EIF4A3-mediated circ_0042881 activates the RAS pathway via miR-217/SOS1 axis to facilitate breast cancer progression

Abstract

Breast cancer (BC) is one of the most frequent cancer-related deaths in women worldwide. Studies have shown the potential impact of circRNAs in multiple human tumorigeneses. Research on the vital signaling pathways and therapeutic targets of circRNAs is indispensable. Here, we aimed to investigate the clinical implications and underlying mechanisms of circ_0042881 in BC. RT-qPCR validated circ_0042881 was notably elevated in BC tissues and plasma, and closely associated with BC clinicopathological features. Functionally, circ_0042881 significantly accelerated the proliferation, migration, and invasion of BC cells in vitro and tumor growth and metastasis in vivo. Mechanistically, circ_0042881 promoted BC progression by sponging miR-217 to relieve its inhibition effect in son of sevenless 1 (SOS1), which further activated RAS protein and initiated downstream signaling cascades, including MEK/ERK pathway and PI3K/AKT pathway. We also demonstrated that treatment of BAY-293, an inhibitor of SOS1 and RAS interaction, attenuated BC progression induced by circ_0042881 overexpression. Furthermore, Eukaryotic initiation factor 4A-III (EIF4A3) could facilitate circ_0042881 circularization. Altogether, we proposed a novel signaling network in which circ_0042881, induced by EIF4A3, influences the process of BC tumorigenesis and metastasis by miR-217/SOS1 axis.

Fig. 1. Circ_0042881 is highly expressed in breast cancer.

A RT-qPCR analysis of the relative circ_0042881 expression in 56 pairs of BC tissues (Tumor) and adjacent non-tumor tissues (Normal). B RT-qPCR analysis of the relative circ_0042881 expression in plasma of BC patients (N = 74) and healthy donors (N = 46). C Relative circ_0042881 expression in BC cell lines and normal mammary epithelial cell line (MCF-10A) was determined by RT-qPCR. D Schematic illustration of circ_0042881 location and formation, with the junction site clarified by Sanger sequencing. E PCR with convergent and divergent primers and agarose gel electrophoresis to verify the circular structure of circ_0042881 in MCF-7; divergent and convergent primers are indicated by the direction of the arrow. F, G The relative expression of NF1 and circ_0042881 after treated with Actinomycin D and RNase R were determined by RT-qPCR. H Representative FISH images of circ_0042881 were shown. The circ_0042881 probe was labeled with Cy3 (red). The nuclei were stained with DAPI (blue). (Magnification x 1000. Scale bar = 10 μm). I Nuclear-cytoplasmic fractionation assay showed the percentage of circ_0042881 levels in nucleus and cytoplasm. U6 was used as the nucleus reference and GAPDH was used as the cytoplasmic reference. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Knockdown of circ_0042881 suppresses BC progression.

A, B RT-qPCR analysis of the relative circ_0042881 and NF1 expression after circ_0042881 knockdown in MDA-MB-231 and MCF-7. C−F The effect of silence circ_0042881 on proliferation in MDA-MB-231 and MCF-7 was tested by CCK-8, EdU and colony formation assays. G Transwell assays were performed to analyze cell migration and invasion ability. H, I Wound healing assays were used to evaluate the cell migration ability of each group. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. Circ_0042881 serves as a miR-217 sponge.

A, B RIP experiments were performed, and RT‐qPCR assays and agarose gel electrophoresis were used to detect the enrichment of circ_0042881 to AGO2. C Venn diagram representing the potential targeted miRNAs of circ_0042881 by CircInteractome, circBANK, Starbase v3.0. D RT-qPCR analyzed the relative expression of 4 candidate miRNAs in MDA-MB-231 and MCF-7 after transfecting with si-circ_0042881. E The relative expression of miR-217 and miR-338-5p was tested by RT-qPCR after circ_0042881 overexpression. F To examine the direct interaction between circ_0042881 and miR-217, the luciferase reporter vectors containing circ_0042881 WT or circ_0042881 MUT were constructed. G The correlation between circ_0042881 and miR-217 in BC tissues was analyzed by Pearson correlation analysis. H The luciferase activities in MDA-MB-231 and MCF-7 cells co-transfected with miR-217 mimic or mimic NC and luciferase reporters containing circ_0042881 WT or circ_0042881 MUT. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. The tumor-inhibition effects of si-circ_0042881 could be reversed by miR-217 inhibitor.

A−C CCK-8, EdU assays were performed to assess cell proliferation ability of each group. D The capacity of cell migration and invasion of each group was determined by transwell assays. E The effect of si-circ_0042881 and miR-217 inhibitor on migration was examined by wound healing assays. The data are presented as mean ± SD. **P < 0.01, ***P < 0.001.

Fig. 5. SOS1 is targeted by miR-217.

A Volcano plot showing differentially expressed genes in MDA-MB-231 cells transfected with mimic NC and miR-217 mimic. B Venn diagram representing the potential targeted mRNAs of miR-217 by miRDB, TargetScan, Starbase v3.0 and RNA-seq results. C, D RT-qPCR analyzed the relative expression of 5 candidate mRNAs in MDA-MB-231 and MCF-7 after transfecting with si-circ_0042881. E, F The luciferase activities in MDA-MB-231 and MCF-7 cells co-transfected with miR-217 mimic or mimic NC and luciferase reporters containing SOS1 3’UTR WT or SOS1 3’UTR MUT. G The correlation between circ_0042881 and SOS1 in BC tissues was analyzed by Pearson correlation analysis. H, I Western blotting analyed the protein levels of SOS1, AKT, p-AKT, ERK, and p-ERK after corresponding treatment. The data are presented as mean ± SD. **P < 0.01, ***P < 0.001.

Fig. 6. Circ_0042881 promotes proliferation and metastasis in a SOS1‐dependent manner.

A−C CCK-8, EdU assays were performed to assess cell proliferation ability of each group. D, E The capacity of cell migration and invasion of each group was determined by transwell assays. F, G The effect of circ_0042881 overexpression, si-circ_0042881 and BAY-293 treatment on migration was examined by wound healing assays. The data are presented as mean ± SD. **P < 0.01, ***P < 0.001.

Fig. 7. RNA binding protein EIF4A3 regulates the expression of circ_0042881.

A The binding sites for EIF4A3 in the flanking sequences of the NF1 pre-mRNA transcript were predicted using CircInteractome and RIP assays validated the binding sites between NF1 and circ_0042881. B The relative expression of EIF4A3 and circ_0042881 after EIF4A3 knockdown was detected by RT-qPCR. C The protein level of EIF4A3 after EIF4A3 knockdown was detected by western blotting. D The correlation between circ_0042881 and EIF4A3 in BC tissues was analyzed by Pearson correlation analysis. E Representative images of immunohistostaining for EIF4A3 of tumor and adjacent non-tumor tissues (n = 30). F−H The proliferation of MDA-MB-231 was detected by CCK-8 and EdU assays. I, J Representative results of transwell and wound healing assays of each group.

Fig. 8. Circ_0042881 promotes the growth and metastasis of BC in vivo.

A Schema of the BC orthotopic mouse models and metastatic models. B Representative images of BC orthotopic mouse models in NC and circ_0042881 groups. C Tumor volume was measured every 5 days. D Tumor weight was examined in each group. E The protein levels of SOS1, AKT, p-AKT, ERK, and p-ERK in each group were verified by western blotting. F Representative images of lung tissues and their corresponding hematoxylin and eosin-stained sections in BC metastatic models. G, H The number of metastasis nodules and the incidence of lung metastasis were measured in NC and circ_0042881 groups. I Schematic depiction of the mechanism underlying EIF4A3-mediated circ_0042881 promotes BC progression by miR-217/SOS1 axis.