The circRNA circAGFG1 acts as a sponge of miR-195-5p to promote triple-negative breast cancer progression through regulating CCNE1 expression

Abstract

Background: In recent years, circular RNAs (circRNAs), a new star of non-coding RNA, have been emerged as vital regulators and gained much attention for involvement of initiation and progression of diverse kinds of human diseases, especially cancer. However, regulatory role, clinical significance and underlying mechanisms of circRNAs in triple-negative breast cancer (TNBC) still remain largely unknown.

Methods: Here, the expression profile of circRNAs in 4 pairs of TNBC tissues and adjacent non-tumor tissues was analyzed by RNA-sequencing. Quantitative real-time PCR and in situ hybridization were used to determine the level and prognostic values of circAGFG1 in two TNBC cohorts. Then, functional experiments in vitro and in vivo were performed to investigate the effects of circAGFG1 on tumor growth and metastasis in TNBC. Mechanistically, fluorescent in situ hybridization, dual luciferase reporter assay, RNA pull-down and RNA immunoprecipitation experiments were performed to confirm the interaction between circAGFG1 and miR-195-5p in TNBC.

Results: We found that circAGFG1 was evidently up-regulated in TNBC, and its level was correlated with clinical stage, pathological grade and poor prognosis of patients with TNBC. The results indicated that circAGFG1 could promote TNBC cell proliferation, mobility and invasion as well as tumorigenesis and metastasis in vivo. Mechanistic analysis showed that circAGFG1 may act as a ceRNA (competing endogenous RNA) of miR-195-5p to relieve the repressive effect of miR-195-5p on its target cyclin E1 (CCNE1).

Conclusions: Our findings suggest that circAGFG1 promotes TNBC progression through circAGFG1/miR-195-5p/CCNE1 axis and it may serve as a new diagnostic marker or target for treatment of TNBC patients.

Keywords: CCNE1; Triple-nagetive breast cancer; circAGFG1; miR-195-5p.

Fig. 1

Expression profiles of circRNA and mRNA in TNBC. a The volcano plot visualizes the expression of circRNA between TNBC tissues and adjacent non-tumor tissues. The red dots and green dots represent upregulated and downregulated circRNAs with statistical significance, respectively. b and c The cluster heat maps displayed the 10 most increased and decreased circRNAs and representative differentially expressed mRNAs. Each column indicates a sample while each row indicates an individual circRNA or mRNA. The red and green strips represent high and low expression, respectively. d KEGG pathway analysis of differentially expressed mRNAs. e GSEA was performed in KEGG gene sets

Fig. 2

circAGFG1 and CCNE1 are up-regulated in TNBC and associated with the progression and poor prognosis. a PCR product of circAGFG1 in agarose gelelectrophoresis and the splicing site verified by DNA sequencing. b Relative expression of circAGFG1 in TNBC tissues (Tumor) and adjacent non-tumor tissues (Normal) was detected by qRT-PCR (n = 40). c Relative expression of circAGFG1 in cell lines was determined by qRT-PCR. d Diagnostic value of circAGFG1 for TNBC was evaluated by ROC curve. e Representative images of circAGFG1 expression in TNBC tissues were detected by ISH assays. Scale bar, 100 μm. f Percentages of specimen with low or high expression of circAGFG1 according to TNM stage. g Kaplan-Meier survival curve of overall survival in 80 patients with TNBC according to the circAGFG1 expression. Patients were stratified into high expression and low expression group by median expression. h Relative expression of CCNE1 in TNBC tissues (Tumor) and adjacent non-tumor tissues (Normal) was detected by qRT-PCR (n = 40). i Pearson correlation analysis of circAGFG1 and CCNE1 expression in 20 TNBC tissues. j Relative expression of CCNE1 in TNBC tissues (Tumor) compared with normal tissues (Normal) was analyzed using TCGA data. k Kaplan-Meier survival analysis of overall survival based on TCGA data (n = 103). Data were showed as mean ± SD, *P < 0.05, ***P < 0.001

Fig. 3

circAGFG1 promotes TNBC cell proliferation. a The schematic illustration of circAGFG1 expression vector and shRNAs. b and c qRT-PCR analysis of circAGFG1 and AGFG1 RNA expression in TNBC cells transfected with circAGFG1 expression vector, mock, sh-circ or sh-NC. d The growth curves of cells transfected with indicated vectors were evaluated by CCK8 assays. e and f EdU assays were conducted in cells after transfection with indicated plasmids (magnification, × 100). Scale bar, 100 μm. g and h Colony formation assays were executed to detect the proliferation of cells transfected with indicated vectors. Data were showed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, N.S, nonsignificant

Fig. 4

circAGFG1 increases TNBC cell migration and invasion and modulates cell cycle and apoptosis. a and b Cell migration capacities were detected by wound healing assays after transfected with indicated vectors (magnification, × 50). Scale bar, 200 μm. c and d Cell invasion abilities were determined by transwell assays after transfection (magnification, × 100). Scale bar, 100 μm. e and f The cell cycle progression was analyzed by flow cytometry after transfected with indicated plasmids. g and h Apoptosis rate was analyzed by flow cytometry after downregulation of circAGFG1. i and j Apoptotic cells were assessed by TUNEL (magnification, × 100, scale bar, 100 μm) and Hoechst 33342 (magnification, × 200, scale bar, 50 μm) assays after knockdown of circAGFG1. k The expression levels of apoptosis-related proteins were determined by western blot. Data were showed as mean ± SD, *P < 0.05, **P < 0.01

Fig. 5

circAGFG1 facilitates tumorigenesis, angiogenesis and metastasis of TNBC cells in vivo. a Representative images of xenograft tumors of each group (n = 3). b Tumor weight was shown. c Growth curves of xenograft tumors which were measured once a week. d and e HE staining of tumor and lung sections displayed microvessels of the tumors and metastatic nodules of the lungs, respectively (magnification, × 100). Scale bar, 100 μm. f The protein level of CCNE1 was detected by western blot. g IHC staining was applied to analyze the protein levels of cell cycle-related molecules (magnification, × 200). Scale bar, 100 μm. Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

circAGFG1 functions as a sponge for miR-195-5p. a The miR-195-5p binding site on circAGFG1 predicted by targetScan and miRanda. b and c FISH was performed to observe the cellular location of circAGFG1 (red) and miR-195-5p (green) in cells (magnification, × 200, scale bar, 50 μm) and tissues (magnification, × 100, scale bar, 100 μm). d Relative expression of miR-195-5p in TNBC tissues (Tumor) and adjacent non-tumor tissues (Normal) was determined by qRT-PCR (n = 40). e Relative expression of miR-195-5p in TNBC tissues (Tumor) compared with normal tissue (normal) was analyzed using TCGA data. f Kaplan-Meier survival analysis of overall survival based on TCGA data (n = 100). g Schematic illustration of circAGFG1-WT and circAGFG1-Mut luciferase reporter vectors. h The relative luciferase activities were detected in 293 T cells after transfection with circAGFG1-WT or circAGFG1-Mut and miR-195-5p mimics or miR-NC, respectively. i and j Anti-AGO2 RIP was executed in MDA-MB-231 cells after transfection with miR-195-5p mimic or miR-NC, followed by western blot and qRT-PCR to detect AGO2 protein, circAGFG1 and miR-195-5p, respectively. k RNA pull-down was executed in MDA-MB-231 cells, followed by qRT-PCR to detect the enrichment of circAGFG1 and miR-195-5p. l The relative expression of miR-195-5p was detected by qRT-PCR after transfection with indicated vectors. m Pearson correlation analysis of circAGFG1 and miR-195-5p expression in 20 TNBC tissues. Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

CCNE1 is directly targeted by miR-195-5p and indirectly regulated by circAGFG1. a Schematic illustration of CCNE1 3’UTR-WT and CCNE1 3’UTR-Mut luciferase reporter vectors. b The relative luciferase activities were detected in 293 T cells after transfected with CCNE1 3’UTR-WT or CCNE1 3’UTR-Mut and miR-195-5p mimics or miR-NC, respectively. c and d Relative mRNA and protein levels of CCNE1 were detected in cells after transfected with miR-NC, miR-195-5p, ihn-NC and inh-195-5p using qRT-PCR and western blot, respectively. e and f Relative expression of CCNE1 was detected by qRT-PCR in cells transfected with indicated vectors, miRNAs or inhibitors. Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

circAGFG1 promotes cell proliferation and invasion through circAGFG1/miR-195-5p/CCNE1 axis. a and b The cell proliferation and invasion were determined after transfection with indicated vectors, miRNAs or inhibitors by EdU and transwell assays, respectively (magnification, × 100, scale bar, 100 μm). c Relative protein levels of CCNE1 in cells were assessed by IF after transfection (magnification, × 100, scale bar, 100 μm). d and e Relative expression of CCNE1 and downstream cell cycle-related molecules at protein level in cells transfected with indicated vectors, miRNAs or inhibitors was determined by western blot. f Schematic diagram of how circAGFG1 promotes TNBC tumorigenesis and progression. Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001