MiR-424 and miR-155 deregulated expression in cytogenetically normal acute myeloid leukaemia: correlation with NPM1 and FLT3 mutation status

Faraoni et al. Journal of Hematology & Oncology 2012, 5:26 http://www.jhoonline.org/content/5/1/26 JOURNAL OF HEMATOLOGY & ONCOLOGY RAPID COMMUNICATION Open Access MiR-424 and miR-155 deregulated expression in cytogenetically normal acute myeloid leukaemia: correlation with NPM1 and FLT3 mutation status Isabella Faraoni1,2*, Serena Laterza1,3, Davide Ardiri1,3, Claudia Ciardi1,3, Francesco Fazi4 and Francesco Lo-Coco1,3 Abstract Background: MicroRNA have a central role in normal haematopoiesis and are deregulated in acute myeloid leukaemia (AML). The purpose of the study was to investigate by qRT-PCR the expression of miRNAs involved in myeloid differentiation (miR-424, miR-155, miR-223, miR-17-5p) in 48 patients with cytogenetically normal AML well characterized for NPM1 and/or FLT3 mutations. Three types of normalization were used for the data validation. Findings: We found that miR-424 was down-modulated in AMLs with NPM1mutA regardless of FLT3 status. On the contrary, miR-155 showed up-regulation in patients with FLT3 internal tandem duplications (ITD) with or without NPM1 mutations. No significant associations were found by analyzing miR-223 and miR-17-5p in relation to FLT3 and NPM1 status. Conclusions: This study supports the view that major genetic subsets of CN-AML are associated with distinct miRNA signatures and suggests that miR-424 and miR-155 deregulation is involved in the pathogenesis of CN-AML with NPM1 and FLT3-ITD mutations, respectively. Keywords: Cytogenetically normal AML, MiR-424, MiR-155, NPM1, FLT3-ITD Introduction Acute myeloid leukaemia (AML) is a heterogeneous disease with recurrent cytogenetic alterations detected in approximately 55% of patients, while no karyotypically visible lesions are detectable in the remaining 45% of cases. This latter subset, otherwise referred to as cytogenetic normal AML (CN-AML), is characterized by a variety of subtle mutations affecting several genes. Of these, nucleophosmin (NPM1) alterations account for up to 60% of CN-AMLs and fms-related tyrosine kinase 3 (FLT3) lesions are detected in almost 30% of patients [1,2]. Abnormalities in these genes are not mutually exclusive as they may partially overlap such that four main categories may be identified, i.e. FLT3wt/NPM1wt, FLT3wt/NPM1+, FLT3+/NPM1wt, FLT3+/NPM1 + . MicroRNAs (miRNAs) are endogenous single-stranded non-coding RNA molecules of 19–24 nucleotides that * Correspondence: 1 Laboratory of Neuro-Oncohematology, Santa Lucia Foundation, Via del Fosso di Fiorano, 64, Rome, Italy 2 Department of Neuroscience, University of Rome “Tor Vergata”, Rome, Italy Full list of author information is available at the end of the article control gene expression mainly at the post-transcriptional level by binding the 3’untraslated region (UTR) of messenger RNAs to regulate their stability and translation. MiRNAs have emerged as key regulators of normal haematopoiesis and profiling studies have shown altered miRNA expression in leukaemias including AML suggesting their role in leukaemogenesis [3]. However, among the largescale miRNA profiling studies on AML only few miRNAs were commonly deregulated. Differences in the reported signatures can be attributed to the analysis of distinct cytogenetic and molecular subgroups and to the type of used controls [3,4]. In the present study we focused on CN-AML subsets well characterized for NPM1 and FLT3 status and restricted our analysis to 4 miRNAs known to be involved in normal granulocytic and/or monocytic differentiation (miR-424, miR-155, miR-223, miR-17-5p) [5]. A number of normal controls (CD34+ progenitors, mature granulocytes and monocytes) were also analyzed in order to elucidate whether the expression patterns of the above miRNAs are associated to myeloid differentiation. We found that deregulated expression of miR-424 and © 2012 Faraoni et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Faraoni et al. Journal of Hematology & Oncology 2012, 5:26 http://www.jhoonline.org/content/5/1/26 miR-155 varies significantly according to NPM1mutA and FLT3-ITD mutational status. Design and methods Fresh primary blast cells were obtained from bone marrow (BM) aspirates of adult patients with newly diagnosed AML admitted at the Department of Biopathology of Tor Vergata University, Rome. All patients provided written informed consent in accordance with the Declaration of Helsinki. BM aspirates with less of 70% of blast infiltration at morphological analysis were discarded. Samples were further enriched for mononuclear cells by Lympholyte Cell Separation Media (Cederlane). FLT3 and NPM1 mutational status was investigated by a multiplex PCR strategy developed in our laboratory and described elsewhere [6]. Samples with FLT3-TKD and nonA type NPM1 mutations were excluded from the study. In order to decrease the level of heterogeneity, only type A mutations for NPM1 and internal tandem duplications (ITD) mutations for FLT3 were included (herein referred to as NPM1+ and FLT3+, respectively). A total of 48 patient samples were selected to identified 4 groups of 12 cases each for the following subsets: FLT3wt/NPM1wt, FLT3wt/NPM1+, FLT3+/NPM1wt, FLT3+/NPM1 + . Mononuclear cells from BM of healthy control subjects were purified by Lympholyte. CD34+ cells were obtained from cord blood samples and purified by positive selection using MACS immunomagnetic separation system (Miltenyi Biotec). Mature cells were purified from whole peripheral blood of healthy subjects. Granulocytes were recovered and purified by Percoll whereas cells retrieved from the Lympholyte ring were plated and monocytes separated by plastic adherence. The purity of granulocytic (90-95% CD15+, CD16+) and monocytic (78-85% CD14+, CD16-) cell fractions was assessed by flow cytometry. Total RNA was isolated from fresh cells using Trizol reagent (Invitrogen). All RNA samples were checked for RNA quality by gel electrophoresis. Quantitative realtime PCR (qRT-PCR) of miRNAs was carried out using TaqMan MicroRNA Reverse Transcription Kit and the Taqman MicroRNA primer/probe Assays (Applied Biosystems). Reverse Transcription (RT) reactions were performed using 10 ng of total RNA as detected with NanoDrop ND-1000 spectrophotometer. qRT-PCR reactions, were performed on ABI 7900 HT Sequence Detection System (SDS; Applied Biosystems) and performed in triplicate. The 2-ΔΔCt relative quantification method was used to calculate relative miRNA expression. A small nuclear RNAs (RNU6B) and a small nucleolar (RNU54) commonly employed in miRNA studies were used for internal normalization. The mean value of a Page 2 of 5 normal BM RNA was used as a calibrator in all plates and for all miRNAs. Group wise comparisons of the distributio (...truncated)