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

Abstract

Background: Circular RNAs (circRNAs) are reported to be key regulators in the progression of human cancers. This work focuses on the function and molecular mechanism of circRNA-BNC2 (circ-BNC2) (also known as hsa_circ_0008732) in ovarian cancer (OC).

Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect circ-BNC2, microRNA-223-3p (miR-223-3p) and F-box and WD repeat domain containing 7 (FBXW7) mRNA expressions in OC tissues and cells. Besides, cell counting kit 8 (CCK-8), transwell assay and cell cycle assays were executed to assess the proliferative, migrative, invasive abilities, and cell cycle progression of OC cells, respectively. Dual-luciferase reporter gene assay and RNA pull-down assay were used to validate the targeting relationships between miR-223-3p and circ-BNC2 or FBXW7. Western blot was adopted to determine FBXW7 protein levels in OC cells.

Results: Circ-BNC2 expression was downregulated in OC tissues and cell lines, which was associated with higher FIGO stage and lymph node metastasis of OC patients. Circ-BNC2 overexpression repressed the proliferation, migration, invasion of OC cells and induced cell cycle arrest, while silencing circ-BNC2 worked oppositely. Mechanistically, circ-BNC2 could upregulate FBXW7 expression in OC cells via sponging miR-223-3p.

Conclusion: Circ-BNC2 suppresses the progression of OC via regulating miR-223-3p / FBXW7 axis. Our findings provided potential biomarker for OC therapy.

Keywords: FBXW7; Ovarian cancer; Proliferation; circ-BNC2; miR-223-3p.

Fig. 1

Circ-BNC2 is down-regulated in OC. A qRT-PCR was used to detect the expression of circ-BNC2 in 40 pairs of OC tissues and normal tissues adjacent to tumors. B-C The association between circ-BNC2 expression and FIGO stage (B), lymph node metastasis (C) in OC tissues. D. Kaplan-Meier curves were used to analyze the correlation between circ-BNC2 expression and the overall survival of OC patients. *P < 0.05, and **P < 0.001

Fig. 2

The effects of circ-BNC2 on the proliferation, migration, invasion and cell cycle of OC cells. A The expression of circ-BNC2 in OC cells (SKOV3, CAOV-3, OVCAR-3, OV90, HO-8910, and ES-2) and normal ovarian epithelial cells IOSE-80 were detected by qRT-PCR. B Circ-BNC2 overexpression plasmids were transfected into SKOV3 cells, and circ-BNC2 siRNAs (si-circ#1 and si-circ#2) were transfected into HO-8910 cells, and the expression of circ-BNC2 was detected by qRT-PCR. C. CCK-8 assay was used to detect the proliferative ability of OC cells after transfection. D and E Transwell assay was used for detecting the migration and invasion of OC cells after transfection. Scale bar: 500 μm. F Cell cycle assays was used to detect the cell cycle distribution in OC cells after transfection. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 3

Circ-BNC2 acts as a sponge of miR-223-3p. A The subcellular fractionation assay was used to detect the expression of circ-BNC2 in the nucleus and cytoplasm of SKOV3 and HO-8910 cells. B Bioinformatics tools was used to predict the binding site of circ-BNC2 and miR-223-3p. C Dual-luciferase reporter gene assay was used to validate the specific binding between circ-BNC2 and miR-223-3p in SKOV3 and HO-8910 cells. D RNA pull-down assay was carried out to verify the interaction between miR-223-3p and circ-BNC2 in SKOV3 and HO-8910 cells. E qRT-PCR was used to detect the expression of miR-223-3p in OC tissues and normal tissues adjacent to tumors. n = 40. F Pearson’s correlation coefficient analysis was performed to analyze the correlation between circ-BNC2 and miR-223-3p expression in OC tissues. G qRT-PCR was used to detect the expression of miR-223-3p in OC cells after silencing or overexpressing circ-BNC2. ***P < 0.001

Fig. 4

Circ-BNC2 inhibits the progression of OC cells by sponging miR-223-3p. A SKOV3 were transfected with negative control (NC), circ-BNC2 overexpression plasmid (Oe-circ), Oe-circ + miR-223-3p mimics, and qRT-PCR was applied to detect the expression of miR-223-3p in SKOV3 cells after transfection. B CCK-8 assay used to detect the proliferation of SKOV3 cells after transfection. C Transwell assay was used to detect the migration and invasion of SKOV3 cells after transfection. Scale bar: 500 μm. D Cell cycle assays used to detect the cell cycle distribution of SKOV3 cells after transfection. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5

Circ-BNC2 increases the expression of FBXW7 by targeting miR-223-3p. A TargetScan online tool was used to predict the binding site between miR-223-3p and FBXW7 3′-UTR. B Dual-luciferase reporter gene assay was used to validate the targeting relationship between miR-223-3p and FBXW7 3′-UTR. C RNA pull-down assay was carried out to verify the interaction between miR-223-3p and FBXW7 in SKOV3 and HO-8910 cells. D-E Western blot assay was used to examine the protein expressios of FBXW7 in SKOV3 and HO-8910 cells after transfection. F qRT-PCR was used to detect the expression of FBXW7 mRNA in OC tissues and normal tissues adjacent to tumors. n = 40. G-H Pearson’s correlation coefficient was used to analyze the correlation between miR-223-3p and FBXW7 mRNA, FBXW7 mRNA and circ-BNC2 expression in OC tissues, respectively. **P < 0.01, and ***P < 0.001

Fig. 6

Circ-BNC2 inhibits the lung metastasis of OC cells in vivo. A-B Metastatic nodules in mice were detected by H&E staining after tail vein injection of circ-BNC2-overexpressing SKOV3 cells. n = 12, scale bar: 50 μm. C-D qRT-PCR was used to detect the expression of miR-223-3p and FBXW7 in the lung tissues of mice in the circ-BNC2 overexpression group and the vector group. **P < 0.01, and ***P < 0.001