Circular RNA circ-BNC2 (hsa_circ_0008732) inhibits the progression of ovarian cancer through microRNA-223-3p/ FBXW7 axis

Ethics statement and patient samples

40 pairs of OC tissues and adjacent tissue samples were collected from patients who were diagnosed with OC and received surgical resection in the Affiliated Hospital of Zunyi Medical University. None of the patients received any anti-cancer treatment before surgery, and all samples were immediately frozen in liquid nitrogen and stored at -80℃. This study, with written informed consent, was supported by the Ethics Committee of Affiliated Hospital of Zunyi Medical University.

Cell culture and transfection

Humans OC cell lines (SKOV3, CAOV-3, OVCAR-3, OV90, HO-8910, ES-2) and normal ovarian epithelial cell line IOSE-80 were available from Shanghai Institute of Cytology, Chinese Academy of Sciences (Shanghai, China) and American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (Gibco, Gran Island, NY, USA), 100 U/mL penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in 5% CO2. Circ-BNC2 overexpression plasmid (oe-circ), pcDNA empty vector (Vector), small interfering RNA (siRNA) targeting circ-BNC2 (si-circ#1 and si-circ#2), siRNA negative control siRNA (si-NC), miR-223-3p mimic and negative control (miR-control) were from Invitrogen. The cell transfection was carried out with Lipofectamine® 3000 according to the manufacture’s protocol.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from tissues and cells using TRIzol reagent (Yeasen Biotech, Shanghai, China). Reverse transcription of miRNA and mRNA were conducted with the Bulge-Loop™ miRNA qRT-PCR kit (RiboBio, Guangzhou, China) and PrimeScript RT Master Mix kit (TaKaRa, Dalian, China), respectively. Besides, qRT-PCR was performed using a SYBR® Premix Ex Taq™ II kit (TaKaRa, Dalian, China) on an Applied Biosystems Prism 7500 Fast Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as the internal references. The relative expression was calculated by 2 − ΔΔCt method. The primers are listed in Table 1.

Cell counting kit-8 (CCK-8) assay

CCK-8 kit (Beyotime, Shanghai, China) was utilized to detect the proliferative ability of OC cell lines. 48 h after transfection, the trasfected cells were seeded in 96-well plates at the density of 2 × 103 cells / well. 10 µL of CCK-8 solution was added into each well at the indicated time (24th h, 48th h, 72nd h and 96th h) and then the cells were incubated for 2 h at 37 °C. Finally, the absorbance at 450 nm wavelength was examined using a microplate reader (Bio-Rad, Hercules, CA, USA).

Transwell assay

The transfected OC cells in the logarithmic growth phase were resuspended with serum-free DMEM, with the density adjusted to 3 × 105 cells/mL. 200 µL of cell suspension was subsequently transferred in the upper chamber of a transwell insert (24-well insert; 8 μm pore size; Corning, NY, USA). 600 µL of DMEM containing 10% FBS was loaded into the lower compartment, and the cells were cultured in 5% CO2 at 37 °C for 24 h. Next, the cells on the upper surface of the filter were gently wiped off with a cotton swab. Subsequently, the cells on the lower surface were accordingly fixed by 4% paraformaldehyde for 15 min and immediately stained with 0.1% crystal violet solution for 10 min. Then the cells were washed by phosphate buffered solution (PBS). Five high-power fields were randomly selected under a light microscope (Nikon, Tokyo, Japan), and the cells passing through the filter were counted. In the invasion assay, the transwell filter was coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), and the remaining steps were the same as the migration assay.

Cell cycle assay

48 h after the transfection, OC cells were fixed with 75% ethanol. Next, the cells were immersed in PBS and subsequently incubated with 0.5% Triton X-100 containing 1 mg / mL RNase A at 37 °C for 30 min. Next, the cells were stained with propidium iodide (PI; 50 µg / mL; Sigma-Aldrich, Louis, MO, USA) for 30 min at ambient temperature in darkness. Ultimately, the cell cycle distribution of the cells was analyzed using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Subcellular localization

Cytoplasmic and nuclear fraction of SKOV3 and HO-8910 cells was isolated using a PARIS™ Kit (Invitrogen, Carlsbad, CA, USA). Briefly, the cells were lysed with cell fractionation buffer and centrifuged at 500 ×g at 4 ℃ for 5 min to separate the nuclear and cytoplasmic cell fractions. The supernatant was transferred to a RNase-free tube. The remaining lysate was washed with cell fractionation buffer and centrifuged again. Then the RNA of cytoplasm and nuclear was eluted with the elution solution. Subsequently, circ-BNC2 expression in the cytoplasm and nucleus of SKOV3 and HO-8910 cells was detected by qRT-PCR, with GAPDH and U6 as cytoplasmic and nuclear controls, respectively.

Dual-luciferase reporter gene assay

The wild type (WT) and mutant (MUT) sequences of circ-BNC2 or FBXW7 3′-UTR containing putative miR-223-3p binding sites were accordingly sub-cloned into the pGL3-Basic luciferase vectors (Promega, Madison, WI, USA) to construct recombinant reporter plasmids. The above luciferase reporter plasmids were co-transfected with miR-control or miR-223-3p mimic into SKOV3 or HO-8910 cells, respectively. 48 h later, the relative luciferase activity was tested by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

RNA pull-down assay

The biotinylated miR-223-3p was labeled using the RNA 3’ End Desthiobiotinylation Kit (Thermo Fisher Scientific, Waltham, MA, USA). The Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Scientific, Waltham, MA, USA) was used to perform RNA pull-down assay. Briefly, SKOV3 or HO-8910 cells were lysed with lysis buffer. The biotin-miR-223-3p, or biotin-negative control (biotin-miR-control) were then incubated with cell lysates, followed by the incubation of M-280 streptavidin magnetic beads (Sigma-Aldrich, Louis, MO, USA). Then, the precipitated RNA on the beads was eluted, and subsequently qRT-PCR was performed.

Western blot assay

Total protein of the transfected OC cells was extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), and the concentration of protein was evaluated by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). After the protein samples were mixed with loading buffer and denatured, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransfered to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% skimmed milk for 2 h at ambient temperature and incubated with specific primary antibodies overnight at 4 °C. Next day, the membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG H&L (1:5000, ab205718, Abcam, Shanghai, China) for 1 h at ambient temperature. The protein bands were visualized by an enhanced chemiluminescence kit (Promega, Madison, WI, USA) and analyzed by Image-Pro Plus software. GAPDH was regarded as an internal reference. The primary antibodies were: anti-FBXW7 antibody (1:1000, ab109617, Abcam, Shanghai, China), anti-GAPDH antibody (1:2000, ab9485, Abcam).

In-vivo model of lung metastasis

The animal experiments were approved by the Animal Care and Use Committee of the Affiliated Hospital of Zunyi Medical University. Six-week-old female BALB/c nude mice (n = 24) were purchased from the Laboratory Animal Center of Guizhou Medical University (Guizhou, China) and maintained in a pathology-free environment. For in-vivo lung metastasis experiments, SKOV3 cells (1 × 106 cells per mouse) without or with circ-BNC2 overexpression or the control cells were injected into the tail vein of the nude mice (12 mice per group). 21 days after injection, the mice were sacrificed, and the lung tissue was harvested. The lung tissues were fixed, formalin, paraffin-embedded, and sectioned before hematoxylin-eosin (H&E) staining.

Statistical analysis

All experiments were repeated for three times, with data presented as mean ± standard deviation (SD). Notably, the statistical analyses were accomplished using SPSS 19.0 (IBM, SPSS, Chicago, IL, USA). Independent sample t-test was executed for comparisons between two groups, and one-way analysis of variance with post-hoc test was conducted for comparisons among multiple groups. Besides, the overall survival analysis was conducted with Kaplan-Meier plots and log-rank tests. The correlation of gene expression was analyzed with Pearson’s correlation coefficient. Statistically, P < 0.05 is significant.

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