MicroRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides length which down-regulate gene expression by annealing with the 3' UTR of target mRNAs, repressing translation and inducing mRNA degradation and de-adenylation. The imprecise miRNA recognition of their mRNA targets allows post-transcriptional regulation over hundreds of target mRNA during homeostasis or in response to stimuli. The microRNA miR-126 represents the most abundant endothelial miRNA upon expression profiling, and is expressed in a pan-endothelial fashion during embryogenesis. We have created knockout mice lacking miR-126 which exhibit 50% embryonic lethality associated with edema, hemorrhage, and angiogenic delay. In surviving miR-126 ko mice, adult angiogenesis is delayed for instance in corneal micropocket assays. Interestingly, miR-126 is present in intron 7 of a host gene, Egfl7. The miR-126 ko phenotype actually recapitulates previously described Egfl7 ko phenotypes, and previously described Egfl7 ko mice are now understood to have inadvertently disrupted miR-126 expression. An essential role for a miRNA during tumor angiogenesis has not been previously demonstrated. The current application explores the role of miR-126 in tumor angiogenesis based upon strong miR-126/Egfl7 expression in tumor endothelium and preliminary data in the MMTV- PyMT transgenic model of breast cancer. Aim 1 investigates whether constitutive genetic deletion of miR-126 inhibits tumor progression and angiogenesis and extends survival in the MMTV-PyMT model. Aim 2 evaluates the therapeutic potential of miR-126 inhibition through temporally conditional miR-126 ko with our floxed mouse allele in pre-established MMTV-PyMT tumors. Conditional miR-126 ko will serve as a reference standard for pharmacologic miR-126 inhibition in the MMTV-PyMT model via both antagomirs, as well as a novel 2'-F/methoxyester anti-miR chemistry. Further, miR-126 inhibition by either genetic deletion or antagomir/anti-mir treatment will be compared and combined with VEGF inhibition. Finally, Aim 3 investigates mechanisms of miR-126 action during breast tumorigenesis through compartment-specific deletion in endothelium, in vitro characterization of miR-126 ko endothelium, and endothelial tip- and stalk cell phenotypes. Overall, these investigations utilize complementary approaches of rigorously characterized miR-126 genetic knockout mice and novel antagomir and 2'-F/methoxyester anti-miR therapeutic strategies to explore the first functional linkages between an endothelial microRNA and tumor angiogenesis. The demonstration of a functional requirement for miR-126 during tumor angiogenesis and progression would have significant implications for design of future anti-angiogenic therapies.