MiR-106b-25 Promotes Chemoresistance in Acute Myeloid Leukemia Via Abolishing Multiple Apoptotic Pathways

Introduction: Chemoresistance and disease relapse remain the main obstacles responsible for treatment failure in leukemia. MicroRNAs (miRNAs) play essential roles in various physiological and pathological processes, including cell proliferation, differentiation, metabolism, and cancer development. The miR-106b-25 cluster consists of three miRNAs: miR-106b, miR-93 and miR-25. We have previously reported that miR-106b-25 was associated with chemoresistance by negatively regulated EP300 in breast cancer, but its role in hematological malignancies has not yet been elucidated. Here, we aim to clarify the biological role and underlying mechanisms of miR-106b-25 on drug resistance in leukemia.

Methods: To see whether the miR-106b-25 was associated with the poor prognosis of AML patients, enriched LSCs (CD34 ⁺ cells) were isolated from the bone marrow of 18 newly diagnosed AML patients, the expression of miR-106b, miR-93, and miR-25 were examined, respectively. The expression levels of miR-106b, miR-93 and miR-25 were further determined in the doxorubicin-resistant leukemia cell line K562/A02 and HL60/ADR, compared with their parental cell lines. In addition, K562 cells were transduced with lentiviral vectors carrying miR-106b-25, and cell proliferation, drug resistance, colony-forming assay, apoptosis assays were performed to explore the function of miR-106b-25 overexpression in leukemia cells in vitro. To investigate the role of miR-106b-25 on tumor growth and overall survival after drug treatment, we performed xenotransplantation in nude mice using miR-106b-25 overexpressed K562 cells. To further clarify the function of each microRNA function in this cluster, K562 cells were also transduced with lentiviral vectors carrying individual miR-25, miR-93, or miR-106b separately. Cell proliferation, colony forming assay and cell apoptosis assay were also carried out subsequently. Simultaneously, RNA-sequencing was performed to reveal the underlying mechanisms of miR-106b-25 in the chemoresistance of myeloid leukemia. To experimentally confirm the direct target of the miR-106b-25 cluster in AMLs, we further performed a dual-luciferase reporter assay.

Results: Upregulated miR-106b, miR-93 and miR-25 expression in enriched LSCs were significantly associated with shortened overall survival of AML patients. We also found miR-106b, miR-93 and miR-25 were significantly upregulated in drug-resistant leukemia cell lines compared with its parental cell lines. Overexpression of miR-106b-25 cluster promoted cell proliferation, led to resistance of K562 cells to doxorubicin, imatinib and ABT-737 (BCL-2 inhibitor) in liquid culture and drug-resistant colony-forming assays. Overexpression of miR-93 or miR-106b accelerated cell growth, and all the three miRNAs can promote drug-resistant colony-forming and inhibit cell apoptosis. RNA-sequencing (RNA-Seq) data revealed that multiple critical genes related to apoptotic pathways were downregulated after overexpressing miR-25, miR-93, miR-106b as well as the whole cluster, such as TP73, BAX, BAK1, Caspase-7, CDKN1A and BTG2. RT-qPCR confirmed that these genes are reduced with or without ABT-737 treatment. Luciferase assay further identified TP73 was a direct target of miR-93 and miR106b, BAK1 was a direct target of miR-25, and CASPASE-7 was a direct target of all these three miRNAs.

Conclusions: In summary, we made the novel observation that miR-106-25 is associated with AML drug-resisitance and disease prognosis and identified TP73, BAK1 caspase-7 as a novel direct target of this cluster. Further studies revealed that the biological effects of miR-106b-25 cluster on leukemic cell proliferation, chemoresistance and apoptosis were mediated through regulation of apoptotic pathway. These findings indicate a promising diagnostic biomarker and a potential target therapeutic strategy for AML patients.