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microRNA (miRNA) genes transcribed by RNA polymerase II generate small noncoding miRNAs of 18 to 24 nucleotides after maturation process. The mature miRNAs and their associated isomirs specifically bind to different mRNA transcripts, resulting in down regulation of multiple genes within the cell in a highly multiplexed way. miRNA expression profiles differ between human cell types suggesting cell-specific impacts of each miRNA on the regulation of different biological processes. Comparison of miRNA profiles of tumor samples and adjacent normal tissues showed that some miRNAs are up- or down- regulated and suggested their implication during tumor progression. However, such a miRNA profiling approach is not sufficient to identify the respective role of each miRNA gene during the tumorigenesis. Here, to assess the individual role of each miRNA gene and its different isomirs in a specific cell environment, we have constructed a lentiviral miRNA expression library containing more than 1100 human known and novel miRNA precursors. The arrayed layout of our library allowed high-throughput screens with a large spectrum of functional read-outs using either normal or tumor cells. To exemplify this approach, the results of three different screens will be presented; i.e. identification of miRNAs that inhibit the BRAF pathway, miRNAs that inhibit tumor angiogenesis and miRNAs that stimulate the mesenchymal to epithelial transition. In addition, beyond this hit identification step, we will present detailed characterization of the role of the identified miRNAs in tumor progression by means of molecular and cellular functional assays. Combining our unique miRNA expression library with a functional screening platform has allowed the identification and the further characterization of several miRNAs able to significantly impact on tumor behavior supporting the therapeutic interest of some candidates. Citation Format: Paula I. van Noort, Negar Babae, Gerald W. Verhaegh, Willemijn M. Gommans, Francesco Cerisoli, Mark Verheul, Raymond M. Schiffelers, Arjan W. Griffioen, Jack A. Schalken, Eugene Berezikov, Edwin Cuppen, Roel Q. J. Schaapveld, Jos B. Poell, Gregoire P. Prevost, Meriem Bourajjaj, Suzanna Vidic, Judy R. van Beijnum, Rick J. van Haastert, Iman Schultz, Thijs de Gunt, Onno van Hooij. Identification of microRNA-based therapeutic candidates using a unique lentiviral microRNA overexpression library [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A12.

Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.