expression is deregulated in MYCN amplified (MNA) tumors relative to MYCN non-amplified tumors (non-MNA), and that miRNA expression signatures are independently predictive of patient survival [12–17]. Moreover, functional studies have also demonstrated that specific miRNAs promote neuroblastoma cell proliferation in in vitro and in vivo assays [18,19], or alternately, act to suppress tumorigenesis by stimulating/reactivating apoptosis [20,21] and differentiation [22] or by inhibiting cell invasion [23]. TH-MYCN is a murine transgenic model of neuroblastoma that is increasingly used for a variety of molecular and pre-clinical studies [24]. Tumorigenesis is driven by neural crest-specific expression of a human MYCN transgene. These animals develop aggressive tumors that replicate major features of MNA high-risk disease [24–26]. The oncogenic action of MYC proteins is strongly enhanced in a variety of cancers by loss of p53, a functional interaction that has been replicated by the introduction of MYC- driven transgenic models in a p53-deficient background [27]. Alterations in p53 signaling are rarely observed in neuroblastoma patients at diagnosis. However abnormalities in multiple p53 pathway members emerge after pharmacological treatment, correlating with therapeutic resistance and reinforcing the notion that strong selective pressure for p53 inactivation exists in MYCN- driven neuroblastoma [28,29]. Indeed, murine neuroblastoma is strongly enhanced by placing TH-MYCN into a p53 insufficient background, and this is concomitant with reduced apoptosis and chemotherapy resistance [25]. The interplay between MYCN and p53, however, is complex. MYCN directly up-regulates p53 transcriptional expression [30], but also inhibits p53 through the direct up-regulation of MDM2, which encodes for a ubiquitin ligase that targets tumor protein p53 for proteasomal degradation [31]. Taken together these observations highlight the utility of these preclinical models for studies of MYCN-p53 control mechanisms [25]. Since miRNA expression levels are associated with clinical tumor grade, metastasis and overall survival in neuroblastoma, miRNAs are potential targets for therapeutic treatment. The availability of transgenic models with altered expression of individual oncogenes and tumor suppressor genes in a genetically homogeneous background present an ideal opportunity to test efficacy of novel miRNA mediated therapeutics [32]. However, knowledge about miRNA expression profiles in the TH-MYCN transgenic mouse model or its variants is lacking, particularly the degree to which murine miRNA expression profiles parallel those of human neuroblastoma. Here, we generated miRNA profiles of murine tumors and adrenal tissues derived from TH-MYCN mice in both wild-type and mutant p53 deficient backgrounds. Our analyses reveal similarities and differences between the miRNA expression profiles of human and mouse neuroblastoma and have allowed us to assess the impact of p53 haploinsuffiency on miRNA expression. By comparing the miRNA profile of murine and human tumors, we demonstrate the extent to which the transgenic mouse model can be used for miRNAs related studies. Results MiRNA expression profiling of murine tumors In order to identify miRNAs associated with neuroblastoma tumorigenesis, we analyzed the expression of 591 murine miRNAs in 22 mouse tumors using TaqMan low density arrays. Nine tumors were derived from TH-MYCN mice (wild-type for p53) and 13 from TH-MYCN mice in a p53 deficient background (TH-MYCN/p53ERTAM). As a control group, we profiled 12 adrenal glands (three from TH-MYCN; five from TH-MYCN/ p53ERTAM mice, and four from wild-type mice) coming from miRNA Expression Profiling of Murine Neuroblastoma young mice (7 days after birth). Adrenals coming from TH- MYCN and wild-type mice were stained with hematoxylin and eosin, confirming that the tissues were histologically normal in the transgenic mice (Figure S1). After primary profiling, the data set was filtered to include 440 miRNAs with expression in at least 10 of the samples (Figure S2). Unsupervised hierarchical clustering based on the expression of the total dataset of 440 miRNAs (Table S1) resolved tumors from adrenal gland controls (Figure 1A). Before using the adrenals as a unique group of controls regardless their genotype, we analyzed the miRNA expression of TH-MYCN (n = 8) and wild type (n = 4) adrenals finding no statistically significant differences. Then we compared all tumors (n = 22) to all adrenal controls (n = 12). Using the Wilcoxon Rank Sum Test corrected for multiple comparisons (n = 440), we found 159 miRNAs differentially regulated (p,0.05) in tumors: 81 were over-expressed (.2-fold) and 78 were under- expressed (.2-fold) (Table S2). Thus, miRNA expression in tumor tissue compared to a developmentally differentiated tissue precursor is significantly differentially regulated. Intriguingly, haploinsufficiency for p53 did not dramatically alter miRNA expression patterns as the tumor samples clustered together regardless their genotype. Since the miRNA profiles did not show any major differences based on the presence of the p53-ERTAM allele, we measured expression of the endogenous p53 transcript in tumors to confirm haploinsufficiency. The primers were designed to detect expression of the endogenous wild-type p53 allele, and not the mutant p53- ERTAM allele (Figure S3A). Five TH-MYCN and 6 TH-MYCN/ p53ERTAM tumors were analyzed through quantitative PCR. All TH-MYCN/p53ERTAM animals showed a reduced level of wild- type p53 mRNA (t-test, p,0.0001) (Figure S3B). These data demonstrate that no compensatory up-regulation of the endoge- nous p53 allele occurs in TH-MYCN/p53ERTAM tumors. Although cluster analysis based on all miRNAs did not distinguish tumors with respect to p53 genotype, miR-125b-3p and miR-676 were significantly down-regulated (p,0.03) in TH- MYCN/p53ERTAM tumors (Figure 1B–C) based on the Wilcoxon Rank Sum Test with p-values corrected for multiple comparisons (n = 440). Cross-Comparison of miRNA expression in human and murine tumors In order to assess the relevance of the mouse model as a representative platform for studies of important miRNA targets modulated in human neuroblastoma, we first compared the set of miRNAs differentially expressed in mouse and human tumors, using previously published miRNA expression profiles of human neuroblastoma [12,16]. In total, 296 mature miRNAs are conserved between mouse and human in the miRBase version 16 (Table S3). Taking into account only conserved miRNAs (Figure S4A), a total of 63 miRNAs have been identified as differentially expressed in human MNA versus non-MNA tumors in at least one published study [12,16]. Among the 63 conserved miRNAs differentially expressed between human MNA versus non-MNA tumors, 29 (46%) were differentially expressed between mouse tumors and adrenals (Figure S4B). This amount of overlap between differentially expressed miRNAs in human versus mouse tumors is statistically significant (p,0.01) according to a hypergeometric distribution [33]. The majority of the miRNAs had alterations in expression that were consistent between the two species, except for miR-323-3p, miR-369-5p, miR-410, miR-411, miR-433, miR-494 and miR-130a, which were expressed discordantly in the tumors from the two different species (Table 1). It is interesting to note that 6 out of 7 of the miRNAs PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28356