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 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% confluences, transfection experiments could be conducted.
LINC00665 shRNA constructs were generated by inserting the LINC00665-targeting shRNA sequences into pENTR/U6 plasmids. The NC mimic and miR-181a-5p mimic were provided by Ribobio corporation (Guangzhou, China). After 6 h, fresh culture medium was added to the CFs with a confluence rate at 70%. After 48-h post-transfection, plasmid transfection efficiency was measured by qRT-PCR.
After 48-h transfection, SKOV-3 and OVCAR-3 were treated with electroporation, and then were inoculated into 96-well plates at 5 × 103 per well. After incubated for 0 hour, 24 hours, 48 hours and 72hours, 10 μL CCK8 solution were added and continuously incubated for 2 hours in the dark.
EdU assay was taken as described . Briefly, after electroporation treatment, we seeded 5 × 103 SKOV-3 and OVCAR-3 into each well of the 96-well plates and cultured the cells for another 72 hours. Then, we incubated the cells with 10 μM EdU for 4 hours, and stained DAPI. Finally, we analyzed the EdU-positive cells by fluorescence microscope (Leica, Hilden, Germany), 200×, and densitometric analyzed the percentage using ImageJ (Bethesda, MD, USA).
Cell cycle and cell apoptosis
After electroporation for transfection, we seeded the SKOV-3 and OVCAR-3 cells into 6-well plates at 5 × 105 per well and cultured the cells for next 72 hours. Then, we harvested the tumor cells. For cell cycle, the cells were first 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 40min, and finally cell cycle detection was analyzed. For apoptosis, the harvested cells were treated with PI (10 µg/mL) and V-fluorescein isothiocyanate (10 µg/mL), respectively.
Wound healing assay
Briefly, 5×105 SKOV-3 or OVCAR-3 were seeded into 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 . After 48-h cell transfection, SKOV-3 or OVCAR-3 were firstly adjusted to the appropriate concentration. Subsequently, the upper chamber was added with adjusted cells, whereas the lower chamber was replaced by a medium supplement 15% FBS. After 24 h, 4% paraformaldehyde was used for fixation for 10 min and 0.1% crystal violet for staining for 30 min, respectively. Five fields were randomly selected under an inverted microscope to represent the migration and invasion ability of cells in each group.
Luciferase reporter assay
The luciferase reporter was constructed 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. Finally, the Dual-Luciferase Reporters’ luciferase were measured activity according to the manufacturer’s protocol.
After 48-h transfection, SKOV-3 and OVCAR-3 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. 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 samples and 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 internal parameter. 2-ΔΔCt methods represented the fold changes of gene expression and the experiments were conducted three times. Primers of LINC00665, miR-181a-5p, U6, FHDC1 and GAPDH were as described in previous studies [12, 16, 17]. 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’.
According to the manufacture’s instruction, the proteins were extracted and its concentration was measured. Subsequently, the prepared protein was separated by polyacrylamide-SDS gels and then transferred onto PVDF membranes (Roche, Switzerland). After blocking, the PVDF membrane was subjected to incubation with primary antibodies: FHDC1 (1:1000; cat. no. NBP1-93579; Novus Biologicals, Littleton, USA), 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). On the following day, the membrane were incubated with the secondary antibody at 37°C for 45 min and the intensity of protein expression was detected by ECL chemiluminescence (Beyotime, Beijing, China).
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. Statistical significance difference was set at p<0.05.