Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 is online at: average * The Journal of Immunology published online 25 July 2011 from submission to initial decision 4 weeks from acceptance to publication http://www.jimmunol.org/content/early/2011/07/25/jimmun ol.1101233 MicroRNA Regulation of Molecular Networks Mapped by Global MicroRNA, mRNA, and Protein Expression in Activated T Lymphocytes Yevgeniy A. Grigoryev, Sunil M. Kurian, Traver Hart, Aleksey A. Nakorchevsky, Caifu Chen, Daniel Campbell, Steven R. Head, John R. Yates III and Daniel. R Salomon J Immunol Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2011/07/25/jimmunol.110123 3.DC1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 24, 2021. Published July 25, 2011, doi:10.4049/jimmunol.1101233 The Journal of Immunology MicroRNA Regulation of Molecular Networks Mapped by Global MicroRNA, mRNA, and Protein Expression in Activated T Lymphocytes Yevgeniy A. Grigoryev,* Sunil M. Kurian,* Traver Hart,* Aleksey A. Nakorchevsky,† Caifu Chen,‡ Daniel Campbell,x Steven R. Head,x John R. Yates, III,† and Daniel. R Salomon* MicroRNAs (miRNAs) regulate specific immune mechanisms, but their genome-wide regulation of T lymphocyte activation is largely unknown. We performed a multidimensional functional genomics analysis to integrate genome-wide differential mRNA, miRNA, and protein expression as a function of human T lymphocyte activation and time. We surveyed expression of 420 human miRNAs in parallel with genome-wide mRNA expression. We identified a unique signature of 71 differentially expressed miRNAs, Downloaded from 57 of which were previously not known as regulators of immune activation. The majority of miRNAs are upregulated, mRNA expression of these target genes is downregulated, and this is a function of binding multiple miRNAs (combinatorial targeting). Our data reveal that consideration of this complex signature, rather than single miRNAs, is necessary to construct a full picture of miRNA-mediated regulation. Molecular network mapping of miRNA targets revealed the regulation of activation-induced immune signaling. In contrast, pathways populated by genes that are not miRNA targets are enriched for metabolism and biosynthesis. Finally, we specifically validated miR-155 (known) and miR-221 (novel in T lymphocytes) using locked nucleic acid inhibitors. http://www.jimmunol.org/ Inhibition of these two highly upregulated miRNAs in CD4+ T cells was shown to increase proliferation by removing suppression of four target genes linked to proliferation and survival. Thus, multiple lines of evidence link top functional networks directly to T lymphocyte immunity, underlining the value of mapping global gene, protein, and miRNA expression. The Journal of Immu- nology, 2011, 187: 000–000. lymphocytes regulate the adaptive immune response by coordinated fashion to achieve a balance among proliferation, serving as Ag-specific effector cells. Activation via TCR memory, and quiescence. T engagement and CD28 costimulation is characterized by MicroRNAs (miRNAs) have emerged as posttranscriptional by guest on September 24, 2021 gene upregulation (1) and is a highly regulated process requiring regulators of gene expression in a variety of biological processes coordination of multiple signaling pathways for proliferation, cyto- (3–7). The mode of miRNA regulation is protein repression via kines, and differentiation. After Ag clearance, some effector cells complementary sequence recognition in the 39 untranslated region must be reduced or eliminated by mechanisms like activation- of the target mRNA and/or degradation of the target transcript (8– induced cell death (2). Thus, activation must be regulated in a 11). A recent paper indicates the major effect of miRNAs is to decrease mRNA levels (12). miRNAs can potentially regulate hundreds of proteins (13) and *Department of Molecular and Experimental Medicine, The Scripps Research In- modulate concentration of proteins over a narrow range in a dose- † stitute, La Jolla, CA 92037; Department of Chemical Physiology, The Scripps Re- dependent manner (14, 15). miRNAs are involved in hematopoi- search Institute, La Jolla, CA 92037; ‡Applied Biosystems, Foster City, CA 94404; and xDNA Microarray Core, The Scripps Research Institute, La Jolla, CA 92037 etic cell function and development (as summarized in Refs. 16– Received for publication May 2, 2011. Accepted for publication June 17, 2011. 64). A few miRNAs have been linked to specific T lymphocyte This work was supported by National Institutes of Health Grants U19 A1063603 and mechanisms—181a (37), 181c (39), 155 (28), 150 (18), 146 (20), R01 AI081757. and 142 (40)—via regulation of T cell sensitivity to Ag stimula- Y.A.G., S.M.K., and D.R.S. conceived and designed the experiments and wrote the tion, regulating transcription factors, and activation-induced cell manuscript; Y.A.G., S.M.K., and A.A.N. performed the experiments; Y.A.G., A.A.N., death. However, at the global level, little is known about the im- and T.H. analyzed the data; and C.C., D.C., S.R.H., and J.R.Y. contributed reagents/ materials/analysis tools. pact of activation-induced miRNAs on mRNA and protein ex- pression in human T lymphocytes, particularly in the context of The sequences presented in this article (entire set of CEL files) have been submitted to National Center for Biotechnology Information Gene Expression Omnibus under mapping miRNA-regulated molecular networks. accession number GSE14352 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= In this study, we show that differentially upregulated miRNAs GSE14352). regulate T lymphocyte activation by targeting highly differentially Address correspondence and reprint requests to Dr. Daniel R. Salomon, Department expressed genes involved in networks critical for cell activation, of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, Mail Code MEM-L55, La Jolla, CA 92037. E-mail address: proliferation, and survival. We used a multidimensional approach [email protected] to integrate genome-wide miRNA, mRNA, and protein expression. The online version of this article contains supplemental material. We surveyed expression for 420 human miRNA sequences at 0, 24, Abbreviations used in this article: Ct, threshold cycle; FDR, false discovery rate; IPA, 48, and 72 h after activation. In parallel, we profiled global mRNA Ingenuity Pathways Analysis; LNA, locked nucleic acid; miRNA, microRNA; qPCR, and protein expression. We found 71 significantly differentially ex- quantitative real-time PCR. pressed miRNAs, of which 57 have not been previously linked to Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 T lymphocyte function. Testing several established miRNA target www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101233 2 microRNA-REGULATED NETWORKS IN T CELL ACTIVATION prediction algorithms, we demonstrated globally that targets of performed for specified pairwise comparisons among all time points of multiple upregulated miRNAs (combinatorial targeting) have de- activation. creased mRNA expression with activation. In validation, we In parallel, we performed our own analysis of differential gene ex- pression to corroborate AltAnalyze results. CEL files for each donor from showed that inhibition of two highly upregulated miRNAs in the 1.0ST HuEx Arrays were normalized by robust multiarray averaging CD4+ T cells increased proliferation by removing suppression of using a custom cumulative distribution function downloaded from the four target genes involved in proliferation and survival. Thus, University of Michigan (http://brainarray.mbni.med.umich.edu/Brainarray/ our studies provide novel evidence for a large number of func- Database/CustomCDF/genomic_curated_CDF.asp). Differential expression was measured with the Limma package (http://www.bioconductor.org/ tional molecular networks populated by downregulated targets of packages/2.6/bioc/html/limma.html) using a two-class model. All calcu- highly upregulated miRNAs. lations were performed in R/Bioconductor. Genes were filtered using a fold-change filter of 1.5 (0.58 in log2) and an FDR filter of 0.01. Genes that were detected as significantly differentially expressed by both AltA- Materials and Methods nalyze and Limma analysis were then selected for further analysis. T lymphocyte isolation The Multidimensional Protein Identification Tool proteomics Blood draw for this study was accepted by our institution’s ethical com- mission, and all subjects gave their written consent according to review The Multidimensional Protein Identification Tool (66) protocol was used board guidelines. CD2+ T lymphocytes were purified from Ficoll-Hypaque as described previously (1). Protein fraction was denatured, alkylated, and density-separated