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miR-20a represses endothelial cell migration by targeting MKK3 and inhibiting p38 MAP kinase activation in response to VEGF

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Abstract

Endothelial cell migration induced in response to vascular endothelial growth factor (VEGF) is a crucial step of angiogenesis and it depends on the activation of the p38 MAP-kinase pathway downstream of VEGFR2. In this study, we investigated the role of microRNAs (miRNAs) in regulating these processes. We found that the VEGF-induced p38 activation and cell migration are modulated by overexpression of Argonaute 2, a key protein in the functioning of miRNAs. Thereafter, we found that miR-20a expression is increased by VEGF and that its ectopic expression inhibits VEGF-induced actin remodeling and cell migration. Moreover, the expression of miR-20a impairs the formation of branched capillaries in a tissue-engineered model of angiogenesis. In addition, the lentivirus-mediated expression of miR-20a precursor (pmiR-20a) is associated with a decrease in the VEGF-induced activation of p38. In contrast, these processes are increased by inhibiting miR-20a with a specific antagomir. Interestingly, miR-20a does not modulate VEGFR2 or p38 protein expression level. miR-20a does not affect either the expression of other known actors of the p38 MAP kinase pathway except MKK3. Indeed, by using quantitative PCR and Western Blot analysis, we found that pmiR-20a decreases the expression of MKK3 and we obtained evidence indicating that miR-20a specifically binds to the 3′UTR region of MKK3 mRNA. In accordance, the VEGF-induced activation of p38 and cell migration are impaired when the MKK3 expression is knocked down by siRNA. We conclude that miR-20a acts in a feedback loop to repress the expression of MKK3 and to negatively regulate the p38 pathway-mediated VEGF-induced endothelial cell migration and angiogenesis.

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Acknowledgments

The authors thank Dr. Karim Ghani and Dr. Manuel Caruso for their help in preparing the lentiviral vectors. They also thank Drs. Sébastien Bonnet, Jacques Landry, Josée N Lavoie, Marc-Etienne Huot, and Gunter Meister for providing some of the reagents used in this study. This study was supported by grants to JH from the Canadian Institutes for Health Research (CIHR), The Heart Stroke Foundation of Canada (HSFC) and The Natural Sciences and Engineering Research Council of Canada (NSERC) and by a CIHR grant to MJS. MJS is a Canadian Institutes of Health Research New Investigator. ALP received a studentship from CRCHUQ.

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  1. Centre de recherche du CHUQ-Hôtel-Dieu de Québec, Université Laval, 9 rue McMahon, Quebec city, QC, G1R 2J6, Canada

    Anne-Laure Pin, François Houle, Maëva Guillonneau, Éric R. Paquet, Martin J. Simard & Jacques Huot

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  1. Anne-Laure Pin
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Correspondence to Jacques Huot.

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Pin, AL., Houle, F., Guillonneau, M. et al. miR-20a represses endothelial cell migration by targeting MKK3 and inhibiting p38 MAP kinase activation in response to VEGF. Angiogenesis 15, 593–608 (2012). https://doi.org/10.1007/s10456-012-9283-z

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