GENETIC AND FUNCTIONAL DISSECTION OF THE MIR-17-92 CLUSTER OF MIRNAS

Background: The miR-17∼92 cluster was among the first cluster of miRNAs to be linked to cancer. It is directly activated by the members of the MYC family of transcription factors and acts as a bona fide oncogene in several types of cancer, including lymphomas. miR-17∼92 is essential for mammalian development. Knockout mice die in utero or at birth with cardiovascular defects and impaired B-cell maturation. Aims: Although miR-17∼92 is important for tumorigenesis and development both the molecular mechanisms and the prominent miRNAs acting in different contexts are still unknown. The main aim of our study was to identify the role played by different members of the miR-17∼92 cluster in B-cell lymphomas and vascular development. Materials and Methods: A mouse model of B-cell lymphomas and a conditional miR-17~92 KO mouse strain were employed to investigate the role of miR-17~92 in Myc-induced cancers. A GFP-based in vitro assay allowed us to rapidly verify the oncogenic potential of each miRNA family and to determine how mutations in the seed/non seed sequence affect miRNA function. Gene-expression profiling, computational prediction and an shRNA-mediated validation screening were used to identify miR-19 targets. miR-17∼92 involvement in vascular development was studied by means of Real time, western blot, ELISA and the CD-31 (PECAM) vascular staining. Results and Conclusions : Our results suggest that the expression of two members of the cluster, miR-19a and miR-19b, is necessary and largely sufficient to recapitulate the oncogenic potential of the full miR-17∼92 cluster in Myc-driven B cell lymphomas. A systematic mutational analysis of miR-19b showed that single point mutation in the seed sequence impaired miR-19b anti-apoptotic activity. Finally, a chimeric miRNA (miR-19b seed/miR-20 non-seed) acted as effectively as miR-19b, suggesting that the specific sequence of non-seed region of miR-19 provides little additional substrate specificity. We identified the tumor suppressor PTEN as the critical miR-19b target and the principal mediator of its pro-survival activity. We also showed that miR- 17~92–null embryos have upregulation of VEGF, although it is unclear at this time whether miR-17~92 suppresses VEGF expression directly or indirectly.