RESUMO
BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Although the introduction of tyrosine kinase inhibitors (TKIs) has substantially improved patients' prognosis, drug resistance remains one of the major challenges in CML therapy. MicroRNAs (miRNAs), a class of short non-coding RNAs acting as post-transcriptional regulators, are implicated in CML progression and drug resistance. The aim of the present study was to analyze the miRNA expression profiles of 45 treatment-naïve CML patients in chronic phase (28 peripheral blood and 17 bone marrow samples) with respect to future response to imatinib therapy. METHODS: TaqMan low density arrays were used to analyze the miRNA expression pattern of the patient samples. For selected microRNAs, reporter gene assays were performed to study their ability to regulate CML associated target genes. RESULTS: Significant lower expression levels of miR-142-5p were identified in both, peripheral blood and bone marrow samples of future non-responders suggesting a potential tumor suppressor role of this miRNA. This was supported by reporter gene assays that identified the survival, proliferation and invasion promoting CML related genes ABL2, cKIT, MCL1 and SRI as targets of miR-142-5p and miR-365a-3p, the latter identified as potential biomarker in peripheral blood samples. CONCLUSION: MiR-142-5p and to a certain extend also miR-365a-3p were able to discriminate treatment-naïve CML patients not responding to imatinib in the course of their treatment from patients, who responded to therapy. However, further large-scale studies should clarify if the identified miRNAs have the potential as predictive biomarkers for TKI resistance.
RESUMO
BACKGROUND: Despite the enormous success of imatinib in chronic myeloid leukemia (CML), therapy resistance has emerged in a significant proportion of patients, partly because of the overexpression of ABC efflux transporters. METHODS: Using an array comprising 667 miRNAs, we investigated whether the expression of microRNAs (miRNAs) is altered in CML K-562 cells becoming resistant to increasing concentrations of imatinib. ABCB1 and ABCG2 mRNA (quantitative real-time PCR) and protein expression (western blot) were quantified under short-term and 4 months' imatinib treatment. Interaction of miR-212 and miR-328 with ABCG2 was investigated by transfection experiments and reporter gene assays using respective miRNA precursors or miRNA inhibitors. RESULTS: Although ABCB1 protein was not expressed, ABCG2 protein was 7.2-fold elevated after long-term treatment with 0.3 µmol/l imatinib and decreased gradually at higher concentrations. miRNAs miR-212 and miR-328 were identified to correlate inversely with ABCG2 expression under these conditions. Short-term treatment also induced ABCG2 protein concentration dependently and caused a downregulation of miR-212, but not of miR-328 at all tested concentrations (P=0.050). Reporter gene assays confirmed miR-212 to target the 3'-UTR region of ABCG2. In contrast, transfection of anti-miR-212 revealed an upregulation of ABCG2 protein expression, whereas the effect of anti-miR-328 was weak. CONCLUSION: Our study suggests an association of imatinib treatment, miRNA downregulation and ABCG2 overexpression, possibly contributing to the mechanisms involved in imatinib distribution and response in CML therapy.
