The circRNA circIFI30 promotes progression of triple-negative breast cancer and correlates with prognosis

Abstract

Growing evidence suggests that circRNAs exert a critical role in tumorigenesis and cancer progression. To date, the molecular mechanisms underlying circRNAs in triple-negative breast cancer (TNBC) are still poorly known. Here, circRNA expression profile was investigated by RNA sequencing in TNBC tissues and matched para-carcinoma tissues. We found that circIFI30 was significantly up-regulated in TNBC tissues and cells using quantitative real-time PCR and in situ hybridization. High circIFI30 expression was positively correlated with clinical TNM stage, pathological grade and poor prognosis of TNBC patients. Functionally, a series of in vivo and in vitro experiments showed that knockdown of circIFI30 could markedly inhibit TNBC cell proliferation, migration, invasion and cell cycle progression, induce apoptosis as well as suppress tumorigenesis and metastasis. Up-regulation of circIFI30 exerted an opposite effect. Mechanistically, we demonstrated that circIFI30 might act as a competing endogenous RNA (ceRNA) of miR-520b-3p to abolish the suppressive effect on target gene CD44 by fluorescent in situ hybridization (FISH), dual luciferase reporter assay, RNA immunoprecipitation and RNA pull-down assays. Therefore, our work uncovers the mechanism by which circIFI30 could promote TNBC progression through circIFI30/miR-520b-3p/CD44 axis and circIFI30 could be a novel diagnostic/prognostic marker and therapeutic target for TNBC patients.

Keywords: CD44; circIFI30; miR-520b-3p; triple-negative breast cancer.

Figure 1

Expression profile of circRNA in TNBC and para-carcinoma tissues by RNA sequencing and characterization of circIFI30. (A) Hierarchical cluster analysis of all target circRNAs in the TNBC and matched para-carcinoma tissues was shown. Each column represents a sample and each row represents a circRNA. Red strip represents high relative expression and green strip represents low relative expression. (B) The cluster heat map showed the top 10 up-regulated and down-regulated circRNAs. (C) The genomic locus of the circIFI30 and the back-spliced junction of circIFI30 were indicated, the back-splice junction sequence was validated by Sanger sequencing. (D) PCR product of circIFI30 was confirmed by agarose gel electrophoresis. (E) CircIFI30, linear IFI30 and GAPDH were amplified from cDNA or gDNA in MDA-MB-231 cells with divergent and convergent primers, respectively. Divergent primers amplified circIFI30 in cDNA but not genomic DNA (gDNA). (F) RNase R treatment was used to evaluate the exonuclease resistance of circIFI30 in MDA-MB-231 cells. GAPDH was measured as a control. (G) Nuclear-cytoplasmic fractionation assay showed that circIFI30 was mainly localized in the cytoplasm of MDA-MB-231 cells. GAPDH was considered as a cytoplasmic control. U6 was used as a nuclear control.

Figure 2

circIFI30 is up-regulated in TNBC and associated with the progression and poor prognosis of TNBC patients. (A) Relative expression of circIFI30 in TNBC tissues and adjacent non-tumor tissues was detected by qRT-PCR (n = 38). (B) Relative expression of circIFI30 in cell lines was determined by qRT-PCR. (C) ROC curve was applied to evaluate the diagnostic value of circIFI30 for TNBC. (D) Representative images of circIFI30 expression in TNBC tissues were detected by ISH assays. Scale bar, 100 μm. (E) Dot distribution graph of circIFI30 ISH staining scores was shown in TNBC patients with different pathological grades. (F) Kaplan-Meier survival curve of overall survival in 78 patients with TNBC according to the circIFI30 expression. Patients were stratified into high expression and low expression group by median expression. Data were showed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

circIFI30 promotes TNBC cell proliferation. (A) Relative expression of circIFI30 was determined in TNBC cells transfected with circIFI30 expression vector, mock, sh-circ or sh-NC by qRT-PCR. (B) The cell viability was measured in TNBC cells transfected with indicated vectors by CCK-8 assay. (C, D) The cell proliferation ability was detected in TNBC cells after transfection with indicated plasmids by EdU assay. Scale bar, 50 μm. (E, F) Cell survival was evaluated in TNBC cells transfected with indicated plasmids by colony formation assay. Data were showed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

circIFI30 increases TNBC cell migration and invasion and modulates cell cycle and apoptosis. (A, B) The cell migration capacity was detected by wound healing assay after transfection with indicated vectors (magnification, × 50). Scale bar, 200 μm. (C, D) The cell invasion ability was determined by transwell assay after overexpression or knockdown of circIFI30 (magnification, × 100). Scale bar, 100 μm. (E, F) The cell cycle progression was analyzed by flow cytometry after transfected with indicated plasmids. (G, H) The apoptosis rate detected by flow cytometry after downregulation of circIFI30. (I, J) The apoptotic cells were observed by Hoechst 33342 (magnification, ×100, scale bar, 100 μm) and TUNEL staining (magnification, × 100, scale bar, 100 μm) assays after knockdown of circIFI30. (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.

Figure 5

circIFI30 facilitates tumorigenesis and metastasis of TNBC cells in vivo. (A, B) Representative images of xenograft tumors of each group and tumor weight analysis were shown. (C) Growth curves of xenograft tumors were measured once a week. (D, E) HE staining of tumor and lung sections were showed. The microvessels of the tumors and metastatic nodules of the lungs were indicated by arrows (magnification, × 100, scale bar, 100 μm). (F) IHC staining was applied to analyze the protein levels of CD44 and EMT-related molecules (magnification, × 200, scale bar, 100 μm). Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

circIFI30 functions as a sponge for miR-520b-3p. (A) The miR-520b-3p binding site on circIFI30 was predicted by targetScan and miRanda. (B) FISH was performed to observe the cellular location of circIFI30 in TNBC cells (magnification, × 200, scale bar, 50 μm) and tissues (magnification, × 100, scale bar, 50 μm). (C) Relative expression of miR-520b-3p in TNBC tissues and adjacent non-tumor tissues was determined by qRT-PCR (n = 38). (D) Schematic illustration of circIFI30-WT and circIFI30-Mut luciferase reporter vectors was shown. (E) The relative luciferase activities were detected in 293 T cells after transfection with circIFI30-WT or circIFI30-Mut and miR-520b-3p mimics or miR-NC, respectively. (F, G) Anti-AGO2 RIP was executed in MDA-MB-231 cells after transfection with miR-520b-3p mimic or miR-NC, followed by western blot and qRT-PCR to detect AGO2 protein, circIFI30 and miR-520b-3p, respectively. (H) RNA pull-down with a biotin-labeled circIFI30 probe was executed in MDA-MB-231 cells, followed by qRT-PCR and RT-PCR to detect the enrichment of circIFI30 and miR-520b-3p. (I) The relative expression of miR-520b-3p was detected by qRT-PCR after transfection with indicated vectors. Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

circIFI30 promotes cell proliferation, migration and invasion through circIFI30/miR-520b-3p/CD44 axis. (A) The cell viability was determined after transfection with indicated vectors, miR-520b-3p mimics or inhibitors by CCK8 assay. (B, C) The cell proliferation was detected after transfection with indicated vectors, miR-520b-3p mimics or inhibitors by EdU assay (magnification, × 100, scale bar, 100 μm). (D, E) The cell survival was measured after transfection with indicated vectors, miR-520b-3p mimics or inhibitors by colony formation assay. (F, G) The cell migration capacity was detected after transfection with indicated vectors, miR-520b-3p mimics or inhibitors by wound healing assays (magnification, × 50). Scale bar, 200 μm. (H, I) The cell invasion ability was determined after transfection with indicated vectors, miR-520b-3p mimics or inhibitors by transwell assays (magnification, × 100, scale bar, 100 μm). (J, K) Relative expressions of CD44 and EMT-related molecules at protein level in cells transfected with indicated vectors, miR-520b-3p mimics or inhibitors were determined by western blot. Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

CD44 is directly targeted by miR-520b-3p and regulated by circIFI30. (A) Schematic illustration of CD44 3’UTR-WT and CD44 3’UTR-Mut luciferase reporter vectors was shown. (B) Luciferase reporter assay demonstrated that CD44 is direct target of miR-520b-3p. (C, D) Relative mRNA and protein levels of CD44 were detected after TNBC cells were transfected with miR-520b-3p mimics or inhibitors using qRT-PCR and western blot, respectively. (E) Pearson correlation analysis between the expression of circIFI30 and CD44 was shown in 38 TNBC tissues. (F) Relative expression of CD44 was detected by qRT-PCR in cells transfected with indicated vectors, miR-520b-3p or inhibitors. (G) The schematic diagram illustrates how circifi30 might promote EMT, tumorigenesis and metastasis of TNBC through circIFI30/miR-520b-3p/CD44 axis. Data were indicated as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.