Impact of microRNA-130a on the neutrophil proteome

Pedersen et al. BMC Immunology (2015) 16:70 DOI 10.1186/s12865-015-0134-8 RESEARCH ARTICLE Open Access Impact of microRNA-130a on the neutrophil proteome Corinna Cavan Pedersen1*, Jan Christian Refsgaard2, Ole Østergaard3, Lars Juhl Jensen2, Niels Henrik Helweg Heegaard3,4, Niels Borregaard1 and Jack Bernard Cowland1* Abstract Background: MicroRNAs (miRNAs) are important for the development and function of neutrophils. miR-130a is highly expressed during early neutrophil development and regulates target proteins important for this process. miRNA targets are often identified by validating putative targets found by in silico prediction algorithms one at a time. However, one miRNA can have many different targets, which may vary depending on the context. Here, we investigated the effect of miR-130a on the proteome of a murine and a human myeloid cell line. Results: Using pulsed stable isotope labelling of amino acids in cell culture and mass spectrometry for protein identification and quantitation, we found 44 and 34 proteins that were significantly regulated following inhibition of miR-130a in a miR-130a-overexpressing 32Dcl3 clone and Kasumi-1 cells, respectively. The level of miR-130a inhibition correlated with the impact on protein levels. We used RAIN, a novel database for miRNA–protein and protein–protein interactions, to identify putative miR-130a targets. In the 32Dcl3 clone, putative targets were more up-regulated than the remaining quantified proteins following miR-130a inhibition, and three significantly derepressed proteins (NFYC, ISOC1, and CAT) are putative miR-130a targets with good RAIN scores. We also created a network including inferred, putative neutrophil miR-130a targets and identified the transcription factors Myb and CBF-β as putative miR-130a targets, which may regulate the primary granule proteins MPO and PRTN3 and other proteins differentially expressed following miR-130a inhibition in the 32Dcl3 clone. Conclusion: We have experimentally identified miR-130a-regulated proteins within the neutrophil proteome. Linking these to putative miR-130a targets, we provide an association network of potential direct and indirect miR-130a targets that expands our knowledge on the role of miR-130a in neutrophil development and is a valuable platform for further experimental studies. Keywords: miR-130a, Neutrophils, pSILAC, Quantitative proteomics, RAIN, miRNA target network Background Neutrophils are the most abundant leukocytes in human blood. They are essential for innate immunity and are the first cells to be recruited for defence against invading microorganisms at sites of infection [1]. Neutrophils are continuously produced in the bone marrow through a tightly regulated process termed granulopoiesis. Different types of granules loaded with microbicidal proteins appear at different stages of neutrophil differentiation, * Correspondence: ; jack.cowland@ regionh.dk 1 The Granulocyte Research Laboratory, Department of Hematology, National University Hospital, University of Copenhagen, 9322, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark Full list of author information is available at the end of the article which is defined by six morphologically distinguishable cellular stages of maturation. The first morphologically identified precursor committed to the neutrophil lineage is the myeloblast. Myeloblasts mature into promyelocytes, in which formation of primary granules containing myeloperoxidase (MPO) takes place, and further to myelocytes where secondary granules are formed. Proliferation ceases as differentiation proceeds via the metamyelocyte, the band cell, and ultimately the mature segmented neutrophil, which is the cell released to blood. Granulopoiesis is strictly regulated by transcription factors including RUNX1, C/EBPα, and C/EBPε [2–4]. MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression on a posttranscriptional level by binding to target mRNAs, thereby © 2015 Pedersen et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pedersen et al. BMC Immunology (2015) 16:70 altering translation of the transcripts or their stability. Target recognition is mediated through base-pairing of the miRNA with its target mRNA, usually between a 6–7 nucleotide seed sequence situated within residues 2–8 of the miRNA and a miRNA recognition element (MRE) in the 3′ untranslated region (3′ UTR) of the mRNA [5, 6]. The resulting changes in protein expression affect widespread cellular processes including development, differentiation, proliferation, survival, and immune responses [7, 8]. Alterations in miRNA levels have been associated with numerous diseases and are implicated in the initiation and progression of human cancers including leukemias [8]. We recently demonstrated that miRNAs are regulators of proteins essential for granulopoiesis [9–11]. We showed that miR-130a suppresses expression of Smad4 and thereby reduces sensitivity to TGF-β1-induced growth inhibition [9]. miR-130a also represses appropriate cell cycle exit and secondary granule protein expression by targeting C/EBPε in neutrophil precursors [9, 11]. These two targets for miR130a were suggested by in silico prediction algorithms based on conservation across species, sequence complementarity, and other miRNA–mRNA binding properties [12, 13]. This is an often-used approach to identify putative targets of miRNAs. However, many predicted targets are false positives, and several factors that may influence the effect of the miRNA on the mRNA are not taken into consideration [14]. These include tissue specificity, expression levels of both RNA molecules, and the surrounding regulatory mechanisms particular to the cell of interest [15, 16]. More direct methods used for finding miRNA targets include identification of changes in mRNA transcript levels induced by altered miRNA expression and immunoprecipitation of miRNA–mRNA complexes [17–21]. However, these methods do not account for the actual reduction in protein levels seen in a particular cell type in response to changes in expression of an endogenous miRNA. Protein turnover times vary, and repression of gene expression by miRNAs can occur solely through translational inhibition, altering only the amount of protein being produced and not the amount of mRNA [9]. Immunoblotting is frequently used to determine the effect of miRNA-mediated reduction on synthesis of selected proteins. As an alt (...truncated)