Data: Additional file 2[Show abstract] ABSTRACT: Figure S1. Confirmation of miR-10a knockdown by anti-10a LNA. A. miR-10a expression 24 h after transfection with Exiqon (LNA) anti-microRNA ASO was assayed by TaqMan microRNA qRT-PCR. RNU6b was used as the reference gene. MiR-10a expression was normalised to that of cells transfected with 100 nM of the non-targeting control specific to each chemistry, with comparison made to untransfected cells The graph depicts the mean miR-10a relative expression of individual experiments for Ambion Anti-miRs (n = 4) and Exiqon LNAs (n = 3), +/-SEM (of fold change values). B. Hela cells were pre-treated with either pcDNA.10a (miR-10a overexpressing plasmid), 30 nM of Pre-miR-10a or were not pre-treated. After 6 h, transfection media was removed and cells washed with PBS. Cells were then transfected with pMIR.HOXA1/pRLCMV and 50 nM of either anti-10a LNA or LNA Control A. After 24 h, Dual Luciferase Assay (Promega) was performed in triplicate. The whole experiment was repeated 5 times. The graph depicts the mean luciferase values for anti-10a LNA treated cells compared to the LNA Control A treated cells (+/-SEM), with the control values corrected to 100. The statistical analysis consists of Student's t-test (paired). NS: not significant; * 0.01 < p < 0.05; ** 0.001 < p < 0.01.
Data: Additional file 3[Show abstract] ABSTRACT: Figure S2. Monocytic differentiation of OCI-AML3 cells is not affected by miR-10a knockdown. 1,25-dihydroxyvitaminD3 (VitD3) was used to induce monocytic differentiation of OCI-AML3 cells over a 96 h period. A. Morphological analysis (Wright Stain) of cytospin samples of OCI-AML3 cells treated with VitD3 demonstrated no observable differences between SCRAM control transfected and anti-miR10a LNA transfected groups. B. Phenotype analysis of CD14 C. CD15 and D. CD11b expression by flow cytometry did not detect a statistically significant difference between SCRAM control or anti-miR10a LNA groups at 48 h or 96 h post, regardless if cells were treated with VitD3 (+) or did not receive treatment (-). N = 3.
Data: Additional file 4[Show abstract] ABSTRACT: Table S2. Patient demographics and AML blast characteristics.
- [Show abstract] 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 (NPM1mut)-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 NPM1mut-AML when compared to NPM1wildtype-AML. Of these, miR-10a is the most differentially expressed in NPM1mut-AML versus NPM1wildtype-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 NPM1mut. OCI-AML3 cells were firstly demonstrated to have a similarly high miR-10a expression to primary NPM1mut-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 NPM1mut-AML. This study provides, for the first time, in vitro evidence of a pro-survival role of miR-10a in NPM1mut-AML, that it may contribute to the pathogenesis of NPM1mut-AML and identifies putative tumorogenic targets.