MicroRNA expression profiling and functional annotation analysis of their targets in patients with type 1 diabetes mellitus Paula Takahashi a , Danilo J. Xavier a , Adriane F. Evangelista a,1 , Fernanda S. Manoel-Caetano a,b , Claudia Macedo a,2 , Cristhianna V.A. Collares a,c , Maria C. Foss-Freitas d , Milton C. Foss d , Diane M. Rassi c , Eduardo A. Donadi a,c , Geraldo A. Passos a,e , Elza T. Sakamoto-Hojo a,b, ⁎ a Department of Genetics, Faculty of Medicine of Ribeirão Preto, University of São Paulo — USP, Av. Bandeirantes 3900, Monte Alegre 14049-900, Ribeirão Preto, SP, Brazil b Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo — USP, Av. Bandeirantes 3900, Monte Alegre 14040-901, Ribeirão Preto, SP, Brazil c Division of Clinical Immunology, Department of Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo — USP, Av. Bandeirantes 3900, Monte Alegre 14048-900, Ribeirão Preto, SP, Brazil d Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo — USP, Av. Bandeirantes 3900, Monte Alegre 14048-900, Ribeirão Preto, SP, Brazil e Disciplines of Genetics and Molecular Biology, Department of Morphology, Faculty of Dentistry of Ribeirão Preto, University of São Paulo — USP, Av. Do Café s/n, Monte Alegre 14040-904, Ribeirão Preto, SP, Brazil abstract article info Article history: Received 2 September 2013 Received in revised form 18 December 2013 Accepted 29 January 2014 Available online 11 February 2014 Keywords: Type 1 diabetes mellitus microRNA Microarray Expression profile T1DM candidate genes Functional annotation analysis Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing pancreatic β-cells, leading to elimination of insulin production. The exact cause of this disorder is still unclear. Although the differential expression of microRNAs (miRNAs), small non-coding RNAs that control gene expression in a post-transcriptional manner, has been identified in many diseases, including T1DM, only scarce information exists concerning miRNA expression profile in T1DM. Thus, we employed the microarray technology to examine the miRNA expression profiles displayed by peripheral blood mononuclear cells (PBMCs) from T1DM patients compared with healthy subjects. Total RNA extracted from PBMCs from 11 T1DM patients and nine healthy sub- jects was hybridized onto Agilent human miRNA microarray slides (V3), 8x15K, and expression data were ana- lyzed on R statistical environment. After applying the rank products statistical test, the receiver-operating characteristic (ROC) curves were generated and the areas under the ROC curves (AUC) were calculated. To exam- ine the functions of the differentially expressed (p-value b 0.01, percentage of false-positives b 0.05) miRNAs that passed the AUC cutoff value ≥0.90, the database miRWalk was used to predict their potential targets, which were afterwards submitted to the functional annotation tool provided by the Database for Annotation, Visualization, and Integrated Discovery (DAVID), version 6.7, using annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. We found 57 probes, corresponding to 44 different miRNAs (35 up- regulated and 9 down-regulated), that were differentially expressed in T1DM and passed the AUC threshold of 0.90. The hierarchical clustering analysis indicated the discriminatory power of those miRNAs, since they were able to clearly distinguish T1DM patients from healthy individuals. Target prediction indicated that 47 candidate genes for T1DM are potentially regulated by the differentially expressed miRNAs. After performing functional annotation analysis of the predicted targets, we observed 22 and 12 annotated KEGG pathways for the induced and repressed miRNAs, respectively. Interestingly, many pathways were enriched for the targets of both up- and down-regulated miRNAs and the majority of those pathways have been previously associated with T1DM, including many cancer-related pathways. In conclusion, our study indicated miRNAs that may be potential biomarkers of T1DM as well as provided new insights into the molecular mechanisms involved in this disorder. © 2014 Elsevier B.V. All rights reserved. Gene 539 (2014) 213–223 Abbreviations: 8-OHdG, 8-hydroxydeoxyguanosine; AGO1, argonaute-1; ALS, amyotrophic lateral sclerosis; AUC, area under the curve; CCL2, C–C motif chemokine 2; CCL3, C–C motif chemokine 3; CCL4, C–C motif chemokine 4; CDS, coding sequence; CTLA4, cytotoxic T-lymphocyte-associated protein 4; CXCL10, C–X–C motif chemokine 10; DAVID, database for anno- tation, visualization, and integrated discovery; DMSO, dimethylsulfoxide; F-actin, filamentous actin; FAK, focal adhesion kinase; GnRH, gonadotropin-releasing hormone; IAC, inter-array correlation; IL2RA, interleukin 2 receptor alpha; INS, insulin; KEGG, Kyoto encyclopedia of genes and genomes; LD, linkage disequilibrium; MAPK, mitogen-activated protein kinase; miRNAs, microRNAs; NOD, nonobese diabetic; NPH, neutral protamine Hagedorn; PBMCs, peripheral blood mononuclear cells; PFP, percentage of false-positives; PTPN22, protein tyrosine phosphatase, non-receptor type 22; RIN, RNA integrity number; ROBO1, roundabout, axon guidance receptor, homologue 1 (Drosophila); ROC, receiver-operating characteristic; SDF-1, stromal cell-derived factor-1; SLIT2, slit homologue 2; T1DM, type 1 diabetes mellitus; TGF-β, transforming growth factor-beta; UTR, untranslated region(s). ⁎ Corresponding author at: Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo — USP, Av. Bandeirantes 3900, Monte Alegre 14040-901, Ribeirão Preto, Brazil. Tel.: +55 16 3602 3827; fax: +55 16 3602 0222. E-mail address: etshojo@usp.br (E.T. Sakamoto-Hojo). 1 Present address: Molecular Oncology Research Center, Barretos Cancer Hospital. R. Antenor Duarte Villela 1331, Paulo Prata 14784-400, Barretos, SP, Brazil. 2 Present address: Department of Dental Materials and Prosthodontics, Faculty of Dentistry of Ribeirão Preto, University of São Paulo — USP, Av. Do Café s/n, Monte Alegre 14040-904, Ribeirão Preto, SP, Brazil. http://dx.doi.org/10.1016/j.gene.2014.01.075 0378-1119/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene