MicroRNAs (miRNAs) are involved in the pathobiology of leukemia. We previously observed that miR-125b, miR-99a and miR-100 were highly expressed in ex vivo vincristine (VCR) resistant leukemic cells of children with precursor B-cell acute lymphoblastic leukemia (ALL) (Schotte et al. Haematologica 2011). VCR is a microtubulin-interfering drug applied in the treatment of children with newly diagnosed ALL. Leukemic cells of TEL-AML1-positive precursor B-cell ALL were found to be resistant to this drug (Ramakers-van Woerden et al. Blood 2000). The expression levels of miR-125b, miR-99a and miR-100 were highly correlated in childhood precursor B-ALL cells. MiR-125b, as the most well-known miRNA among these three, was shown to contribute to drug resistance in different types of solid tumors. Down-regulation of miR-125b was also shown to sensitize the TEL-AML1-positive Reh cell line to doxorubicin and staurosporine (Gefen et al. Leukemia 2010). However, recent studies suggest that miRNAs co-operate and the combination of multiple distinct miRNAs co-regulates expression of associated target genes. The current study addressed whether miR-125b alone or in combination with miR-99a and/or miR-100 contributed to VCR resistance in childhood TEL-AML1-positive ALL cells.
MiRNAs were over-expressed in TEL-AML1-positive Reh cells using lentiviral particles and/or miRNA precursors. Expression level of mature (and hence processed precursor) miRNAs was analyzed by qRT-PCR. The cell cycle distribution and the amount of leukemic cells in apoptosis were determined by flow cytometry of propidium iodide stained nuclei and Annexin V-propidium iodide stained cells, respectively. Leukemic cells were incubated with 9 ng/mL VCR and after 3 days of exposure the cellular response to VCR was measured by an MTT-based cell survival assay.
The expression level of mature miR-125b, miR-99a and miR-100 was raised >100-fold upon lentiviral and precursor miRNA transduction compared to basal expression levels of TEL-AML1-positive Reh cells. Over-expression of miR-125b, miR-99a or miR-100 as single miRNAs did not significantly affect cellular survival after 3 days of exposure to VCR (20±5%, 29±17% and 29±17% viable cells, respectively) compared to the cells transduced with a scrambled miR-control (10±4% viable cells, p>0.05 each) when cell viability in absence of VCR was set to 100%. Over-expression of the combination of miR-100 and miR-99a only had a limited protective effect on cell viability (30±7% viable cells compared to 10±4% viable cells for the scrambled miR-control, p<0.05). In contrast, co-expression of miR-99a or miR-100 together with miR-125b or the combination of miR-99a and miR-100 together with miR-125b strongly and significantly induced resistance to VCR in TEL-AML- positive Reh cells; 91±4% of miR-99a/miR-125b-transduced, 93±5% of miR-100/miR-125b-transduced and 82±17% of miR-99a/miR-100/miR-125b-transduced cells remained alive upon VCR exposure compared to only 38±13% of miR-125b-transduced cells (p<0.05). The combination of these miRNAs did not change the cell cycle distribution or the amount of apoptotic cells in the absence of VCR. This suggests that these 3 miRNAs synergize in the development of resistance to VCR. Computational miRNA target prediction algorithms (TargetScan and EIMMo3) predicted 14 possible protein-coding target genes for the combination of miR-125b, miR-99a and miR-100. Gene expression profiling of ex vivo VCR-resistant (n=20) and VCR-sensitive (n=10) leukemic cells of TEL-AML1-positive ALL patients revealed that the expression levels of these 14 predicted target genes did not differ between resistant and sensitive cases (p>0.05). Ongoing studies currently explore which other protein-coding genes can be directly targeted by the miR125b, miR-99a and/or miR100 combination using functional gene expression studies.
In conclusion, miR-125b, miR-99a or miR-100 as single factors were not effective to induce VCR resistance. In contrast, miR-125b in combination with miR-99a and/or miR-100 strongly increased VCR resistance in leukemic cells of TEL-AML1-positive precursor B-ALL. Discovery of directly regulated protein-coding target genes of these 3 miRNAs may point to ways to modulate resistance to VCR in children with ALL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.