In Vivo Monitoring of Angiogenesis Inhibition via Down-Regulation of Mir-21 in a VEGFR2-Luc Murine Breast Cancer Model Using Bioluminescent Imaging

Dongliang Zhao,Lin Wan,Yingfeng Tu,Tao Huang,Baozhong Shen,Xilin Sun,Lihong Bu,Kai Wang,Marc Tjwa

doi:10.1371/journal.pone.0071472

Abstract

MicroRNA-21 (miR-21) is overexpressed in a wide range of cancers and involved in tumor proliferation and metastasis. However, the potential function of miR-21 in regulating tumor angiogenesis has been little disclosed. In this study, we treated the cultured 4T1 murine breast cancer cells and human umbilical vein endothelial cells (HUVECs) with miR-21 mimic, antagomir-21 or negative control (scramble), which were subjected to MTT, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), quantitative Reverse Transcriptase PCR (qRT-PCR) and immunoblotting analysis. In addition, 4T1 cells were implanted beneath the right breast fat pad of the VEGFR2-luc transgenic mice, which were randomly divided into three groups and received saline, antagomir-21 or scramble treatment once respectively after tumor model establishment. Bioluminescent imaging was used to monitor tumor growth and angiogenesis in vivo at 0d, 3d, 5d, 7d, 10d, and 14d after treatment. Mice were killed at the end of study and tumor tissues were collected for use. The results showed that knockdown of miR-21 by antagomir-21 decreased cell proliferation and induced apoptosis via targeting PTEN both in 4T1 cells and HUVECs. We also found the anti-angiogenesis and anti-tumor effects of antagomir-21 in the VEGFR2-luc transgenic mouse model using bioluminescent imaging. Moreover, the Western blotting data revealed that antagomir-21 inhibited tumor angiogenesis through suppressing HIF-1α/VEGF/VEGFR2-associated signaling pathway. In conclusion, the results from current study demonstrate that antagomir-21 can effectively suppress tumor growth and angiogenesis in VEGFR2-luc mouse breast tumor model and bioluminescent imaging can be used as a tool for noninvasively and continuously monitoring tumor angiogenesis in vivo.

Highlights

Tumor angiogenesis is a vital process resulting in the formation of new blood vessels, which has been identified to play a critical role in tumor growth, invasion, and metastasis in recent cancer researches [1]
We evaluated the expression of VEGFR2 in mouse breast tumor, which was closely associated with tumor angiogenesis
The major findings of our study are as follows: firstly, we demonstrated that antagomir-21 could down-regulate the miR-21 expression in 4T1 cells and human umbilical vein endothelial cells (HUVECs); secondly, we found that antagomir-21 could suppress 4T1 cells proliferation in vitro and in vivo; thirdly, antagomir-21 induces 4T1 cells and HUVECs apoptosis via targeting PTEN

Summary

Introduction

Tumor angiogenesis is a vital process resulting in the formation of new blood vessels, which has been identified to play a critical role in tumor growth, invasion, and metastasis in recent cancer researches [1]. Recent research has demonstrated that HIF-1a/VEGF/VEGFR2 signaling pathway is involved in endothelial cell proliferation, differentiation, migration, as well as vascular permeability [3]. Recent studies showed that miRNAs act as tumor suppressors or oncogenes and play critical roles in tumor cells proliferation and apoptosis [5,6]. MiR-21 has been demonstrated to modulate apoptosis in HUVECs by down-regulation of PTEN [17] Taken together, these data support that miR-21 is closely related to tumor angiogenesis, which prompts us to disclose the underlying role of miR-21 in breast angiogenesis and tumor growth

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