miR-126 Is Downregulated in Cystic Fibrosis Airway Epithelial Cells and Regulates TOM1 Expression This information is current as Irene K. Oglesby, Isabella M. Bray, Sanjay H. Chotirmall, of September 27, 2021. Raymond L. Stallings, Shane J. O'Neill, Noel G. McElvaney and Catherine M. Greene J Immunol published online 18 January 2010 http://www.jimmunol.org/content/early/2010/01/18/jimmun ol.0902669 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/01/13/jimmunol.090266 Material 9.DC1 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 by guest on September 27, 2021 *average 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 18, 2010, doi:10.4049/jimmunol.0902669 The Journal of Immunology miR-126 Is Downregulated in Cystic Fibrosis Airway Epithelial Cells and Regulates TOM1 Expression Irene K. Oglesby,* Isabella M. Bray,† Sanjay H. Chotirmall,* Raymond L. Stallings,† Shane J. O’Neill,* Noel G. McElvaney,*,1 and Catherine M. Greene*,1 Cystic fibrosis (CF) is one of the most common lethal genetic diseases in which the role of microRNAs has yet to be explored. Predicted to be regulated by miR-126, TOM1 (target of Myb1) has been shown to interact with Toll-interacting protein, forming a complex to regulate endosomal trafficking of ubiquitinated proteins. TOM1 has also been proposed as a negative regulator of IL- 1b and TNF-a–induced signaling pathways. MiR-126 is highly expressed in the lung, and we now show for the first time differential expression of miR-126 in CF versus non-CF airway epithelial cells both in vitro and in vivo. MiR-126 downregulation in CF bronchial epithelial cells correlated with a significant upregulation of TOM1 mRNA, both in vitro and in vivo when compared with their non-CF counterparts. Introduction of synthetic pre–miR-126 inhibited luciferase activity in a reporter Downloaded from system containing the full length 39-untranslated region of TOM1 and resulted in decreased TOM1 protein production in CF bronchial epithelial cells. Following stimulation with LPS or IL-1b, overexpression of TOM1 was found to downregulate NF-kB luciferase activity. Conversely, TOM1 knockdown resulted in a significant increase in NF-kB regulated IL-8 secretion. These data show that miR-126 is differentially regulated in CF versus non-CF airway epithelial cells and that TOM1 is a miR-126 target that may have an important role in regulating innate immune responses in the CF lung. To our knowledge, this study is the first to report of a role for TOM1 in the TLR2/4 signaling pathways and the first to describe microRNA involvement in CF. The Journal http://www.jimmunol.org/ of Immunology, 2010, 184: 000–000. ystic fibrosis (CF) is an inherited disorder characterized by flammatory cytokines (2). Therefore, TLRs and their signaling chronic airway inflammation. Bronchial epithelial cells intermediates represent potential therapeutic targets for CF. Despite C contribute significantly to the pulmonary inflammation significant advances in treatment regimes, CF remains a condition evident in CF. LPS and IL-1b, which bind to TLR4 and the IL-1RI for which there is no effective cure. Therefore, investigating the respectively, also play a pivotal role in this process. These agonists expression and function of microRNAs (miRNAs) in CF will shed can activate the innate immune response culminating in proin- light on previously unidentified regulatory mechanisms controlling flammatory gene expression leading to neutrophil-dominated air- changes in gene expression and direct the development of future by guest on September 27, 2021 way inflammation and tissue damage in the CF lung. IL-1RI and therapeutic strategies for this debilitating and fatal disorder. TLRs are present on a variety of cell types, including both immune Expanding interest in miRNAs over the past decade has uncovered cells and epithelial cells within the lung. In the context of CF, airway their importance in several biological processes and has identified epithelial cells have been shown to promote proinflammatory gene diseasestateswithalteredmiRNAexpressionpatterns.miRNAsare20– transcription following stimulation with their cognate agonists (1, 25 nucleotides long and negatively regulate gene expression at a post- 2). For example, in airway epithelial cells of non-CF and CF origin transcriptional level. Within each miRNA there exists a 2–8-nucleotide triacylated lipopeptide, LPS or unmethylated CpG DNA can induce seed region thought to be critical for target selection (3). Mature IL-6, IL-8, and TNF-a production via TLRs 2, 4, and 9 (1). Simi- miRNAs use this seed region to bind selectively to miRNA recognition larly, IL-1b can upregulate production of a plethora of proin- elements (MREs) within the 39UTR of target mRNAs. Different target genes may have several MREs and therefore be regulated by numerous miRNAs. The number of and distance between MREs are considered *Respiratory Research Division, Department of Medicine, Royal College of Sur- important for the biological activity of miRNAs. Relatively few † geons in Ireland, Beaumont Hospital; and Cancer Genetics Group, Royal College miRNAs have been studied in detail, and hence the biological rele- of Surgeons in Ireland, Dublin, Ireland vance of the majority remains to be uncovered. Expression levels vary 1N.G.M. and C.M.G. share joint senior authorship. greatly among tissues, and it is believed that dysregulation of miRNA Received for publication August 13, 2009. Accepted for publication December 3, 2009. can contribute to pathologic disease (4). Therefore, we considered it plausible to investigate whether unique miRNA expression profiles This work was supported by the Children’s Medical & Research Foundation. exist in CF, particularly in CF bronchial epithelial cells, and explore Address correspondence and reprint requests to Dr. Catherine M. Greene, Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland, their effects on influencing signaling pathways. Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland. E-mail We performed expression profiling comparing miRNA expression address: [email protected] in CF and non-CF bronchial brushings. Based on these studies, we The online version of this article contains supplemental material. selected miR-126 for further investigation, given that its expression is Abbreviations used in this paper: Cf, Canis familiaris; CF, cystic fibrosis; CFTR, CF known to be highest invascularized tissues such as the lung, heart, and transmembrane conductance regulator; Ct, cycle threshold; ER, endoplasmic reticu- lum; Hs, Homo sapiens; miRNA, microRNA; Mm, Mus musculus; MRE, microRNA kidney (5–7), and as it has been shown to be present in bronchial recognition element; NTC, no-template control; qRT-PCR, quantitative real-time epithelium (8). miR-126 is 21 nucleotides in length, located on PCR; Rn, Rattus norvicus; Scr, scrambled control; siRNA, small interfering RNA; chromosome 9q34.3 and is contained within intron 5 of its host gene Tollip, Toll-interacting protein. epidermal growth factor like-7 (6, 9). In recent studies, miR-126 has Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 been shown to have functional roles in angiogenesis (10, 11), to be www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902669 2 miR-126 REGULATION OF TOM1 IN CF AIRWAY EPITHELIUM downregulated in a number of malignancies (8, 12) and to act as profiling method using the Taqman MicroRNA Arrays v2.0 (released June a tumor suppressor in breast cancer (13). In silico analysis of a number 2009) from Applied Biosystems (Foster City, CA). of miRNA target prediction databases shows that TOM1 is a potential The content is derived from the miRBase (microRNA database) miRNA registry, providing comprehensive coverage of miRNAs from release 10.0, target of miR-126. TOM1 is a member of a family of proteins con- using the most up-to-date TaqMan MicroRNA Assays. Two array cards (A taining an N-terminal VHS (Vps27p/Hrs/STAM) domain reported to and B) for each sample were run on the Applied Biosystems 7900HT fast be involved in intracellular trafficking (14). Previous studies have real time PCR system, which measured expression levels of 667 different shown that TOM1 forms a complex with Toll-interacting protein human miRNAs in each sample and three positive and one negative control per card. RNA (30 ng) from clinical samples was reverse transcribed with (Tollip), a negative regulator of TLR2, TLR4, and IL-1RI signaling. the Megaplex primer pool (Applied Biosystems), allowing simultaneous This complex regulates endosomal trafficking of ubiquitinated pro- reverse transcription of 430 miRNAs and 36 endogenous controls in one teins (15). Moreover, this complex has been shown to traffic IL-1RI to RT pool (18). A preamplification step was performed on the Megaplex RT the endosome for degradation (16). TOM1 has also been proposed as product (5 ml) using TaqMan PreAmp Master Mix (23) and PreAmp 3 a negative regulator of IL-1b-andTNF-a–induced signaling path- Primer Mix (5 ; Applied Biosystems). The PreAmp primer pool con- tained forward primers specific for each miRNA and a universal reverse ways, whereby its overexpression can suppress the activity of the primer (Applied Biosystems). All miRNAs with cycle threshold (Ct) val- transcription factors NF-kB and AP-1 (17). In this study, we explore ues .35 were considered nonamplified or not expressed and were excluded the presence of miRNA in CF for the first time.