miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death

Abstract

Acute myeloid leukaemia (AML) with nucleophosmin-1 (NPM1) mutation is a major subtype of AML. The NPM1 mutation induces a myeloproliferative disorder, but evidence indicates that other insults are necessary for the development of AML. We utilised microRNA microarrays and functional assays to determine if microRNA dysregulation could be involved in the pathogenesis of in NPM1 mutated (NPM1 mut )-AML. We used a stringent locked nucleic acid (LNA) based microRNA microarray platform to profile bone marrow samples of patients with normal karyotype AML. A panel of five microRNAs dichotomised AML patients according to their NPM1 mutational status. miR-10a, let-7b and let-7c were significantly over-expressed, while miR-130a and miR-335 were under-expressed in NPM1 mut -AML when compared to NPM1 wildtype -AML. Of these, miR-10a is the most differentially expressed in NPM1 mut -AML versus NPM1 wildtype -AML (> 10 fold higher as confirmed by qRT-PCR). To investigate the functions of miR-10a, the OCI-AML3 cell line was utilised, which is the only commercially available cell line bearing NPM1 mut . OCI-AML3 cells were firstly demonstrated to have a similarly high miR-10a expression to primary NPM1 mut -AML patient samples. Inhibition of miR-10a expression by miRCURY LNA Inhibitors (Exiqon) in these cells resulted in increased cell death as assessed by MTS, cell cycle and Annexin-V assays and reduced clonogenic capacity, indicative of an involvement in leukaemic cell survival. In silico filtering of bioinformatically predicted targets of miR-10a identified a number of potential mRNA targets with annotated functions in haematopoiesis, cell growth and apoptosis. Lucferase reporter assays confirmed a number of these putative tumorogenic genes that are miR-10a suppressible including KLF4 and RB1CC1. This provides a potential mechanism for the pathogenic role of miR-10a in NPM1 mut -AML. This study provides, for the first time, in vitro evidence of a pro-survival role of miR-10a in NPM1 mut -AML, that it may contribute to the pathogenesis of NPM1 mut -AML and identifies putative tumorogenic targets.