Downregulation of miR-217 correlates with resistance of Ph(+) leukemia cells to ABL tyrosine kinase inhibitors.
Bottom Line: This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibitors (TKI) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMT) and a decrease in levels of microRNA miR-217.Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3'-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI.In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib.
Affiliation: Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Japan.Show MeSH
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Mentions: Recent studies show that miR-29b and miR-140 regulate expression of DNMT, including DNMT1 and DNMT3A.15–18 However, the role of miR-217 in the regulation of DNMT remains unknown. Computational miRNA target analysis from miRNA databases identified homology between miR-217 and the 3′-UTR of the human DNMT3A gene, but not DNMT1 genes, prompting us to elucidate the link between miR-217 and DNMT3A (Fig.2a). We explored the effect of miR-217 on transcriptional activity of DNMT3A by using DNMT3A 3′-UTR luciferase reporter vector (Fig.2b). The luciferase activity in miR-217 stably expressing K562DR cells were less than that in control miRNA transfected cells (Fig.2b, Fig. S2). We further deleted four nucleotides (CAUG) from the miR-217 binding site of DNMT3A 3′-UTR and transfected this mutant construct in miR-217 stably expressing K562DR cells (Fig.2b). This mutant abrogated the miR-217/DNMT3A interaction, as evidenced by unchanged luciferase activity (Fig.2b). These results suggest that miR-217 negatively regulates expression of DNMT3A in leukemia cells. In fact, downregulated levels of DNMT3A mRNA and protein were noted in miR-217 stably expressing K562DR cells (Fig.2c,d). Intriguingly, forced expression of miR-217 in K562DR cells sensitized these cells to dasatinib (5 or 10 nM, 96 h), as measured by the MTT assay (Fig.2e). To explore the involvement of the elevated levels of DNMT3A in the acquisition of drug resistance of K562DR cells, we suppressed DNMT3A by siRNA and tested the sensitivity of these cells to dasatinib. Both DNMT3A siRNA1 and DNMT3A siRNA2 effectively downregulated the levels of DNMT3A in K562DR cells (Fig.2f). As expected, DNMT3A-depleted K562DR cells regained sensitivity to dasatinib (Fig.2g).
Affiliation: Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Japan.