Cell lines and culture
The OC cell lines SKOV3 and A2780 and the normal ovarian cell line ISOE-80 were purchased from ATCC. 293T cell lines were maintained in our laboratory. The SKOV3, A2780, and ISOE-80 cells were cultured in RPMI-1640 (Gibco, USA), and 293 cell lines were cultured in DMEM (Gibco, USA) at 37 ℃ in 5% CO2. The media were supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Gibco, USA).
Plasmid construction and cell transfection
To construct the circMAN1A2 overexpression vector, full-length human circMAN1A2 was subcloned into the pLO5ciR vector (Geenseed Biotech, China). To knockdown circMAN1A2, siRNAs targeting the back splice junction of circMAN1A2 (siRNA1, siRNA2, siRNA3) and siRNA-NC were synthesized (GenePharma Biotech, China). The most effective siRNA1 and si-NC molecules were subcloned into the lentivirus vector (pLKO.1) to construct sh-circMAN1A2 and sh-NC vectors. The circMAN1A2 vector, sh-circMAN1A2, and their control plasmids were transfected into 293T cells for lentivirus preparation, and the viruses were collected to infect A2780 and SKOV3 cells, followed by selection on puromycin. miR-139a-3p mimics, mimics-NC, inhibitor, and inhibitor-NC were purchased from GenePharma (Shanghai, China). All transfections were performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. The sequences of siRNAs, shRNAs, miR-139a-3p mimics, mimics-NC, inhibitor, and inhibitor-NC are listed in Table S1.
Polymerase chain reaction (PCR) and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA from the tumor tissue samples or cell lines with or without 4 U/µg of RNase R (ab286929, Abcam) was extracted using TRIzol reagent (Thermo Fisher, USA). Nuclear and cytoplasmic RNA from the OC cells was separated using NE-PER™ Kit (Thermo Fisher, USA) following the manufacturer’s instructions. Subsequently, the RNA was reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA), Genomic DNA(gDNA) is extracted with DNA extraction kit (TIANGEN, China), For PCR, cDNA and gDNA were used as templates to amplify the target sequence according to PCR Amplification Kit(Takara, Japen), and 2% agarose gel was used to detect the amplification results. For RT-qPCR, the target genes were amplified in a 20 µL reaction using GoTaq® qPCR Master Mix (A6002, Promega). The relative gene expression levels were quantified using the 2 − ΔΔCt method. Primer sequences are listed in Table S2.
Western blotting
Total protein was extracted from cell lysates (Beyotime, China). Equal amounts of denatured proteins (30 µg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to polyvinylidene fluoride membranes (Millipore, USA) and probed using IL1RAP (1:1,000; 73070, Cell Signaling Technology), HGF (1:1,000; 52445, Cell Signaling Technology), TAK1 (1:1,000; 5206, Cell Signaling Technology), and p-TAK1 (1:1,000; 9339, Cell Signaling Technology) antibodies for 1.5 h. The membranes were then incubated with goat anti-rabbit antibody (1:5,000; SA00001-2, Proteintech) for 1 h. The blots were visualized after chemical development using an electrochemiluminescence reagent ( Thermo Scientific, Massachusetts, USA). GAPDH was used as an internal reference antibody (1:5,000; 10494-1-AP, Proteintech).
Fluorescence in situ hybridization (FISH)
FITC-labeled circMAN1A2 (5-CTTCCTCTTCCTCAAATTCAC-3) and Cy3-labeled hsa-miR-135a-3p probes (5-CGCCACGGCUCCAAUCCCUAUA-3) (Geneseed, Guangzhou, China) were used to observe colocalization of circMAN1A2 and hsa-miR-135a-3p in OC cells. FISH assay was performed using Fluorescent in Situ Hybridization Kit (GenePharma, China), according to the manufacturer’s instructions. The cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, China). Images were photographed using a fluorescence microscope (Leica, Germany).
Cell Counting Kit-8 (CCK8) assay
Briefly, a single-cell suspension was prepared using trypsin and inoculated in 96-well plates at a density of 3 × 103 cells/well. Next, 10 µL of CCK8 reagent (Solarbio) was added and the cells incubated for 60 min at 72 h. The optical density (OD) at 490 nM was measured using a microplate reader (51119670DP, Thermo Scientific). All the experiments were performed in triplicate.
EdU staining
A single-cell suspension was prepared using trypsin and inoculated in six-well plates at a density of 5 × 105 cells/well. EdU staining was performed using an EdU staining kit (BeyoClick™ EdU-594, Beyotime) according to the manufacturer’s instructions. Images were photographed using a fluorescence microscope (Leica, Germany).
Clone formation assay
Cells were digested using 0.25% trypsin and resuspended by centrifugation to obtain a single-cell suspension; 200 cells/well were inoculated in a six-well plate. The cells were then fixed for 15 min in 5 ml of 4% paraformaldehyde, an appropriate amount of 1X GIMSA dye solution (G1015, Solarbio) was applied, and the cells were incubated for 10–30 min at room temperature. The dishes were air-dried, and the clones were counted directly with the naked eye.
Cell cycle assay
Cells were harvested and fixed in pre-cooled 70% ethanol at 4°C overnight, stained with propidium iodide (PI), and measured using flow cytometry (Becon Dickinson FACS Calibur, USA).
Wound healing assay
Approximately 1 × 106 cells were added to a six-well plate, and the next day, the pipette tip was used to scratch parallel lines on the cell layer. The cells were then washed three times with phosphate-buffered saline, the streaked cells were removed, and serum-free medium was added. The remaining cells were then placed in a 37°C, 5% CO2 incubator and cultivated. Images were acquired after 0 and 24 h.
Dual luciferase assay
The sequences of circMAN1A2 or IL1RAP 3′UTR containing the wild-type or mutant binding site of hsa-miR-135a-3p were designed and synthesized by GenePharma (Shanghai, China). These sequences were transfected into the psiCHECK2 vector (Promega, USA). 293T cells were co-transfected with the psiCHECK2 plasmids containing Insertion sequence and hsa-miR-135a-3p mimics/mimics-NC or hsa-miR-135a-3p inhibitors/inhibitor-NC using Lipofectamine 2000 (Invitrogen, USA). After 48 h of incubation, the activities of firefly and Renilla luciferase were measured using Dual Luciferase Reporter Assay Kit (Promega, USA), Firefly fluorescence as an internal reference.
RNA pull-down assay
a biotinylated circMAN1A2 probe (5-CTTCCTCTTCCTCAAATTCAC-3) synthesized by RiboBio (Guangzhou, China) was used; an oligo probe (5-CCCCACTTTTATCTATACCCT-3) was used as a control. Approximately 1 × 107 OC cells were transfected with the biotin-labeled circMAN1A2 probe. Subsequently, the biotin-coupled RNA complex was purified through streptavidin-coated magnetic bead adsorption. The enriched circMAN1A2 and miR-135a-3p were analyzed using qRT-PCR.
Animals
Four-week-old female BALB/c nude mice were chosen for the xenograft experiments. All procedures were approved by Hainan Women and Children's Medical Center Animal Care and Use Committee. The mice were subcutaneously inoculated with SKOV3 cells infected with the circMAN1A2 overexpression/mock plasmid or A2780 cells infected with lentiviruses carrying sh-circMAN1A2/sh-NC plasmid (2.5 × 106 cells; 200 ml). After 28 days, the mice were sacrificed and hotographed. Tumors are extracted and photographed. The tumor volume was calculated in accordance with the formula (length × width2/2), and the volume was measured weekly. The tumor weight was also determined.
Statistical analysis
Data are expressed as mean ± standard deviation. Multiple comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. All statistical analyses were performed using SPSS (version 19.0; IBM Corp.). Statistical significance was set at P < 0.05.