Skip to main content
main-content

01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

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

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Adam Bryant, Catalina A Palma, Vivek Jayaswal, Yee Wa Yang, Mark Lutherborrow, David DF Ma
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-8) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

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.

Abstract

Background

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.

Results

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.

Conclusions

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.
Zusatzmaterial
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 1
12943_2011_991_MOESM5_ESM.tiff
Authors’ original file for figure 2
12943_2011_991_MOESM6_ESM.pdf
Literatur
Über diesen Artikel

Weitere Artikel der Ausgabe 1/2012

Molecular Cancer 1/2012 Zur Ausgabe

Neu im Fachgebiet Onkologie

Mail Icon II Newsletter

Bestellen Sie unseren kostenlosen Newsletter Update Onkologie und bleiben Sie gut informiert – ganz bequem per eMail.

Bildnachweise