A role for MicroRNAs in Normal Karyotype Acute Myeloid Leukaemia

MicroRNAs are short approximately 22 nucleotide sequences of RNA which fine-tune gene expression by reducing the translation of messenger RNA (mRNA) into protein. They are newly emerging as important regulators of normal cellular functions such as haematopoiesis, and potentially may participate in the dysregulated transcriptional pathways that characterise acute myeloid leukaemia (AML).microRNA expression profiling, using a stringent locked nucleic acid (LNA) platform, was performed on 28 cases of normal karyotype AML (NK-AML). 19 (67%) were NPM1-mutated (NPM1mut-AML). Cluster analysis demonstrated that a five microRNA expression signature distinguished NPM1mut-AML from NPM1 wild type cases, with Let-7B, let-7C and miR-10a over-expressed in NPM1mut-AML, while miR-130a and miR-335 were significantly under-expressed. As distinct from prior reports, miR-10a is the dominantly expressed species in NPM1mut-AML, rather than miR-10b. Similar to NPM1mut primary AML cells, the NPM1mut bearing OCI-AML3 cell line had high miR-10a expression when compared to other malignant cell lines. Target prediction analysis identified almost 1000 genes to be potential miR-10a targets. Reporter analysis of a refined subset of these predicted targets confirmed a number of crucial pathways with genes that are miR-10a suppressible including apoptosis-related BCL2L11 (Bim), myeloid differentiation related KLF4 and the cell cycle protein RB1CC1.To explore for a possible leukaemogenic effect of miR-10a overexpression, a system of LNA knockdown of miR-10a was optimised in NPM1mut bearing OCI-AML3 cells (the only publicly available cell line bearing the NPM1 mutation). Knockdown of miR-10a lead to a decreased cell count after 48 hours. Increased Annexin-V positive events but no change in cell cycling or proliferation were noted at 24 and 48 hours. Furthermore, miR-10a knockdown in OCI-AML3 cells resulted in reduced clonogenic potential, suggesting a possible role for miR-10a in self renewal pathways. Technical challenges related to knockdown of microRNAs in primary AML cells meant that the findings in OCI-AML3 cells were not able to be replicated in an alternative system, though this will be required.In summary, miR-10a is strongly over-expressed in NPM1mut-AML and appears to have a pro-survival role. If further substantiated in other systems, miR-10a knockdown could represent a model for targeted therapy in this common AML subtype