, 1990) (Figures 4D and 4E). In contrast, SHEP-TR-miR-17-92 cells persisted much longer and showed statistically higer luciferase signals Itraconazole
at 7, 14, and 21?days, indicating that, although tumorigenesis decreases, miR-17-92 activation significantly prolongs the engraftment of SHEP cells, probably through increased proliferation and decreased apoptosis, activities previously ascribed to miR-17-92 overexpression (Fontana et?al., 2008). Together, these results confirm the relation between miR-17-92 activation and cell proliferation and reveal a role for miR-17-92 in the regulation of cell adhesion, hereby confirming the GSEA results. GSEA analysis identified three TGF-��-responsive gene sets (Padua et?al., 2008?and?Verrecchia et?al., 2001) among the proteins downregulated upon miR-17-92 activation in the SHEP-TR-miR-17-92 cells (Figure?5A). To exclude the possibility that repression of TGF-��-responsive genes is an artifact of miRNA overexpression, we analyzed eight published protein expression data sets of miRNA overexpression (Baek et?al., 2008?and?Selbach et?al., 2008) using GSEA. None of the TGF-�� gene lists were significantly enriched in any of the data sets, suggesting the observed effect to be related to miR-17-92. For a subset of the TGF-��-responsive genes, the measured protein repression was confirmed on the mRNA level using RT-qPCR (Figure?S5). We next Verteporfin price
evaluated this TGF-�� signature in NB tumor samples using the pathway activity score of all genes that significantly contributed to the GSEA results (n?= 21). For this purpose, we used the larger Oberthuer data set (Oberthuer et?al., 2006) (data set D2, Table S1) to CYC202 mw
increase the power of our analysis. TGF-�� pathway activity was significantly downregulated in MNA NB tumors that are characterized by high miR-17-92 expression (Mann Whitney, p?< 0.001) (Figure?5B), and showed a negative correlation to MYC pathway activity (Spearman's Rank p?< 0.01, rho?= ?0.460). In addition, Kaplan-Meier survival analysis indicates that tumors with low TGF-�� pathway activity are characterized by poor event-free survival (log-rank, p?< 0.0001) (Figure?5C). To further substantiate the inverse relation between TGF-�� target gene expression and miR-17-92 expression, we performed an expression correlation analysis in a subset of 40 of the 95 NB tumors for which also mRNA expression was available (data set D3, Table S1) (Mestdagh et?al., 2009a). Hierarchical clustering of the correlation coefficients revealed that, indeed, miR-17-92 expression inversely correlates to TGF-�� target gene expression (Figure?S6A). These results confirm that TGF-�� signaling is downregulated in aggressive NB tumors with high miR-17-92 expression and underscore the potential importance of TGF-�� activity in NB tumor biology.