Figure 1.
Unsupervised clustering analysis of miRNA expression in K562 cells as compared to a pooled sample of healthy blood.
Heat maps illustrating unsupervised clustering of miRNAs that were differentially expressed between blood and K562 samples. The red and blue colors indicate relatively high and low fold-change of expression, respectively. Missing values are indicated in gray. The 8 miRNAs we focused on are indicated with arrows at the bottom of the cluster. Blue arrows represent miRNAs whose expression was downregulated in K562 vs blood, the black arrow represent miRNA whose expression was upregulated in K562 vs blood. Two main miRNAs clusters are shown on top (cluster 1; purple and cluster 2, orange), correlated with the expression patterns described above.
Table 1.
Summary of miRNAs downregulated in K562 cells as compared to healthy blood samples.
Table 2.
Summary of miRNAs upregulated in K562 cells as compared to healthy blood samples.
Figure 2.
Real-time RT-PCR validation of microarray results, presenting changes in miRNA expression in K562 cells as compared to a pooled sample of healthy blood.
The expression of A. miR-31, B. miR-34a, D. miR-143, E. miR-145, F. miR-155, G. miR-196b and H. miR-564 was downregulated whereas the expression of C. miR-128 was upregulated in K562 cells compared to healthy blood. The histograms show percentage of miRNA expression normalized to U6-snRNA±SEM from at least 3 experiments. Insets show microarray expression results for the specified miRNAs (Blood: red circles; K562: blue circles).
Figure 3.
Expression analysis of miRNAs in CML-derived cell lines as compared to a pooled sample of healthy blood.
Expression analysis of miR-31, miR-34a, miR-143, miR-145, miR-155 and miR-564 in Meg-01 and K562 cell lines and in healthy blood samples was analyzed by miR–qRT-PCR. Shown is the percentage of miRNA expression normalized to U6-snRNA±SEM from at least 3 experiments.
Figure 4.
Expression analysis of miRNAs in a CML patient before and during treatment with imatinib.
Expression analysis of miR-31, miR-155 and miR-564 in a CML patient at diagnosis an on day 5 of imatinib treatment at a dose of 100 mg/day (for the first week of treatment) and on day 30 of imatinib treatment at a dose of 400 mg/day (from the fourth week of treatment and thereafter). Expression was analyzed by miR–qRT-PCR. Shown is the percentage of miRNA expression normalized to U6-snRNA±SEM from at least 3 experiments.
Table 3.
Putative target genes of the deregulated miRNAs as predicted by Target Scan analysis.
Figure 5.
Expression of putative targets of the derugulated miRNAs.
Expression of CBL, E2F3, cyclin D1, K-ras and Akt2 was analyzed by real-time PCR in K562 cells and in healthy blood samples. Shown is the percentage of miRNA expression normalized to GUSB RNA±SEM from at least 3 experiments.
Table 4.
Ingenuity analysis of miRNA predictive pathways.
Figure 6.
KEGG pathways of deregulated miRNAs predicted targets in CML.
This figure presents cell survival and growth signaling pathways within a CML cell. Red squares represent proteins encoded by genes targeted by the deregulated miRNAs. We have shown that the miRNAs regulating these target genes are downregulated in CML. As a result, these target genes are probably upregulated leading to increased cell survival and induced of uncontrolled cell proliferation.
Figure 7.
Overlap of predicted target genes.
A Venn diagram showing the overlap between predicted targets of CML-related miRNAs and miRNAs related to the different pathways that are predicted to be deregulated in our study.