Circular RNA circBCBM1 promotes breast cancer brain metastasis by modulating miR-125a/BRD4 axis

Abstract

Circular RNAs (circRNAs) play critical roles in tumorigenesis and the progression of various cancers. We previously identified a novel upregulated circRNA, circBCBM1 (hsa_circ_0001944), in the context of breast cancer brain metastasis. However, the potential biological function and molecular mechanism of circBCBM1 in breast cancer brain metastasis remain largely unknown. In this study, we confirmed that circBCBM1 was a stable and cytoplasmic circRNA. Functionally, circBCBM1 promoted the proliferation and migration of 231-BR cells in vitro and growth and brain metastasis in vivo. Mechanistically, circBCBM1 acted as an endogenous miR-125a sponge to inhibit miR-125a activity, resulting in the upregulation of BRD4 (bromodomain containing 4) and subsequent upregulation of MMP9 (matrix metallopeptidase 9) through Sonic hedgehog (SHH) signaling pathway. Importantly, circBCBM1 was markedly upregulated in the breast cancer brain metastasis cells and clinical tissue and plasma samples; besides, circBCBM1 overexpression in primary cancerous tissues was associated with shorter brain metastasis-free survival (BMFS) of breast cancer patients. These findings indicate that circBCBM1 is involved in breast cancer brain metastasis via circBCBM1/miR-125a/BRD4 axis. CircBCBM1 may serve as a novel diagnostic and prognostic biomarker and potential therapeutic target for breast cancer brain metastasis.

Keywords: BRD4; biomarker; breast cancer brain metastasis; circBCBM1; miR-125a; therapeutic target.

Figure 1

Characterization of circBCBM1 in 231-BR cells. (A) The schematic diagram of genomic location and splicing pattern of circBCBM1. The splice junction was verified by Sanger sequencing. (B) RT-qPCR analysis for the abundance of circBCBM1 and its linear counterpart after treatment with RNase R in 231-BR cells. The amount of circBCBM1 and its linear counterpart were normalized to the value measured in the mock treatment. (C) The expression level of circBCBM1 and its linear counterpart after treatment with Actinomycin D (2.5 μg/ml) at the indicated time points in 231-BR cells. (D) Cellular RNA fractionation analysis. CircBCBM1 was mainly located in the cytoplasm of 231-BR cells. (E) RNA fluorescence in situ hybridization (FISH) for circBCBM1. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 25 μm. Data are presented as means ± SEM (B-D).

Figure 2

Silencing of circBCBM1 inhibits proliferation and migration and induces apoptosis of 231-BR cells in vitro. 231-BR cells were either transfected with si-circBCBM1 or siRNA Control. (A) RT-qPCR for the expression analysis of circBCBM1 and its linear counterpart. The relative expression levels were normalized to the values in the siRNA-control group. (B) CCK-8 assay. (C) Colony formation assay. Scale bar, 5 mm. (D) Annexin V-PE/ 7-AAD staining and FACS quantified the apoptotic cells percentage. (E) Wound-healing assay. (F) Transwell migration assay. Data are presented as means ± SEM (A-F). si-circBCBM1, siRNA against circBCBM1; siRNA Control, siRNA for negative control.

Figure 3

Silencing of circBCBM1 inhibits growth and brain metastasis of 231-BR cells in vivo. Nude mice were subcutaneously injected 231-BR cells with stable transfection of negative control (shRNA Control) or shRNA-circBCBM1 (n = 7) (A-D). (A) Tumor growth curves of subcutaneous models. (B) Images of the subcutaneous xenograft models on the 15th day. (C) Images of the dissected subcutaneous tumors. (D) Weights of the dissected subcutaneous tumors. Brain metastasis model was generated by injection of 231-BR cells (2 × 10⁵ per mouse) with stable transfection of negative control (shRNA Control) or shRNA-circBCBM1 into the left cardiac ventricle of mouse heart. After 4 weeks, the brains were collected and metastatic nodules were counted after H&E staining (n = 6) (E-G). (E) Representative H&E images and quantification of brain metastases. (F) An enlargement of the corresponding area (black rectangle) of H&E image in (E). The metastasis nodules were categorized into large- (> 50 μm²) and micro-metastases (≤ 50 μm²). Ovals, large-metastases; Circles, micro-metastases. (G) Quantification of the counts of large-metastases and micro-metastases. Data are presented as means ± SEM (A, D, E and G).

Figure 4

CircBCBM1 directly interacts with miR-125a in 231-BR cells. (A) RNA immunoprecipitation (RIP) and RT-qPCR assays were conducted to analyze the binding of circBCBM1 to Ago2 protein. (B) Biotinylated miRNA pull-down assay. RNA was affinity-isolated by biotinylated miR-125a, miR-1306, miR-34c, miR-26a, miR-10399, miR-661 or the negative control miRNA, and the circBCBM1 and GAPDH mRNA levels were quantified by RT-qPCR. The relative level of circBCBM1 was normalized to input. (C) RIP and RT-qPCR assays were performed to analyze the binding of miR-125a to Ago2 protein. (D) Biotinylated circBCBM1 pull-down assay. RNA was affinity-isolated by biotinylated circBCBM1 or the control probe, and the circBCBM1 and U6 levels were analyzed by RT-qPCR. The relative level of miR-125a was normalized to input. (E) The relative level of miR-125a in 231-BR cells versus the parental MDA-MB-231 cells. (F) The expression correlation analysis between circBCBM1 and miR-125a in 13 pairs of breast cancer and adjacent normal breast tissues. (G) FISH assay of circBCBM1 and miR-125a in the brain slices of brain metastasis mice. Left panel, H&E staining. Blue, DAPI; Red, circBCBM1; Green, miR-125a. Scale bar: H&E, 100 μm; FISH, 50 μm. Data are presented as means ± SEM (A-E).

Figure 5

BRD4 is the downstream target of the circBCBM1/miR-125a axis. (A) Volcano plots assessing the differentially expressed mRNAs between 231-BR (BCBM) and its parental MDA-MB-231 cell (BC) groups. (B) RT-qPCR assays analyzed the expression of BRD4 in 231-BR cells after treatment with si-circBCBM1. (C) Putative miR-125a binding site in BRD4. The potential complementary residues are shown in red. (D) Relative luciferase activity of BRD4 wild-type (BRD4-WT) and 3ʹ-UTR mutant (BRD4-MUT) constructs co-transfected with miR-125a mimics or miRNA negative control. (E) Western blot analysis of BRD4 expression in 231-BR cells versus the parental MDA-MB-231 cells. (F and G) Western blot assays examined the expression levels of BRD4 in 231-BR cells transduced with miR-125a mimics (F) or miR-125a inhibitors (G). MiRNA mimic control (F) and miRNA inhibitor control (G) were used as the miRNA mimic and inhibitor negative control, respectively. Data are presented as means ± SEM (B, D-G).

Figure 6

CircBCBM1 promotes cell migration via circBCBM1/miR-125a/BRD4 axis in 231-BR cells. (A) Western blot analysis of BRD4 expression in 231-BR cells after treatment with si-circBCBM1. (B) Western blot analysis of BRD4 expression in 231-BR cells transfected with control vector (Control group) or circBCBM1 over-expression plasmid (circBCBM1 group). (C) Transduction of 231-BR cells with circBCBM1 overexpressing lentivirus significantly increased miR-125a-inhibited BRD4 expression as determined by western blot analysis. (D) Transduction of 231-BR cells with circBCBM1 siRNA significantly inhibited miR-125a inhibitor induced BRD4 expression as determined by western blot analysis. (E) CircBCBM1 overexpression rescued the migration of 231-BR cells after transfection with miR-125a mimics as determined by transwell migration assay. OE: overexpression; Con: control. Data are presented as means ± SEM (A-E).

Figure 7

BRD4 promotes MMP9 expression through SHH signaling pathway in 231-BR cells. (A and B) Western blot analyses of BRD4, Shh, Gli and MMP9 expression in 231-BR cells transfected with si-BRD4 (A) or BRD4 over-expression plasmid (BRD4 group) (B). The relative expression level was normalized to that of the siRNA control group (A) or control vector group (B). Data are presented as means ± SEM. (C) Schematic representation of the mechanism and function of circBCBM1 in breast cancer brain metastasis.

Figure 8

The potential diagnostic and prognostic value of circBCBM1. (A) RT-qPCR analysis of circBCBM1 expression in 231-BR cells versus other breast cancer cells (MDA-MB-231, BT-474 and T47D). The relative expression level was normalized to that of MDA-MB-231 cells. (B and C) RT-qPCR analyses of circBCBM1 expression level in tissue (B) and plasma (C) samples. For (B), NBT, adjacent normal breast tissues, n = 13; BC, breast cancertissues, n = 13; BCBM, breast cancer brain metastasis tissues, n = 6. For (C), BC, breast cancer plasmas, n = 20; BCBM, breast cancer brain metastasisplasmas, n = 20. Data are presented as means ± SEM (A-C). (D) Kaplan-Meier analysis for brain metastasis-free survival (BMFS) of 53 BCBM patients. Patients were divided into two groups based on the expression of circBCBM1 in the patients' primary tumors. P-value was calculated using the log-rank test.