Table 1. miRNAs differentially expressed in mouse and human tumors. MIRNA NAME MOUSE TUMORS HUMAN MNA TUMORS Bray et al. Mestdagh et al. miR-130b miR-17 miR-18a miR-19a miR-19b miR-20a miR-20b miR-25 miR-9 miR-9* miR-93 miR-323-3p miR-369-5p miR-410 miR-411 miR-433 miR-494 miR-152 miR-204 miR-26a miR-26b miR-30a miR-30a* miR-30d miR-30e miR-30e* miR-328 miR-491 miR-130a Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down Down Down Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down Down Down Up Up Up Up Up Down Down Down Down Down Down Down Up Up = Over-expressed in MNA tumors; Down = under-expressed in MNA tumors. doi:10.1371/journal.pone.0028356.t001 current study were included in our prior study and by the non- conservation of many miRNAs between the species. 155 miRNAs were profiled in both murine and human tumors profiled by Bray et al. [12]. Unsupervised hierarchical cluster analysis with this set of 155 miRNAs of murine and human samples (n = 180 samples) indicated that murine adrenals and tumors clustered closer together relative to human tumors (Figure 2). However, hierar- chical cluster analysis maintains the distinction between murine tumors and adrenals. Interestingly, this subset of miRNAs was differentially expressed in MNA versus non-MNA human tumors (x2-test, p,0.001). The MNA branch of the dendrogram contained 31/36 (86%) of the MNA tumors, while the non- MNA branch contained 5/36 (14%) of the MNA tumors. Since the mouse model over-expresses human MYCN, we hypothesized that there might be similarities with human MNA tumors for MYCN regulated miRNAs. Hierarchical clustering based on the conserved miRNAs differentially expressed between human MNA and non-MNA tumors [12] revealed that mouse tumors more closely resemble adrenals than human MNA tumors, miRNA Expression Profiling of Murine Neuroblastoma (Figure 3A). As expected, there was a branch of human tumors significantly enriched (p,0.005) for MNA human tumors 22/37 (60%). Principal component analysis (PCA) was carried out in order to identify miRNAs that could account for the distinction between human and mouse tumors and between human MNA and non- MNA tumors. Figure 3B depicts the two top ranking principal components, PC1 and PC2. The distribution of the samples in PCA discriminates the two species mainly based on PC1, while the PC2 axis highlighted a distribution of the samples according to MYCN expression. The human tumors segregated by line b (Figure 3B) were significantly enriched for MNA (25/36, 69%; x2- test; p,0.001). To identify the miRNAs involved in these events, we analyzed the miRNAs of PC1 and PC2 (Table S4). The miRNAs that contributed most prominently to PC1 (human - mouse split) were miR-93 and miR-19a, with a lesser contribution from miR-19b, miR-20a and miR-130b, while the miRNAs that contributed most significantly to PC2 (MYCN high versus low expression) were miR-17, miR-25, miR-20b and miR-15b. Interestingly, 8 of the top 20 PC2 miRNAs were deregulated in both human and mouse tumors (Table 1). Moreover, 11 of the first 20 miRNAs contributing to each component were common to PC1 and PC2, clarifying how both the shifts, due to species and to human MYCN expression, involved the two axis. These results suggest that human MYCN may regulate similar miRNAs in human and mouse. We finally restricted our analysis to conserved miRNAs with differential expression between murine tumors and adrenals. Interestingly, the hierarchical clustering based on this subset of miRNAs (n = 63) distinguished tumors and adrenals independent of species. Unlike previous analyses, these miRNAs delineated murine tumor tissue from adrenal with higher specificity (Figure 4A). The analysis did not clearly distinguish human MNA and non-MNA tumors, however it identified a group of samples enriched (p,0.01) for non-MNA 68/75 (91%) allowing the mouse tumors to cluster closer to the human MNA samples. PCA again revealed an interspecies difference (PC2 axis), while the PC1 axis discriminated tumors and controls (Figure 4B). To identify the miRNAs underlying these differences, we considered the loadings of the two principal components (Table S5). The most significant contribution to PC1 was from miR-10a, miR-30a, miR- 30d, let-7f, miR-22 and miR-23b, ranked in order. Consistent with the interspecies shift based on PC2, 3 out of the first 5 implicated miRNAs (miR-411, miR-410, miR-382, miR-495 and miR-494) were differentially modulated in human and mouse samples (Table 1). Discussion The importance of highly representative murine models for studying human pathologies and for developing new pharmaco- logical treatments is established [32], however it is necessary to determine on a case-by-case basis the degree to which each model faithfully mimics human pathology. The TH-MYCN murine model for neuroblastoma has been well-characterized from the histopathological, gene expression and genome instability point of view [24–26,34]. Here, we identify miRNAs that are differentially expressed in this model (relative to adrenal controls). Cross- comparison between this and previous human studies reveals similarities and differences between the mouse model and the human pathology with regard to miRNA expression. The miRNA profile across murine tumors is more homogeneous than that observed in human tumors, perhaps a reflection of the inbred genetic tumor environment in murine versus human tumors. This PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28356