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MYB effects on miRNA profile of Ph+ leukemia cells
expression of the miR-17-92 cluster (Figure 4D). Expression levels of BIM, PTEN and THBS1 mRNA, after 24 h Doxy treatment, were assessed also in the SUP-B15 ShMYB cells analysis. This revealed a statistically signifi- cant (P≤0.05) increase of BIM and THBS1 in MYB-silenced SUP-B15 cells compared to untreated cells (Online Supplementary Figure S3).
The expression of p21 and E2F1 genes, two other exper- imentally validated miR-17-92 targets,40 was also assessed in MYB-silenced BV173 cells. MYB silencing induced an increase in the expression of p21 but this increase was not blocked by overexpression of the miR-17-92 cluster. In contrast, expression of E2F1 was down-modulated after MYB silencing and was not affected by overexpression of the miR-17-92 cluster (Online Supplementary Figure S4). These results suggest that MYB silencing modulates p21 and E2F1 expression independently of its effect on the miR-17-92 cluster expression.
Since our goal was to investigate novel miR-17-92 tar- gets, potentially involved in the “MYB addiction” of Ph+ leukemia cells, we focused on FRZB because ectopic expression of the miR-17-92 cluster blocked the increased expression of FRZB mRNA but not of PBX2 mRNA (Figure 4E) induced by MYB silencing in BV173-ShMYB cells (Figure 4B). FRZB is the founding member of the secreted Frizzled-related protein (SFRP) family of Wnt inhibitors32,41 and suppresses Wnt signaling thus preventing the accu- mulation of β-catenin into the nucleus.42 Then, we used miRwalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mir- walk2/), TargetScan 5.2 (http://www.targetscan.org), and miRanda (http://www.microrna.org/microrna) algorithms to assess the presence of putative miR-17-92-binding sites within the 3’untranslated region (3’UTR) of FRZB-mRNA. This analysis identified one putative miR-17-92 binding site for miR-19a (seed sequences: 76-81 bp) and one for miR-17 and -20a (seed sequences:1091-1097 bp) (Figure 4F,
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Figure 5. Expression of the miR-17-92 cluster and its target FRZB correlates with MYB levels in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cells. (A) MiR-17-92 expression levels evaluated by stem-loop qRT-PCR in primary leukemia cells (Patient 1: p210BCR/ABL chronic myeloid leukemia (CML)-myeloid blast crisis) compared to normal CD34+ cells from a healthy subject [Control (Ctrl) CD34+] and Patient 2 (p190BCR/ABL ALL) compared to normal peripheral blood mononuclear (PBMC) cells (Ctrl/PB). Samples were normalized for RNU44 small-nucleolar RNA expression using the comparative Ct method. Data are the average of three experiments; error bars indicate Standard Deviation (SD). (B) mRNA quantification of MYB and FRZB, by SYBR Green-based qRT-PCR, in Patient 1 (p210BCR/ABL CML-myeloid blast crisis) and Patient 2 (p190BCR-ABL ALL) compared to normal CD34+ cells and PBMC cells from healthy donors (Ctrl/CD34+ and Ctrl/PB), respectively. Values are reported as 2-ΔCt normalizing to GAPDH gene expression. (C) mRNA expression by microarray of MYB or FRZB in normal B cells or Ph+ ALL cells. (Values represent the sum of all probes signals for each gene and are derived from dataset GSE13159).
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