CircAGFG1 Promotes Ovarian Cancer Progression Through the miR-409-3 p/ZEB1 Axis

Abstract

Objectives: Circular RNAs (circRNAs) serve a crucial regulatory role in ovarian cancer (OC). Circular RNA ArfGAP with FG repeats 1 (circAGFG1) has been shown to be involved in promoting the progression of several cancers, containing triple-negative breast cancer, esophageal cancer and colorectal cancer. However, the function of circAGFG1 in OC is unclear.

Methods: Quantitative real-time reverse transcription PCR (RT-qPCR) was conducted to determine the expression levels of circAGFG1 and miR-409-3p. The proliferation and metastasis of cells were determined by colony formation assays, EdU assays, transwell assays and wound healing assays. In addition, a dual-luciferase reporter assay was performed to validate the mechanism between circAGFG1, miR-409-3p, and ZEB1.

Results: Our data suggested that circAGFG1 was significantly overexpressed in OC tissues compared to normal ovarian epithelial tissues. Overexpression of circAGFG1 was correlated with intraperitoneal metastasis, tumor recurrence and advanced stage. Additionally, circAGFG1 overexpression revealed a poor prognosis in OC patients. Knockdown of circAGFG1 suppressed the proliferation, invasion and migration of OC cells. Mechanistically, circAGFG1 acted as a sponge of miR-409-3p to enhance the expression level of zinc finger E-box binding homeobox 1 (ZEB1), thereby conferring OC cell proliferation, invasion and migration. Importantly, re-expression of ZEB1 effectively reversed the effects of circAGFG1 knockdown on OC cells.

Conclusions: In summary, our study indicated that circAGFG1 may act as a prognostic biomarker and potential therapeutic target for patients with OC.

Keywords: ZEB1; circAGFG1; miR-409-3p; ovarian cancer; progression.

Figure 1.

circAGFG1 up-regulation is correlated with poor prognosis in OC. (A) circAGFG1 expression was explored in 30 pairs of OC tissues and normal epithelial ovarian tissues controls. (B) Expression levels of circAGFG1 in OC tissues with different stages. (C) Expression levels of circAGFG1 in OC tissues with distant metastasis or not. (D) Expression levels of circAGFG1 in OC cell lines and normal epithelial ovarian cell. (E, F) Overall survival rate and recurrence-free survival rate in OC patients were determined using the Kaplan–Meier curve. *P < 0.05, ns indicates no significance. Each error bar represents the mean ± SD of three independent experiments.

Figure 2.

circAGFG1 sponges miR-409-3p in OC. (A, B) Nuclear/cytoplasmic fractionation analysis for circAGFG1 expression. (C, D) circAGFG1 expression was reduced by using two shRNAs targeting circAGFG1. (E) circAGFG1 knockdown caused upregulated expression of miR-409-3p (F) Negative correlation between circAGFG1 and miR-409-3p in OS tissues. (G, H) Luciferase reporter assay indicated circAGFG1-wt activity was impaired by miR-409-3p. (I) Biotin-wt-miR-93-3p interacted with AWPPH by pull-down assay. *P < 0.05, ns indicates no significance. Each error bar represents the mean ± SD of three independent experiments.

Figure 3.

circAGFG1 promotes OC progression through suppressing miR-409-3p. (A, B) CCK8 assay was performed to test cell proliferation in the indicated cells. (C, D) colony formation assay was performed to explore cell proliferation in the indicated cells. (E) EdU incorporation assay was conducted to examine cell proliferation in the indicated cells. (F, G) Transwell assay was conducted to analyze migration. A2780 and SKOV3 cells were transfected with NC shRNA, sh circAGFG1 or sh circAGFG1 + miR-409-3p inhibitor. (H, I) Wound healing experiments was conducted in the indicated A2780 and SKOV3 cells. *P < 0.05, ns indicates no significance. Each error bar represents the mean ± SD of three independent experiments.

Figure 4.

ZEB1 up-regulation by circAGFG1-induced miR-409-3p suppression promoted metastasis of OC cells. (A, B) Luciferase reporter assay showed miR-409-3p directly targeted ZEB1. (C) Negative correlation between ZEB1and miR-409-3p in OC tissues. (D) Expression of ZEB1 was measured by qRT-PCR in OC cells transfected with the indicated plasmids. (E–L) Expression of ZEB1 and its related genes (E-cadherin, Vementin and FN1) in the indicated cells. *P < 0.05, ns indicates no significance. Each error bar represents the mean ± SD of three independent experiments.