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Mir-19A-3P and Mir-125B-5P 5-Lipoxygenase Is a Direct Target Of 5-Lipoxygenase Is a Direct Target of miR-19a-3p and miR-125b-5p Saskia Busch, Eileen Auth, Friederike Scholl, Sabine Huenecke, Ulrike Koehl, Beatrix Suess and Dieter This information is current as Steinhilber of October 2, 2021. J Immunol published online 14 January 2015 http://www.jimmunol.org/content/early/2015/01/14/jimmun ol.1402163 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/01/14/jimmunol.140216 Material 3.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 14, 2015, doi:10.4049/jimmunol.1402163 The Journal of Immunology 5-Lipoxygenase Is a Direct Target of miR-19a-3p and miR-125b-5p Saskia Busch,* Eileen Auth,† Friederike Scholl,* Sabine Huenecke,† Ulrike Koehl,‡ Beatrix Suess,x and Dieter Steinhilber* 5-Lipoxygenase (5-LO) is the key enzyme in leukotriene biosynthesis. Leukotrienes are mediators of the innate immune system and inflammatory processes, and they might also be involved in cancer development. MicroRNAs (miRNAs) are important translational regulators and have been shown to be involved in development, differentiation, and cancer. Unraveling the miRNA network is im- portant for understanding the cellular regulation processes. We identified two new miRNAs, miR-19a-3p and miR-125b-5p, regu- lating 5-LO and confirmed direct interaction by reporter gene assays. Furthermore, we investigated the regulation of 5-LO by these two miRNAs in several cell types. Inhibition of both miRNAs by antagomirs during differentiation of the myeloid cell line Mono Mac 6 led to a significant increase in 5-LO protein expression. Stimulation of human T lymphocytes with PHA resulted in a strong down- Downloaded from regulation of 5-LO mRNA expression and in the induction of miR-19a-3p. The inhibition of miR-19a-3p with an antagomir led to a significant increase in 5-LO mRNA expression in T lymphocytes. Taken together, our data reveal that miR-19a-3p and miR-125b-5p target 5-LO in a cell type– and stimulus-specific manner. The Journal of Immunology, 2015, 194: 000–000. eukotrienes are important mediators for inflammatory, al- transcribed by RNA polymerase II and metabolized in the nucleus lergic, and immunologic reactions. 5-Lipoxygenase (5-LO) by Drosha to pre-miRNA with a length of ∼70 nt. The pre- L is the key enzyme in leukotriene biosynthesis, catalyzing miRNAs, once in the cytoplasm, are further processed by Dicer http://www.jimmunol.org/ the conversion of arachidonic acid to leukotriene A4 (1). Depending to ∼21 nt short, double-stranded miRNA. Only one strand of the on the cell type, leukotriene A4 is either metabolized to leukotriene miRNA duplex, the guide strand, is then incorporated into the B4 or the cysteinyl leukotrienes (leukotrienes C4,D4,andE4). RNA-induced silencing complex and directs this complex to the 39 Leukocytes are the major producers of leukotrienes, leading to in- untranslated region (39UTR) of its target genes. duction and recruitment of macrophages and monocytes as well as Even though regulation of gene expression by miRNAs has been dendritic cell maturation and migration from the peripheral site of studied intensively in recent years, not much is known about the action to the lymphoid nodes (2). As part of the adaptive immune regulation of the 5-LO pathway by miRNAs. In 2010, miR-135a and response, leukotriene B4 is a chemoattractant for T cells (3, 4). miR-199a-5p were the first two miRNAs identified to target the 5-LO by guest on October 2, 2021 Moreover, 5-LO is involved in the regulation of Ig production in activating protein (10). miR-219-2 was the first and only miRNA B cells (5). The expression of the 5-LO can mainly be found in the identified so far as a regulator of 5-LO in macrophages (11). myeloid lineage of the hematopoietic cells (monocytes, macro- Recently, it was shown that inhibition of the leukotriene receptors phages, granulocytes, neutrophils, mast cells, and dendritic cells) as cysteinyl leukotriene 1 or leukotriene B4 receptor-1 (BLT1) receptor or well as B lymphocytes (6, 7), where the expression and activity a knockdown of the BLT1 receptor led to a downregulation of several depend on stimulation and differentiation status of the cell (8). miRNAs (12). Among them were miR-19a-3p and miR-125b-5p. MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs. Overexpression of miR-125b-5p in the myeloid cell line HL-60 pre- miRNAs can induce mRNA degradation or translational repression vented their differentiation into macrophages or granulocytes by ei- of their target genes (9). Their precursors, the pri-miRNAs, are ther DMSO or GM-CSF (13). Alternatively, it could be shown that high expression of 5-LO requires cell differentiation, for example, by DMSO, GM-CSF, or TGF-b (14–17), which provides a possible link *Institute of Pharmaceutical Chemistry, Goethe University, 60438 Frankfurt/Main, Germany; †Laboratory for Stem Cell Transplantation and Immunotherapy, Pediatric between miR-125 and the 5-LO pathway. Mir-19a-3p is a member of Hematology and Oncology, University Hospital Frankfurt, 60596 Frankfurt, Germany; the miR-17∼92 cluster, which has been shown to be expressed in ‡Integrated Research and Treatment Center Transplantation, Institute of Cellular Ther- apeutics, Hannover Medical School, 30625 Hannover, Germany; and xDepartment of B cells (18). Within B cell development, the 5-LO is downregulated Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany in germinal center cells (8, 19), whereas the miR-17∼92 cluster as well Received for publication August 22, 2014. Accepted for publication December 10, as miR-125b-5p are upregulated (20–23). Furthermore, it has been 2014. shown that the miR-17∼92 cluster regulates T cell proliferation (24, This work was supported by Merck, Deutsche Forschungsgemeinschaft Grant GRK 25) and that miR-19a-3p plays a role in promoting Th1 responses (26). 1172, and by the LOEWE Center for Cell and Gene Therapy. All of these data suggest a possible role of miR-19a-3p and miR- Address correspondence and reprint requests to Prof. Dieter Steinhilber or Prof. 125b-5p in the regulation of leukocyte functions and immune Beatrix Suess, Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue- Strasse 9, 60438 Frankfurt/Main, Germany (D.S.) or Department of Biology, Technical responses by regulation of the 5-LO pathway. In this study, we show University of Darmstadt, 64287 Darmstadt, Germany (B.S.). E-mail addresses: that miR-19a-3p and miR-125b-5p directly regulate 5-LO ex- [email protected] (D.S.) or [email protected] (B.S.) pression in myeloid cells and in T lymphocytes. The online version of this article contains supplemental material. Abbreviations used in this article: BLT1, leukotriene B4 receptor-1; 5-LO, 5-lipoxy- Materials and Methods genase; miRNA, microRNA; MM6, Mono Mac 6; SAC, Staphylococcus aureus Cell lines Cowan strain protein A; 39UTR, 39 untranslated region. Cos-7 cells were cultivated in DMEM medium (Invitrogen) supplemented Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 with 10% FCS (Biochrom), 100 mg/ml streptomycin, and 100 U/ml www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402163 2 CONTROL OF 5-LIPOXYGENASE BY miRNAs penicillin (Invitrogen). The cells were detached using trypsin-EDTA For cDNA synthesis the high-capacity RNA-to-cDNA kit (Invitrogen) (Invitrogen) and split at a ratio of 5:1 twice a week. Mono Mac 6 was used according to the manufacturer’s protocol. All quantitative PCR (MM6) cells were cultivated in RPMI 1640 medium (Invitrogen) sup- experiments were carried out with a StepOne Plus device (Applied Bio- plemented with 10% FCS (Biochrom), 100 mg/ml streptomycin, 100 systems). The endogenous control used for the MM6 cells was b-actin and U/ml penicillin (Invitrogen), 1 mM sodium pyruvate (Invitrogen), for the primary cells RNU48. 13 nonessential amino acids (Sigma-Aldrich), 1 mM oxaloacetate For the specific cDNA synthesis of hsa-miR-19a-3p, 100 ng RNA was (AppliChem), and 10 mg/ml human insulin (Sanofi-Aventis). The cells were transcribed into specific cDNA using stem-loop primers, designed as pre- split twice a week to a density of 0.35 3 106/ml. For the time course ex- viously described (28). Consequently, the quantitative PCR was carried out periment, the cells were seeded at a density of 0.3 3 106/ml and were using a specific forward primer, a universal reverse primer, and Universal cultivated for 4 d. MM6 cells were seeded at a density of 0.3 3 106/ml for ProbeLibrary probe no. 21 (Roche Diagnostics). RNU48 served as the en- untreated time courses and 0.2 3 106/ml for differentiation experiments. For dogenous control. All primer sequences are available upon request. Hsa- differentiation, the cells were stimulated with 50 nM calcitriol (Sigma- miR-125b-5p was detected using the TaqMan miRNA assay kit according Aldrich) and 1 ng/ml TGF-b (isolated and purified in-house) for up to 4 d.
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