Enforced Expression Of MiR-24, MiR-126 and MiR-365 Does Not Alter Cellular Viability In TCF3-Rearranged Leukemia

MicroRNAs (miRNAs) are involved in fine-tuning of the several cellular processes in human healthy and malignant tissues. In various types of tumors, miR-24, miR-126 and miR-365 were shown to regulate cell cycle progression and apoptosis. Interestingly, these three miRNAs were downregulated in pediatric TCF3-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL) (Schotte et al. Haematologica 2011). Here, we investigated whether these three miRNAs, individually or in combination, may alter cell cycle distribution and amount of apoptosis in TCF3-rearranged leukemia. In addition, we used a new miRNA target identification method (van Iterson et al. Nucleic Acid Res 2013) which integrates the mRNA and miRNA expression profiles of 37 cases with childhood BCP-ALL (4 BCR-ABL1-positive, 3 TCF3-rearranged, 7 ETV6-RUNX1-positive, 13 hyperdiploid (>50 chromosomes) and 10 unclassified B-others negative for the above mentioned genetic aberrations). Following overexpression of miRNAs in TCF3-rearranged leukemic cells, changes in the expression levels of predicted candidate target genes in our miRNA-mRNA integration cohort were analyzed to discover functional relationships between these miRNAs and their predicted candidate target genes in BCP-ALL.

MiRNAs were over-expressed in three TCF3-rearranged leukemic cells; i.e. 697, KASUMI-2 and MHH-CALL-3 cells, using miRNA precursors transfection. Expression level of mature miRNAs and candidate target genes were 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.

Our data revealed that the expression level of mature miR-24, miR-126 and miR-365 was raised >100-fold upon precursor miRNA transduction compared to basal expression levels of Three TCF3-rearranged leukemic cell lines. Individual or combined overexpression of miR-24, miR-126 and miR-365 did not alter cell cycle progression and amount of apoptosis in these three different leukemic cell lines. Integration of miRNA and mRNA expression levels of 37 newly diagnosed children with BCP-ALL revealed significant association between the expression of 29 miRNAs and their predicted target genes (FDR<0.05, p<0.005), including miR-24 (ranked at position #5), miR-126 (ranked at position #17) and miR-365 (ranked at position #20). Candidate target genes were selected for further analysis: ELL, EBF3 and IRF4 for miR-24 (Pearson r <-0.6, p<0.005), PITPNC1 for miR-126 (Pearson r <-0.5, p<0.01) and ZAP-70 for miR-365 (Pearson r <-0.4, p<0.01). However, miRNAs overexpression in MHH-CALL-3 cells did not reduce the expression levels of these selected candidate target genes. Considering the presence of only 3 patients with TCF3-rearranged ALL in our miRNA-mRNA expression data integration cohort, the observed significant inverse correlation between miRNA-mRNA pairs might mainly originate from other subtypes with higher number of cases in this cohort, such as hyperdiploid (13 cases) or unclassified B-others BCP-ALL (10 cases). Interestingly, expression level of AURKB – a validated target for miR-24 – was reduced by ∼4-fold upon miR-24 overexpression in hepatocarcinoma HEP-G2 cells, while overexpression of similar amount of miR-24 cannot alter AURKB expression levels in MHH-CALL-3 leukemic cells. Together, our findings indicate that individual and combined expression of miR-24, miR-126 and/or miR-365 does not affect the amount of apoptosis and cell cycle distribution in TCF3-rearranged leukemia. Moreover, our data suggest that miRNAs function is highly cell type-dependent and a defined biological target gene or function of one miRNA in a specific cellular context cannot be generalized for all types of cells/tissues.