Abstract 896: Identification of Pro- And Anti-angiogenic MicroRNAs
MicroRNAs (miR) are small noncoding RNAs that regulate gene expression by binding to the target mRNA leading either to translational repression or degradation. Dicer and Drosha are the miR processing enzymes, which are required for the maturation of miR and endothelial sprout formation. Increasing evidence indicates that miR have distinct expression profiles and play crucial roles, e.g. in cardiogenesis and stem cell expansion. However, the individual role of miR in endothelial cell (EC) biology is poorly understood. Based on screening analysis of 160 human miR using real-time-PCR, we focused on miR-92, mir-27b and let-7f for further characterization. In order to analyze the functional role of these miR in endothelial cells, we performed a three-dimensional spheroid assay. Knockdown of mir-92 using specific 2‘O-methyl antisense oligoribonucleotides increased endothelial cell sprouting (128 ± 7% control, n = 4), while mir-27b and let-7f inhibitors significantly reduced the sprouting capacity (mir-27b: 51 ± 1%, let-7f: 51 ± 6% control, n = 4, p < 0.05). In contrast, overexpression of mir-92 by transfection with precursor-mir-92 oligonucleotides reduced capillary sprouting (39% control) and network formation (62% control). Having shown that mir-92, mir-27b and let-7f regulate in vitro angiogenesis, we next performed in silico analysis to identify potential miR targets. The endogenous angiogenesis inhibitor semaphorin 6A is a potential target for mir-27b and let-7f, whereas predicted targets of miR-92 involved in endothelial cell biology include the proangiogenic factors Wnt5a and SIRT1. Indeed, the knockdown of miR-92 significantly increased SIRT1 protein concentration (233 ± 41% control, n = 3, p < 0.05), while preliminary data demonstrate a decreased SIRT1 and eNOS expression in precursor-mir-92 transfected cells. Luciferase reporter gene assays will reveal whether SIRT1and eNOS are direct targets of mir-92. Taken together, we identified mir-92 as negative and mir-27b and let-7f as positive regulators of endothelial cell sprouting. Whereas the down-stream targets of mir-27b and let-7f have to be determined, mir-92 down-regulates the expression of SIRT1 and eNOS.