2.1 Specimen

A total of 70 paired tumor tissues and adjacent normal tissues were obtained from OC patients at Yi chang Maternal and Child Health Hospital. Samples were confirmed by pathological diagnosis and were stored at −80 °C. Patients all signed informed consent forms, and the study was ratified by the Ethics Committee of Yi chang Maternal and Child Health Hospital in accordance with the Declaration of Helsinki. Inclusion criteria: age > 18 years; no participant had received anti-tumor therapies (such as radiotherapy or chemotherapy); signed written informed consent before the surgery; OC confirmed by pathological examination after surgery; completed clinical and pathological information. Exclusion criteria: patients with other cancers or a history of treatment for other cancers; patients with other ovarian diseases; severe complicated diseases or severe infectious diseases.

2.2 Cell culture and transfection

OC cell lines (SKOV3, A2780, CAOV3, and OVCAR3) and normal ovarian epithelial cells (IOSE-80) (Biobw, Beijing, China) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin solution (15,140,122; Gibco, Grand Island, NY, USA).

Small interfering RNA against circ_0001741 (si-circ_0001741), miR-491-3p mimic or inhibitor (anti-miR-491-3p), and negative controls (si-NC, miR-NC and anti-miR-NC) were synthesized by RiBoBio (Guangzhou, China). The pcDNA3.1 PRSS8 overexpression vector was structured by inserting the PCR products of PRSS8 into pcDNA3.1 vector. Cell transfection was performed using Lipo6000™ (C0526-1.5 mL; Beyotime, Shanghai, China). For lentiviral transduction in animal study, SKOV3 cells were infected by lentivirus circ_0001741 shRNA (sh-circ_0001741) or its control (sh-NC) in media containing 8 µg/ mL of polybrene. After 24 h, the stably infected SKOV3 cells were selected by 1 µg/mL of puromycin over 72 h.

2.3 Quantitative real-time PCR (qRT-PCR)

As previously described [14], total RNAs were extracted using TRIzol reagent (15596026CN; Invitrogen, Carlsbad, CA, USA), and cDNA was obtained using PrimeScriptTM RT reagent kit (RR716; TaKaRa, Dalian, China). SYBR Green (RR820A; TaKaRa) was used for PCR amplification with specific primers (Table 1) and cDNA. Relative expression was calculated using 2−ΔΔCt method with β-actin (for circ_0001741, PRSS8, and TNPO3) or U6 (for miR-491-5p) as internal control. Cytoplasmic & nuclear RNA purification kit (NGB-21000; Norgen, Ontario, Canada) was used to isolate nuclear and cytoplasm RNAs and then performed qRT-PCR. Besides, the extracted RNA was incubated with RNase R and then used for qRT-PCR to measure circ_0001741 and TNPO3 mRNA levels.

Table 1 Primer sequences used for qRT-PCR2.4 Cell counting kit 8 (CCK8) assay

As previously described [15], the digested OC cells were re-suspended and seeded into 96-well plates (2 × 103 cells). CCK8 solution (C0037; Beyotime) was incubated with cells at each time point (0, 24, 48 and 72 h) for 2 h. The absorbance was examined at 450 nm using a microplate reader.

2.5 Edu assay

Edu In Vitro Kit (C10310-1; RiBoBio) was used for measuring cell proliferation according to the previous study described [16]. OC cells were inoculated into 96-well plates (2 × 104 cells), and then incubated with Edu solution and DAPI solution. Edu positive (Edu+) cells were counted under fluorescent microscope with ImageJ software.

2.6 Flow cytometry

As previously described [17], OC cells (2 × 105 cells) were collected and then stained with Annexin V-FITC and PI solution (A211; Vazyme, Nanjing, China). Cell apoptosis rate was analyzed by FACScalibur flow cytometer with CellQuest Pro software.

2.7 Cell migration and invasion assays

Cell suspended with serum-free medium was added to the upper of Transwell chambers (5 × 105 cells/well) pre-coated with or without matrigel (356,255; Corning Inc., Corning, NY, USA), while completed medium was added into lower chamber. After incubation for 24 h, cells were fixed with 4% paraformaldehyde and stained by crystal violet (C0121-100 mL; Beyotime), and the migrated cells and invaded cells were counted with a microscope with ImageJ software.

2.8 Western blot

Western blot analysis was performed as previously described [15]. Briefly, proteins were extracted by RIPA buffer (P0013B; Beyotime), separated by SDS-PAGE gel and transferred to PVDF membranes. After blockage, membranes were incubated with specific primary antibodies (Abcam, Cambridge, MA, USA) including anti-Bax (1:1000, ab53154), anti-Bcl-2 (1:1000, ab196495), anti-Snail (1:1000, ab216347), anti-MMP9 (1:1000, ab38898), anti-N-cadherin (1:1000, ab245117), anti-PRSS8 (1:1000, ab200736), anti-β-actin (1:2000, ab8227) at 4 °C overnight, and then incubated with secondary antibody (1:2000) at room temperature for 1 h. Then, membrane was treated with ECL luminescent solution (P0018S; Beyotime), and image grayscale value was analyzed by Image J software.

2.9 Dual-luciferase reporter assay

As previously described [14], circ_0001741 containing miR-491-5p binding sequence (circ_0001741-WT) and its mutant sequence (circ_0001741-MUT), PRSS8 3'UTR sequence (PRSS8 3'UTR-WT) and its mutant sequence (PRSS8 3'UTR-MUT) were inserted into pmirGLO vector. The vector was co-transfected with miR-NC/miR-491-5p into SKOV3 and A2780 cells. After 48 h, cells were assayed for detecting luciferase activity.

2.10 RIP assay

RIP test was performed according to the Magna RIP kit (17–700; Millipore, Billerica, MA, USA) instructions. As previously described [18], OC cells were lysed with RIP buffer, and then the cell lysates were incubated with magnetic beads and anti-IgG or anti-Ago2 overnight. Immunoprecipitated RNA was collected to detect circ_0001741, miR-491-5p and PRSS8 enrichments by qRT-PCR.

2.11 Animal experiments

According to the previous study [18], BALB/c nude mice were subcutaneously injected with 1 × 107 SKOV3 cells stably infected with lentiviral sh-circ_0001741 or sh-NC (N = 6). Tumor volumes were measured weekly after cell injection. Mice were sacrificed by cervical dislocation under 2% isoflurane anesthesia at 35 days, and tumors were excised, photographed, and weighed. Animal research was approved by the Animal Ethics Committee of Yi chang Maternal and Child Health Hospital and was performed in compliance with the ARRIVE guidelines.

2.12 Statistical analysis

GraphPad Prism 7.0 analysis software was used to analyze the data, and the data were expressed as mean ± SD. Values between groups were analyzed using Student's t-test and ANOVA. P < 0.05 was considered statistically significant.