MiRNA-29b Targets MCL-1 and Is Down-Regulated in Chemotherapy-Resistant Acute Myeloid Leukemia (AML). (2007)

Abstract AML is a heterogeneous disorder that includes several differently biologic entities. Patients with intermediate and poor-risk cytogenetics represent the majority of AML and chemotherapy-based regimens fail to cure most of these patients that are either resistant to or relapse shortly after treatment. Therefore, it is crucial to understand the biology of chemotherapy resistance in AML. Here, we proposed to investigate the relation between chemoresistance and miRNA expression in AML. First, using a miRNA microarray platform, we analyzed the miRNA profiles in pretreatment samples from a group of 24 patients with newly diagnosed AML treated with idarubicin 12mg/m2/day (days 1–3) and cytarabine 1500 mg/m2/d (days 1–4). After background subtraction and normalization to a set of housekeeping genes, Significance Analysis of Microarrays (SAM) was performed. Among others miRNAs, miR-29b was found significantly down-regulated in pts with resistant disease (n=13) compared to those who achieved CR (n=11)(SAM score −2.92). To validate these results, we measured miR-29b levels by quantitative(q)RT-PCR in an independent set of 36 AML patients treated with the same chemotherapy regimen as described above and with no statistical differences in baseline pretreatment characteristics. We found that miR-29b was down-regulated in pts with resistant disease compared with pts who achieved complete remission (p=0.015, t-test). Interestingly miR-29b is predicted to target MCL1, an oncogene whose over-expression has been associated with resistance to chemotherapy. An inverse correlation was indeed observed between MCL1 protein levels measured by immunoblotting and miR-29b expression measured by miRNA microarray in AML patients (n=9). Next, we inserted the 3′ untranslated region (3′UTR) of the MCL1 gene containing the putative interaction sites of miR-29b into a luciferase reporter. When the miR-29b precursor oligonucleotide was transfected into MEG-O1 cells along with the MCL1 reporter construct, a 70% reduction of luciferase activity was observed. In contrast, no changes in the luciferase activity was observed when co-transfection was performed with controls inckluding a construct containing a mutated 3′UTR sequence of MCL1. To validate MCL1 as a target of miR-29b, a synthetic miR-29b oligonucleotide was transfected by nucleoporation into K562 cells, which overexpress MCL1 and lack endogenous miR-29b. This resulted in a marked reduction of MCL1 protein levels compared with controls. Finally, to explore the functional impact of restoring miR-29b and downregulating MCL1 in AML cells, we measure the apoptosis rates in K562 cells transfected with miR-29b or scrambled oligonucleotides by annexin V staining. A 3-fold increase in apoptosis was found in miR-29b transfected cells compared with scrambled oligonucleotides-treated controls. Consistent with these results, miR-29b-treated K562 cells exhibited increased chemosensitivity to ARA-C compared with scrambled oligonucleotides-treated cells. In conclusion, we demonstrate here that miR-29b down-regulation is associated with chemotherapy resistance, which might occur as the results of MCL1 overexpression. Restoring miR-29b expression levels down modulates MCL1, induces apoptosis and enhances chemosensitivity in AML cells, thereby suggesting miRNA-based therapy as a novel pro-apoptotic approach to increase response in high-risk AML.