miRNA Expression Profiling of Murine Neuroblastoma Figure 2. Overlapping of miRNAs deregulated in human and mouse tumors and hierarchical clustering on all miRNAs. Hierarchical clustering based on the overall miRNAs conserved and present in both the studies (n = 155). Cluster ‘‘Mouse’’ contains all the murine samples split in two branches (‘‘Tumors’’ and controls ‘‘Adrenals’’). Cluster ‘‘Human’’ includes all the human tumors split (p,0.001) in a ‘‘MNA’’ and a ‘‘non-MNA’’ branch with an enrichment (52.5%) and an under-representation (5.7%) of MNA samples (red), respectively. doi:10.1371/journal.pone.0028356.g002 supports the use of mouse models for studying pathological events in a more defined genetic background, with reduced heterogeneity due to genetic variation that often affects human studies. In total, 22 conserved miRNAs were differentially expressed across both murine tumor versus adrenal and human MNA versus non-MNA tumors (as identified in at least one of two independent studies on human tumors [12,16]). This degree of similarity between human and mouse tumors is consistent with interspecies comparisons for other types of tumors [35]. Notably, several members of the miR-17-92 polycistronic cluster, which are highly up-regulated in human MNA tumors [36], were up-regulated in mouse tumors relative to the adrenal glands. This polycistronic cluster has demonstrated oncogenic functions in neuroblastoma through targeting multiple genes of the TGFB signaling pathway [18,19]. Similarly, miR-9, which is positively regulated by MYCN in human neuroblastoma [37], was also significantly up-regulated in the mouse tumors. In addition, miR-152 is down regulated in both mouse and human neuroblastoma, having a documented tumor suppressor function in human neuroblastoma [38]. Overall, our results in the mouse model indicate that the over-expression of MYCN strongly deregulates miRNAs expression, similar to the situation in human neuroblastoma. The major tumor suppressor miRNA, miR-34a, which is down- regulated in human neuroblastoma, was not changed in the mouse tumors and represents an example of where the mouse model is inconsistent with human neuroblastoma. Many factors are likely to account for the differences in miRNA expression between human and mouse neuroblastoma. For example, in addition to MYCN, large-scale genomic imbalances have been demonstrated to influence miRNA expression in human tumors [12]. Although some similarities in genomic imbalances have been demonstrated between murine and human tumors [34], there are also extensive differences between human and mouse genomes resulting from the accumulation of approximately 200 chromosomal rearrangements since the evolutionary divergence of rodent and primate lineages. It is also important to realize that neuroblastoma originate from fetal neuroblasts [39], so that some differences in miRNA expression between tumors and adrenal glands (from 7 day old mice) could relate to differentiation associated changes rather than cancer specific alterations. This possibility requires further testing by profiling micro-dissected fetal neuroblasts, a technically challenging endeavor. P53 is a critical tumor suppressor gene inactivated in a variety of cancers. In neuroblastoma, p53 is rarely inactivated in PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28356