MiR-21 Induced Angiogenesis through AKT and ERK Activation and HIF-1α Expression

Lihui Lai,Ling-Zhi Liu,Yue Jiang,Hsiang-Fu Kung,Alfons Navarro,Bing-Hua Jiang,Richard Carpenter,Yi Jing,Chongyong Li,Qi Chen

doi:10.1371/journal.pone.0019139

Abstract

MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that play important roles in various cellular functions and tumor development. Recent studies have indicated that miR-21 is one of the important miRNAs associated with tumor growth and metastasis, but the role and molecular mechanism of miR-21 in regulating tumor angiogenesis remain to be elucidated. In this study, miR-21 was overexpressed by transfecting pre-miR-21 into human prostate cancer cells and tumor angiogenesis was assayed using chicken chorioallantoic membrane (CAM). We found that overexpression of miR-21 in DU145 cells increased the expression of HIF-1α and VEGF, and induced tumor angiogenesis. AKT and extracellular regulated kinases (ERK) 1/2 are activated by miR-21. Inhibition of miR-21 by the antigomir blocked this process. Overexpression of the miR-21 target, PTEN, also inhibited tumor angiogenesis by partially inactivating AKT and ERK and decreasing the expression of HIF-1 and VEGF. The AKT and ERK inhibitors, LY294002 and U0126, suppressed HIF-1α and VEGF expression and angiogenesis. Moreover, inhibition of HIF-1α expression alone abolished miR-21-inducing tumor angiogenesis, indicating that HIF-1α is required for miR-21-upregulated angiogenesis. Therefore, we demonstrate that miR-21 induces tumor angiogenesis through targeting PTEN, leading to activate AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression; HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.

Highlights

MicroRNAs are a family of small non-coding RNAs with the length of 21- to 25-nucleotides that regulate the expression of various kinds of cellular proteins by targeting their mRNA expression levels. miRNAs act as gene regulators through translational repression or mRNA degradation via binding of miRNAs to target sites in the 39-untranslated regions (UTR) of protein-coding transcripts
The number of branches of microvessels in the premiR-21-transfected cells increased to 3.5-fold of the control (Figs. 1E and F). These results suggest that miR-21 induces tumor angiogenesis by upregulating the expression of HIF-1a and Vascular endothelial growth factor (VEGF)
To determine the signaling molecules that are involved in miR-21-inducing HIF-1a and VEGF expression and angiogenesis, we found that overexpression of miR-21 induced AKT and ERK1/2 activation when compared to the negative control of precursor miRNA group by Western blotting (Fig. 4A)

Summary

Introduction

MicroRNAs (miRNAs) are a family of small non-coding RNAs with the length of 21- to 25-nucleotides that regulate the expression of various kinds of cellular proteins by targeting their mRNA expression levels. miRNAs act as gene regulators through translational repression or mRNA degradation via binding of miRNAs to target sites in the 39-untranslated regions (UTR) of protein-coding transcripts. 500 miRNA genes have been identified in the human genome, which are involved in regulating development, differentiation, apoptosis and proliferation [1,2] Among these miRNAs, miR-21 is one of the well characterized miRNAs and overexpressed in various solid tumors including prostate cancers [3]. Anti-miR-21 suppressed both cell growth of breast cancer in vitro and tumor growth in the xenograft model partially through downregulating of the antiapoptotic factor, B-cell lymphoma 2 (Bcl-2) [12]. These data, taken together, support an important role of altered miR-21 expression during tumor development

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Results

Conclusion