We have recently generated conditional and constitutive loss of function alleles for three related miRNA clusters in the mouse. These clusters are miR-17~92, miR-106b~25 and miR-106a~363. MiR-17~92 is also known as oncomir-1 because of its oncogenic properties in humans and mice. Our initial characterization of these null alleles in the mouse has revealed a critical role of miR-17~92 in controlling B cell survival and B cell development, as well as in regulating lung and heart development. Experiments performed on compound mutant animals for miR-17~92 and miR-106b~25 (one of its two paralogs) also suggest that these two miRNA clusters strongly synergizes in regulating early embryonic development.
To extend the functional characterization of these three miRNA clusters, we are trying to address four main questions:
1) Degree of functional overlap and cooperation among related miRNAs.
By generating double and triple KO animals for miR-17~92 and its two paralogs, we are studying the degree of functional overlap and cooperation among them. This is a very relevant question in the miRNA field, given the frequent occurrence of multiple miRNA sharing identical or nearly identical sequence.
2) Functions of miR-17~92 and its paralogs in mammalian development.
By using spatially and temporally regulated Cre-expressing mice in combination with the conditional miR-17~92 allele, we are investigating the functions of this cluster in specific tissues and developmental stages. By employing a combination of gene targeting and in vitro experiments we are attempting to identify the functions of individual members of each miRNA clusters.
3) Targets identification and generation of improved "prediction" algorithms.
A critical question regards the identification of functionally relevant miRNA targets. A variety of computational methods exist for their prediction based on sequence complementarity, evolutionary conservation and thermodynamic considerations. Unfortunately these methods are still imperfect and the number of false negative and false positives is still very high. We are employing cells and tissues derived from the knockout animals to experimentally identify functionally relevant targets in an unbiased way. To do so we are combinining proteomic approaches (SILAC in particular) and gene expression profiling methods to compare wild type and miR-17~92 mutant cells. Candidate targets will be functionally tested in vitro and in vivo.
4) miR-17~92 and its paralogs as novel anticancer targets.
The role of miR-17~92 in the pathogenesis of human B-cell lymphomas is supported by studies in transgenic animals. We are using the conditional miR-17~92 allele to determine whether this cluster is not only required for tumor initiation, but also for tumor maintenance. This study will determine whether miR-17~92 is a relevant novel anticancer target and might lead to improved treatments. The availability of highly specific means for the down-regulation of miRNAs in vivo (i.e. antagomirs and similar antisense strategies) may provide a promising way to follow-up on these experiments in pre-clinical trials.
In addition to miR-17~92, we are beginning experiments aimed at investigating other cancer associated miRNAs, in particular those that appear to be endowed with tumor suppressive properties.
