MiR-199b-5p Suppresses Tumor Angiogenesis Mediated by Vascular Endothelial Cells in Breast Cancer by Targeting ALK1

Abstract

Angiogenesis is a crucial event during cancer progression that regulates tumor growth and metastasis. Activin receptor-like kinase 1 (ALK1), predominantly expressed in endothelial cells, plays a key role in the organization of neo-angiogenic vessels. Therapeutic targeting of ALK1 has been proposed as a promising strategy for cancer treatment, and microRNAs (miRNAs) are increasingly being explored as modulators of angiogenesis. However, the regulation of ALK1 by miRNAs is unclear. In this study, we identified that ALK1 is directly targeted by miR-199b-5p, which was able to inhibit angiogenesis in vitro and in vivo. Moreover, it was found that miR-199b-5p was repressed in breast cancer cells and its expression was decreased during the VEGF-induced angiogenesis process of human umbilical vein endothelial cells (HUVECs). Overexpression of miR-199b-5p inhibited the formation of capillary-like tubular structures and migration of HUVECs. Furthermore, overexpression of miR-199b-5p inhibited the mRNA and protein expression of ALK1 in HUVECs by directly binding to its 3'UTR. Additionally, overexpression of miR-199b-5p attenuated the induction of ALK1/Smad/Id1 pathway by BMP9 in HUVECs. Finally, overexpression of miR-199b-5p reduced tumor growth and angiogenesis in in vivo. Taken together, these findings demonstrate the anti-angiogenic role of miR-199b-5p, which directly targets ALK1, suggesting that miR-199b-5p might be a potential anti-angiogenic target for cancer therapy.

Keywords: ALK1; HUVECs; angiogenesis; breast cancer; miR-199b-5p; tumor growth.

Figure 1

MiR-199b-5p was downregulated during the angiogenesis process of HUVECs. (A) Real-time PCR analysis of the knockdown efficiency of ALK1 using siALK1-1, siALK1-2, siALK1-3 and their combination (siALK1-pool) (N = 3). (B) The sequences of siALK1-1, siALK1-2, and siALK1-3. (C) HUVECs transfected with siALK1-2 were subjected to the tube formation assay in matrigel and culture plate, respectively (N = 3). Representative images of tube formation are presented. Scale bars = 500 μm. (D) Real-time PCR analysis of candidate miRNAs possibly targeting ALK1 including miR-7, miR-96, miR-145, miR-181a-5p, miR-181b-5p, miR-181c-5p, miR-181d-5p, miR-199b-5p, miR-324-5p, miR-399-5p, miR-874-3p and miR-4262 in breast cancer cell lines (T47D, MDA-MB-231, MCF-7, and BT474) and a normal breast cell line (HBL-100) (N = 3). (E) Enlarged figure of Real-time PCR analysis of miR-199b-5p in breast cancer cell lines (T47D, MDA-MB-231, MCF-7, and BT474) and a normal breast cell line (HBL-100) (N = 3). (F) The Kaplan–Meier plot of overall survival and disease-free survival in TCGA database was analyzed according to miR-199b-5p expression. (G) Real-time PCR analysis of the expression of miR-199b-5p in HUVECs induced with VEGF (0, 1, and 2.5 ng/ml) for 24 h (N = 3). The data are presented as the means ± SD; *P < 0.05 and **P < 0.01 versus the control group. HUVECs, human umbilical vein endothelial cells; ALK1, Activin receptor-like kinase 1.

Figure 2

MiR-199b-5p suppresses the tube formation of HUVECs. (A) Real-time PCR analysis of miR-199b-5p expression in HUVECs transfected with scramble control and agomiR-199b-5p (N = 3). (B) HUVECs transfected with agomiR-199b-5p were subjected to the tube formation assay on matrigel and culture plate, respectively (N = 3). Representative images of tube formation are presented. Scale bars = 500 μm. (C) Quantification of tube length and number of intersections in (B) using Image J. (D) Real-time PCR analysis of miR-199b-5p expression in HUVECs transfected with scramble control and antagomiR-199b-5p (N = 3). (E) HUVECs transfected with antagomiR-199b-5p were subjected to the tube formation assay on matrigel and culture plate, respectively (N = 3). Representative images of tube formation are presented. Scale bars = 500 μm. (F) Quantification of tube length and number of intersections in (E) using Image J. Three independent experiments were performed in triplicate, and the data are presented as the means ± SD; *P < 0.05 and **P < 0.01 versus the control group. HUVECs, human umbilical vein endothelial cells.

Figure 3

MiR-199b-5p represses the migration of HUVECs. (A) Wound healing assay of HUVECs was performed 48 h after transfection with agomiR-199b-5p, antagomiR-199b-5p, and scramble control, respectively. The distance between the wound edges was observed and photographed at 0, 6, 12, and 24 h, respectively (N = 3). Scale bars = 500 μm. (B, C) The change of the width of injury was evaluated using Image J. (D, E) Representative and quantified results of the transwell migration assay in HUVECs transfected with agomiR-199b-5p and scramble control. Scale bars = 100 μm. (F, G) Representative and quantified results of the transwell migration assay in HUVECs transfected with antagomiR-199b-5p and scramble control (N = 3). Scale bars = 100 μm. Three independent experiments were performed in triplicate, and the data are presented as the means ± SD; **P < 0.01 versus the control group. HUVECs, human umbilical vein endothelial cells.

Figure 4

MiR-199-5p directly targets ALK1 and regulate downstream genes. (A, C) Real-time PCR (A) and western blot (C) analysis of ALK1 expression in HUVECs transfected with scramble control and agomiR-199b-5p (N = 3). (B, E) Real-time PCR (B) and western blot (E) analysis of ALK1 expression in HUVECs transfected with scramble control and antagomiR-199b-5p (N = 3). (D) Quantification of relative protein expression in (C) using Image J. (F) Quantification of relative protein expression in (E) using Image J. (G) Effects of agomiR-199b-5p on the ALK1 3’UTR luciferase reporters in HEK293T cells. Luciferase activity was measured at 36 hours post transfection (N = 6). (H) Western blot analysis of the expression of ALK1, p-Smad1/5/8, Smad1, and Id1 in BMP9-induced HUVECs transfected with agomiR-199b-5p, antagomiR-199b-5p, and scramble control (N = 3). (I) HUVECs transfected with agomiR-199b-5p, agomiR-199b-5p with pCMV3-ALK1, and scramble control, were subjected to the tube formation assay on matrigel, respectively (N = 3). Representative images of tube formation are presented. Scale bars = 200 μm. (J) Quantification of tube length and number of intersections in (I) using Image J. (K) Real-time PCR analysis of Id1 expression in HUVECs transfected with agomiR-199b-5p, agomiR-199b-5p with pCMV3-ALK1, and scramble control on matrigel (N = 3). The data are presented as the means ± SD; *P < 0.05 and **P < 0.01 versus the control group. HUVECs, human umbilical vein endothelial cells; ALK1, Activin receptor-like kinase 1.

Figure 5

MiR-199-5p reduces tumor growth and angiogenesis in vivo. (A) The body weight of mice from the scramble control treatment and agomiR-199b-5p treatment was measured every three days (N = 6). (B) A comparison of tumor size between the agomiR-199b-5p group and the scramble control group (N = 6). (C) Tumor weights of mice from the scramble control treatment and agomiR-199b-5p treatment groups were measured after three weekly injections (N = 6). (D) Representative images of tumors isolated from mice treated with scramble control and agomiR-199b-5p by surgical excision on the final day of the experiment. (E) Representative images of capillary tubes within tissues adjacent to the tumor on the final day of the experiment. (F, G) Representative images of immunofluorescence detection of blood vessels (red, CD31 staining) in tumors (F) and adjacent tissues (G) using an Aperio AT2 Digital Whole Slide Scanner. The data are presented as the means ± SD; *P < 0.05 and **P < 0.01 versus the control group.

Figure 6

Proposed regulatory model depicting the mechanism of miR-199b-5p in suppressing tumor angiogenesis by targeting ALK1. HUVECs, human umbilical vein endothelial cells; ALK1, Activin receptor-like kinase 1.