCK8 solution were added and incubated for 2 hours in the dark. Then, absorbance at 450 nm was determined using a VarioskanTM LUX microplate reader (Thermo Fisher Scientific).
EdU assay was performed as described (1). Briefly, after electroporation treatment, 5 × 103 SKOV-3 and OVCAR-3 cells were seeded into each well of the 96-well plates and cultured for another 72 hours. Then, the cells were incubated with 10 μM EdU for 4 hours and stained DAPI. Finally, the EdU-positive cells were analyzed by fluorescence microscope (Leica, Hilden, Germany), 200×, and densitometric analyzed the percentage using ImageJ (Bethesda, MD, USA).
Cell cycle and cell apoptosis
After cell electroporation, SKOV-3 and OVCAR-3 cells were seeded into 6-well plates at 5 × 105 per well and cultured cells for the next 72 hours. Then, the tumor cells were harvested for cell cycle and apoptosis analysis.
For cell cycle analysis, the cells were incubated in ice-cold ethanol for 2 hours and then treated with RNase A (0.2 mg/mL, Sigma-Aldrich). Next, the cells were incubated with propidium iodide (2 µL) at room temperature for 40 minutes, and finally, cell cycle detection was analyzed.
For analysis, the tumor cells with indicated treatments were washed once with PBS. Then, the cells were resuspended in 100 µL binding buffer and incubated with 10 µg/mL Annexin V-fluorescein isothiocyanate and 10 µg/mL PI (both Sigma-Aldrich) at room temperature for 30 minutes. Apoptosis analysis was performed using a FACScan flow cytometer (Becton Dickinson).
Wound healing assay
To study the migration of SKOV-3 and OVCAR-3 cells transfected with different reagents. The wound-healing assay was performed as described (18). Briefly, 5×105 transfected SKOV-3 or OVCAR-3 cells were seeded into each well in a 6-well plate. When the cells were cultured to 90% confluence, a wound was made by a 100 μl size pipette tip. After another 48 hours, the wound recovery area was evaluated under a light microscope.
To detect the capacity of migration, the Transwell assay was performed as described (13). Briefly, 3×104 transfected SKOV-3 or OVCAR-3 cells were seeded into the upper chamber of Matrigel-precoated transwell and cultured in RPMI-1640 medium. The lower chamber contained 10% FBS growth medium. After 48 hours of culture, the transwell cells present in the lower chamber were fixed and stained with crystal violet. The pictures were taken under a light microscope, and individual cell colonies were counted.
Luciferase reporter assay
The Dual-Luciferase system was generated by inserting the cDNA fragments containing the putative miR-181a-5p binding site from LINC00665 or FHDC1 3’-UTR into pmirGLO Dual-Luciferase miRNA Target Expression Vectors (Promega, Madison, WI, USA). PmirGLO/LINC00665 or pmirGLO/ FHDC1 3’-UTR constructs along with miR-181a-5p mimics were co-transfected into SKOV-3 and OVCAR-3 cells; then the cells were seeded into a 24-well plate for about 48 hours culture. Finally, the cells were lysed to measure the Dual-Luciferase Reporters’ luciferase activity according to the manufacturer’s protocol.
After cell transfection, SKOV-3 and OVCAR-3 cells were seeded in 96-well plates at 5 × 103 per well and cultured for the next 72 hours. Then, the cells were fixed in 4% paraformaldehyde and permeabilized with xylene. Blocked with 5% BSA in PBS, the cells were immunolabeled with primary antibody: FHDC (1:100; Proteintech) and then incubated with FITC-conjugated secondary antibody after washing by xxx. The nuclei were counterstained using DAPI (Invitrogen), and the cells were observed under a fluorescence microscope (Olympus, Japan).
TRIpure reagent (Invitrogen, USA) was used to isolate the total RNA from the cultured cells. PrimeScript RT kit (TaKaRa, Otsu, Japan) was used for reverse transcription. After the sample was prepared, the expression level was detected with SYBR green, and GAPDH was controlled as the internal parameter. Fold changes of gene experiments were measured by 2-ΔΔCt methods. Primers of LINC00665, miR-181a-5p, U6, FHDC1 and GAPDH were as described in previous studies: LINC00665 sense, 5’-AGCACCCCTAGTGTCAGTCA-3’ and antisense, 5’-TGGTCTCTAGGGAGGCAGAA-3’; miR-181a-5p sense, 5’-AACATTCAACGCTGTCGGTGAGT-3’ and antisense, 5’-GTGCAGGGTCCGAGGT-3’; U6 sense, 5’-CTCGCTTCGGCAGCACA-3’ and antisense, 5’-AACGCTTCACGAATTTGCGT-3’; FHDC1 sense, 5’- ACATCCAGCGGGATGGTGAACT-3’ and antisense, 5’-GGAGCTCTTGTTTCCAGCATTCC-3’.
The cultured cells were harvested in lysis buffer and incubated on ice for 30 min. The supernatant was collected by centrifugation with 15,000 g for 5 min at 4 °C. Then the proteins (40 μg) were separated on 12% SDS-PAGE and transferred onto PVDF membranes (Millipore) using a MiniGenie blotting system (Bio-Rad). Next, the membranes were blocked for 1 hour with TBST containing 1% skimmed milk powder and then incubated overnight at 4 °C with rabbit antibodies against human FHDC1 (1:1000; cat. no. NBP1-93579; Novus Biologicals), MMP2 (1:1000; cat. no. 40994S; Cell Signaling Technology, Danvers, MA, USA), MMP9 (1:1000; cat. no. 13667S; Cell Signaling Technology), Cyclin D1 (1:1000; cat. no. 55506S; Cell Signaling Technology), p21 (1:1000; cat. no. 2947S; Cell Signaling Technology), Bcl-2 (1:1000; cat. no. ab185002; Abcam), Bax (1:500; cat. no. ab53154; Abcam), Cleaved Caspase-3 (1:500; cat. no. ab49822; Abcam), Cleaved Caspase-9 (1:1000; cat. no. 20750S; Cell Signaling Technology) or GAPDH (1:1000; cat. no. 5174S; Cell Signaling Technology). Next, the membranes were washed with TBST and incubated with goat-anti-rabbit secondary antibody (1:10000; cat. no. 14708S; Cell Signaling Technology). Finally, the protein bands on the membranes were visualized with an enhanced chemiluminescence (ECL) system. Densitometric analysis of the bands was done using The ImageJ.
All experiments were done in triplicates. GraphPad Prism (Version 6.01 for Windows) statistical software was used to perform statistical analysis. Student t-tests were employed to identify the significant differences between groups. One-way ANOVA and Tukey test were used to identify differences among three or more groups. Statistical significance difference was set at p<0.05.