miR-153 inhibits the migration and the tube formation of endothelial cells by blocking the paracrine of angiopoietin 1 in breast cancer cells

Abstract

The sprouting of endothelial cells is the first step of tumor angiogenesis. Our previous study suggests that miR-153 suppresses breast tumor angiogenesis partially through targeting hypoxia-induced factor (HIF1α). In this study, we demonstrated that miR-153 also suppresses the migration and the tube formation of endothelial cells through directly targeting angiopoietin 1 (ANG1) in breast cancer cells. There was a negative correlation between miR-153 and ANG1 levels in breast cancer. miR-153 blocked the expression and secretion of ANG1 in breast cancer cells through binding to ANG1 mRNA. Conditioned medium from the breast cancer cell, MCF7, treated with miR-153 had no effect on the proliferation of HUVECs, but significantly inhibited the migration and tube formation of HUVECs, which could be rescued by overexpression of ANG1. In addition, miR-153 also directly inhibited the proliferation and migration of MCF7 through downregulation of ANG1. These findings suggest that miR-153 suppresses the activity of tumor cells and the migration and tube formation of endothelial cells by silencing ANG1.

Keywords: Angiopoietin 1; Breast cancer; Endothelial cell; MiR-153; Tumor angiogenesis.

Fig. 1

ANG1 is highly expressed in clinical breast cancer tissues. a Protein levels of ANG1 in breast cancer and matched adjacent tissues were detected by Western blot. b The quantitative results of a. *p < 0.05, t test. c The correlative analysis of miR-153 and ANG1 protein levels in breast cancer and matched adjacent tissues. There is a negative correlative trend between miR-153 and ANG1 at protein level in seven breast cancer tissues. d The correlation analysis of miR-153 and ANG1 mRNA levels in breast cancer and matched adjacent tissues. There is a negative correlative trend between miR-153 and ANG1 mRNA in seven breast cancer tissues. e Immunohistochemical staining of ANG1 in breast cancer and matched adjacent tissues. Representative images are shown. Scale bar, 100 µm

Fig. 2

miR-153 suppresses the expression and secretion of ANG1 in breast cancer. a Expression levels of miR-153 and ANG1 mRNA in the breast cancer cell lines was detected by RT-qPCR. b The correlative analysis of miR-153 and ANG1 mRNA in a. c Protein levels of ANG1 in breast cancer cell lines were examined by Western blot. d The correlation analysis of miR-153 and ANG1 protein levels for c. e The predicted binding sequence and the mutant sequences of miR-153 at the 3′UTR of ANG1 mRNA. f miR-153 mimics (50 nM) significantly inhibited the luciferase activity of the pMIR-ANG1-3′UTR-WT but not the pMIR-ANG1-3′UTR-Mut3 in the HEK293T cell line. **p < 0.01, t test. g miR-153 mimics (50 nM) downregulated the mRNA levels of ANG1 in three breast cancer cell lines (MCF7, MDA-MB-231, and HCC1937). **p < 0.01, t test. h miR-153 mimics (50 nM) downregulated protein levels of ANG1 including the non-glycosylated (~ 55 kDa) and the glycosylated (~ 70 kDa) formats in the MCF7, MDA-MB-231, and HCC1937 cell lines. i miR-153 mimics (50 nM) significantly decreased the secretion of ANG1 in MCF7 and MDA-MB-231 cell lines. *p < 0.05, **p < 0.01, t test

Fig. 3

miR-153 suppresses MCF7 proliferation and migration by silencing ANG1. a A schematic illustration of cell treatment, collection and detection. b Downregulation of ANG1 protein levels after ANG1 siRNA (50 nM) treatment for 72 h in MCF7 cells as detected by Western blot. c Knockdown of ANG1 by siRNA decreased cellular viability of MCF7 cells as characterized by the SRB assay. **p < 0.01, t test. d Knockdown of ANG1 with siRNA inhibited the migration of MCF7 cells characterized with the transwell assy. The representative images are shown, and the scale bar is 100 µm. *p < 0.05, **p < 0.01, t test. e Ectopic expression of ANG1 in MCF7 without the miR-153 binding site. f Ectopic expression of ANG1 increased cellular viability of MCF7 cells and rescued the inhibition of miR-153, shown with the SRB assay. *p < 0.05, t test. g Ectopic expression of ANG1 increased the migration of MCF7 cells and rescued the inhibition of miR-153, shown using the transwell assay. Representative images are shown, and the scale bar is 100 µm. **p < 0.01, t test

Fig. 4

ANG1 from MCF7-conditioned media has no influence on HUVEC proliferation. a Downregulation of ANG1 protein levels after miR-153 and ANG1 siRNAs treatment. b MCF7-conditioned media from cells treated with the miR-153 or the ANG1 siRNAs had no effect on the proliferation of the primary HUVECs, characterized with the EdU assay. Representative images are shown. “CM” means the conditioned medium. c The quantitative results of b. d Ectopic expression of ANG1 without the binding site of miR-153 in MCF7 cells. e Ectopic ANG1 expression in MCF7 cells was detected using the ELISA assay. **p < 0.01, t test. f MCF7-conditioned media with ectopic ANG1 had no influence on the proliferation of primary HUVECs as characterized by EdU assay. Representative images are shown. g The quantitative results of f

Fig. 5

miR-153 inhibits the migration of primary HUVECs through suppression of the secretion of ANG1 from MCF7 cells. a MCF7-conditioned media from cells treated with the miR-153 or the ANG1 siRNAs inhibited the migration of the primary HUVECs in a wound healing assay. Representative images are shown. The “CM” means the conditioned medium. b The quantitative results of a. *p < 0.05, **p < 0.01, t test. c The MCF7-conditioned medium with the ectopic ANG1 expression increased the migration of the primary HUVECs and rescued the inhibition of miR-153 by the wound healing assay. The representative images are shown. d The quantitative results of c. *p < 0.05, t test

Fig. 6

miR-153 inhibits the tube formation of the primary HUVECs through suppressing the secretion of ANG1 from MCF7 cells. a MCF7-conditioned medium with the miR-153 or the ANG1 siRNAs treatment inhibited the tube formation of the primary HUVECs by the tube formation assay. The representative images are shown. The “CM” means the conditioned medium. b The quantitative results of a. *p < 0.05, **p < 0.01, t test. c MCF7-conditioned medium from cells treated with the ectopic ANG1 increased the tube formation of the primary HUVECs and rescued the inhibition of miR-153 as characterized with a tube formation assay. Representative images are shown. d The quantitative results of c. *p < 0.05, **p < 0.01, t test