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MYB effects on miRNA profile of Ph+ leukemia cells
Table 1. Predicted down-and up-regulated target genes in BV173-ShMYB and K562-ShMYB cells after gene expression and miR-17-92 cluster analyses.
Down-regulated genes
BAZ1B
BUB1 CASP6 CNOT6L CPT1A EFNB2 GBE1 HDAC4 HRH2
ID2 ITGA4 ITGA4 MAD2L1 MAP3K1 MYO10 NR3C1 NRP2 PDE3B PIBF1 PRKRA REST RFC3 RPS6KA5 SCML2 SDC2 SERPINB8 TFRC TLE4 TNFAIP3
BV173 K562 Up-regulated genes DFCtest DFCtest
2.26E-10 0.0239 ABCA1 0.025 0.0033 ADARB1 0.001 0.0043 ARHGAP1 0.027 4.65E-05 BPNT1
0.02 0.026 CD22 0.046 0.0188 COL1A1
4.74E-22 0.0034 FRZB 0.004 0.0014 KIAA0513
5.14E-09 0.0222 PBX2 0.031 0.0053 PEX10 2.53E-09 0.0152 PTP4A3 2.53E-09 0.0004 RAB13 1.76E-08 0.0472 RPL19
1.71E-09 0.0346 SPIB 2.59E-10 0.0362 THBS1
0.03 0.0109 0.002 0.0133 0.014 0.0059 0.047 0.0063 0.017 0.0087 0.027 0.0256
8.99E-11 0.025 0.028 0.0372 0.03 0.0475 0.014 0.0012 7.04E-05 0.0111
5.72E-10 1.72E-05 7.65E-14 0.0017 7.89E-19 0.0026
Reference
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37
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35
BV173 K562 DFCtest DFCtest
0.009 0.006
1.01E-19 0.026 0.008 0.034 0.013 0.002
4.97E-15 0.003 0.014 0.018 0.0001 0.036 1.55E-17 0.007 0.0003 0.012 0.032 0.016 8.36E-17 0.041 4.56E-14 0.032 0.005 0.007
0.003 0.01 0.001 0.023
Doxy-treated BV173-ShMYB over-expressing miR-17-92 cells had less apoptosis than Doxy-treated BV173- ShMYB-EV cells, as indicated by the lower frequency of Annexin V-positive cells (9% vs. 15%, after 96 h Doxy treatment) (Figure 3D, left) and the increased expression of uncleaved PARP and BCL-2 (48% and 14%, respectively) (Figure 3D, middle and right panels).
Integrative analysis of gene expression profiles of MYB-silenced cells and predicted miRNA-regulated genes identifies novel putative miR-17-92 targets
We used gene expression profiling of MYB-silenced cells to identify MYB target genes potentially regulated by the miR-17-92 cluster. The miRWalk 2.0 database was used to investigate potential interactions of the miR-17-92 cluster with genes regulated by MYB silencing in BV173 and K562 cells. From this analysis, we found that 44 genes modulated by MYB silencing (15 up-regulated and 29 down-regulated) are predicted targets of the miR-17-92 cluster (Table 1 and Figure 4A). We focused on the up-reg- ulated genes since the decreased expression of the miR-
17-92 cluster in MYB-silenced cells should increase the levels of its putative targets. Thus, we performed qRT- PCR to assess the expression of two candidate targets, PBX2 and FRZB, involved in the regulation of proliferation and apoptosis.31,32 Such analysis revealed a statistically sig- nificant (P≤0.05) increase of PBX2 and FRZB expression in MYB-silenced Ph+ ALL BV173 and SUP-B15 or K562 cells (Figure 4B and C).
FRZB is a potential effector of the miR-17-92 cluster in the “MYB addiction” of Ph+ leukemia cells
The oncogenic effect of the miR-17-92 cluster is caused by the co-operation of its members in targeting tumor- suppressive pathways.28,33 Several studies have shown that the miR-17-92 cluster directly targets “pro-apoptotic” genes such as Phosphatase and tensin homolog (PTEN), the apoptosis facilitator BCL2L11 (BIM) and the anti- angiogenic factor thrombospondin-1 (THBS1) in normal lymphopoiesis,34-37 in MYC-driven lymphomas38 and in immunodeficiency or lymphoproliferative states.39 To assess whether the expression of validated miR-17-92 tar-
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