miR-20a represses endothelial cell migration by targeting MKK3 and inhibiting p38 MAP kinase activation in response to VEGF.
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.
Centre de recherche du CHUQ-Hôtel-Dieu de Québec, Université Laval, 9 rue McMahon, Quebec city, QC, G1R 2J6, Canada.
Anne-Laure
AL
Pin
François
F
Houle
Maëva
M
Guillonneau
Eric R
ER
Paquet
Martin J
MJ
Simard
Jacques
J
Huot
D001483:Base Sequence; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation; D006801:Humans; D048371:MAP Kinase Kinase 3 / Q000378:metabolism*; D035683:MicroRNAs / Q000502:physiology*; D008856:Microscopy, Fluorescence; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D017931:DNA Primers; C534451:MIRN20a microRNA, human; D035683:MicroRNAs; D042461:Vascular Endothelial Growth Factor A; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; C482530:MAP2K3 protein, human