The online version of this article (doi:10.1186/1476-4598-11-8) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
AB carried out qRT-PCR, apoptosis, cell cycle and cell growth assays, microRNA target prediction and validation by luciferace assay, and wrote the manuscript. CAP carried out the clonogenic, BrdU, differentiation assays and contributed to the apoptosis assays and wrote the manuscript. VY performed the bioinformatic microarray analysis. YWY contributed to statistical analysis. ML carried out the microarrays, participated in the design of the study and wrote the manuscript. DM conceived of the study, participated in its design, supervised the experiments and helped to write the manuscript. All authors read and approved the final manuscript.
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.
Additional file 1: Table S1. List of significantly (p < .05) differentially expressed probes on microarray comparison of NK-AML samples versus normal bone marrow. Only those microRNAs with a FC of ≥ 2 in either direction were included in this table. There were 26 overexpressed probes and 11 under expressed probes in NK-AML versus normal BM. The microRNAs depicted in bold represent proprietary miRPlusTM probe whose sequences have subsequently been annotated on miRBase. Those probes denoted as miRPlusTM have not been further annotated by the release of miRBase release 15. (PDF 81 KB)12943_2011_991_MOESM1_ESM.PDF
Additional file 2: 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. (PDF 97 KB)12943_2011_991_MOESM2_ESM.PDF
Additional file 3: 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. (PDF 685 KB)12943_2011_991_MOESM3_ESM.PDF
Additional file 4: Table S2. Patient demographics and AML blast characteristics. (PDF 95 KB)12943_2011_991_MOESM4_ESM.PDF
Authors’ original file for figure 112943_2011_991_MOESM5_ESM.tiff
Authors’ original file for figure 212943_2011_991_MOESM6_ESM.pdf
Dohner H, Estey EH, Amadori S, Appelbaum FR, Buchner T, Burnett AK, Dombret H, Fenaux P, Grimwade D, Larson RA: Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010, 115: 453-474. 10.1182/blood-2009-07-235358 CrossRefPubMed
Palma CA, Tonna EJ, Ma DF, Lutherborrow M: MicroRNA control of myelopoiesis and the differentiation block in Acute Myeloid Leukaemia. J Cell Mol Med. 2012, [Epub ahead of print],
Dickstein J, Senyuk V, Premanand K, Laricchia-Robbio L, Xu P, Cattaneo F, Fazzina R, Nucifora G: Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome. Proc Natl Acad Sci USA. 2010, 107: 9783-9788. 10.1073/pnas.1004297107 PubMedCentralCrossRefPubMed
Pulikkan JA, Dengler V, Peramangalam PS, Peer Zada AA, Muller-Tidow C, Bohlander SK, Tenen DG, Behre G: Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia. Blood. 2010, 115: 1768-1778. 10.1182/blood-2009-08-240101 PubMedCentralCrossRefPubMed
Klein U, Lia M, Crespo M, Siegel R, Shen Q, Mo T, Ambesi-Impiombato A, Califano A, Migliazza A, Bhagat G, Dalla-Favera R: The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell. 2010, 17: 28-40. 10.1016/j.ccr.2009.11.019 CrossRefPubMed
Garzon R, Liu S, Fabbri M, Liu Z, Heaphy CE, Callegari E, Schwind S, Pang J, Yu J, Muthusamy N: MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1. Blood. 2009, 113: 6411-6418. 10.1182/blood-2008-07-170589 PubMedCentralCrossRefPubMed
Alcalay M, Tiacci E, Bergomas R, Bigerna B, Venturini E, Minardi SP, Meani N, Diverio D, Bernard L, Tizzoni L: Acute myeloid leukemia bearing cytoplasmic nucleophosmin (NPMc+ AML) shows a distinct gene expression profile characterized by up-regulation of genes involved in stem-cell maintenance. Blood. 2005, 106: 899-902. 10.1182/blood-2005-02-0560 CrossRefPubMed
Mullighan CG, Kennedy A, Zhou X, Radtke I, Phillips LA, Shurtleff SA, Downing JR: Pediatric acute myeloid leukemia with NPM1 mutations is characterized by a gene expression profile with dysregulated HOX gene expression distinct from MLL-rearranged leukemias. Leukemia. 2007, 21: 2000-2009. 10.1038/sj.leu.2404808 CrossRefPubMed
Verhaak RG, Goudswaard CS, van Putten W, Bijl MA, Sanders MA, Hugens W, Uitterlinden AG, Erpelinck CA, Delwel R, Lowenberg B, Valk PJ: Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance. Blood. 2005, 106: 3747-3754. 10.1182/blood-2005-05-2168 CrossRefPubMed
Silber J, Lim DA, Petritsch C, Persson AI, Maunakea AK, Yu M, Vandenberg SR, Ginzinger DG, James CD, Costello JF: miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med. 2008, 6: 14- 10.1186/1741-7015-6-14 PubMedCentralCrossRefPubMed
Veerla S, Lindgren D, Kvist A, Frigyesi A, Staaf J, Persson H, Liedberg F, Chebil G, Gudjonsson S, Borg A: MiRNA expression in urothelial carcinomas: important roles of miR-10a, miR-222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, and frequent homozygous losses of miR-31. Int J Cancer. 2009, 124: 2236-2242. 10.1002/ijc.24183 CrossRefPubMed
Agirre X, Jimenez-Velasco A, San Jose-Eneriz E, Garate L, Bandres E, Cordeu L, Aparicio O, Saez B, Navarro G, Vilas-Zornoza A: Down-regulation of hsa-miR-10a in chronic myeloid leukemia CD34+ cells increases USF2-mediated cell growth. Mol Cancer Res. 2008, 6: 1830-1840. 10.1158/1541-7786.MCR-08-0167 CrossRefPubMed
Debernardi S, Skoulakis S, Molloy G, Chaplin T, Dixon-McIver A, Young BD: MicroRNA miR-181a correlates with morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis. Leukemia. 2007, 21: 912-916. PubMed
Debernardi S, Lillington DM, Chaplin T, Tomlinson S, Amess J, Rohatiner A, Lister TA, Young BD: Genome-wide analysis of acute myeloid leukemia with normal karyotype reveals a unique pattern of homeobox gene expression distinct from those with translocation-mediated fusion events. Genes Chromosomes Cancer. 2003, 37: 149-158. 10.1002/gcc.10198 CrossRefPubMed
Cammarata G, Augugliaro L, Salemi D, Agueli C, La Rosa M, Dagnino L, Civiletto G, Messana F, Marfia A, Bica MG: Differential expression of specific microRNA and their targets in acute myeloid leukemia. Am J Hematol. 2010, 85: 331-339. PubMed
Garzon R, Garofalo M, Martelli MP, Briesewitz R, Wang L, Fernandez-Cymering C, Volinia S, Liu CG, Schnittger S, Haferlach T: Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin. Proc Natl Acad Sci USA. 2008, 105: 3945-3950. 10.1073/pnas.0800135105 PubMedCentralCrossRefPubMed
Ovcharenko D, Stolzel F, Poitz D, Fierro F, Schaich M, Neubauer A, Kelnar K, Davison T, Muller-Tidow C, Thiede C: miR-10a overexpression is associated with NPM1 mutations and MDM4 downregulation in intermediate-risk acute myeloid leukemia. Exp Hematol. 2011, 39 (1030-1042): e1037-
Mansfield JH, Harfe BD, Nissen R, Obenauer J, Srineel J, Chaudhuri A, Farzan-Kashani R, Zuker M, Pasquinelli AE, Ruvkun G: MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat Genet. 2004, 36: 1079-1083. 10.1038/ng1421 CrossRefPubMed
Han L, Witmer PD, Casey E, Valle D, Sukumar S: DNA methylation regulates MicroRNA expression. Cancer Biol Ther. 2007, 6: 1284-1288. PubMed
Bullinger L, Kronke J, Schon C, Radtke I, Urlbauer K, Botzenhardt U, Gaidzik V, Cario A, Senger C, Schlenk RF: Identification of acquired copy number alterations and uniparental disomies in cytogenetically normal acute myeloid leukemia using high-resolution single-nucleotide polymorphism analysis. Leukemia. 2010, 24: 438-449. 10.1038/leu.2009.263 CrossRefPubMed
Smyth G: Limma Linear Models for microarray data. Bioinformatics and Computational Biology Solutions using R and Bioconductor. Edited by: Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W. 2005, New York: Springer,
Huang DW, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009, 4: 44-57. CrossRef
- miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death
Catalina A Palma
Yee Wa Yang
David DF Ma
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II