Role of Dicer and Drosha for endothelial microRNA expression and angiogenesis.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to the cellular transcript leading to translational repression or degradation of the target mRNA. Dicer and Drosha are the miRNA processing enzymes that are required for the maturation of miRNAs. Here, we investigated the role of Dicer and Drosha for angiogenesis. Endothelial cells were transfected with siRNA against Dicer and Drosha to inhibit miRNA biogenesis. Genetic silencing of Dicer and Drosha significantly reduced capillary sprouting of endothelial cells and tube forming activity. Migration of endothelial cells was significantly decreased in Dicer siRNA-transfected cells, whereas Drosha siRNA had no effect. Silencing of Dicer but not of Drosha reduced angiogenesis in vivo. Next, we attempted to identify miRNAs expressed in endothelial cells. A screening analysis of 168 human miRNAs using real-time PCR revealed that members of the let-7 family, mir-21, mir-126, mir-221, and mir-222 are highly expressed in endothelial cells. Dicer and Drosha siRNA reduced lef-7f and mir-27b expression. Inhibitors against let-7f and mir-27b also reduced sprout formation indicating that let-7f and mir-27b promote angiogenesis by targeting antiangiogenic genes. In silico analysis of predicted targets for let-7 cluster identified the endogenous angiogenesis inhibitor thrombospondin-1. Indeed, Dicer and Drosha siRNA significantly increased the expression of thrombospondin-1. Taken together, transient reduction of the miRNA-regulating enzyme Dicer impairs angiogenesis in vitro and in vivo, whereas Drosha siRNA induced a minor antiangiogenic effect in vitro and was not effective in vivo. The let-7 family and mir-27b appear to be attractive targets for modulating angiogenesis.
Kuehbacher
Angelika
A
Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Urbich
Carmen
C
Zeiher
Andreas M
AM
Dimmeler
Stefanie
S
D002465:Cell Movement / Q000235:genetics; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics*; D018919:Neovascularization, Physiologic / Q000235:genetics; D043244:Ribonuclease III / Q000502:physiology*
D035683:MicroRNAs; C517918:DROSHA protein, human; D043244:Ribonuclease III