Circulating microRNAs as biomarkers of adult Crohn’s disease Michael D. Jensen a , Rikke F. Andersen b , Henry Christensen b , Torben Nathan a , Jens Kjeldsen c and Jonna S. Madsen b Objective Previous studies have found a differential expression of microRNAs (miRNAs) in the blood of patients with Crohn’s disease (CD) compared with healthy controls. The aim of this study was to identify circulating miRNAs expressed in CD and assess their performance as biomarkers in patients with clinically suspected or known CD. Methods The study consisted of two parts: (a) miRNA profiling: The miRNA expression pattern was examined in six patients with CD and six controls using OpenArray miRNA profiling, and the best miRNAs were selected according to their P-value. (b) Validation cohort: In a well-characterized cohort of 102 patients with suspected or known CD, miRNAs identified by miRNA profiling or selected from previously published studies (hsa-miR-16, hsa-miR-21, hsa-miR-106a, and hsa-miR-140-3p) were measured in plasma using reverse transcription PCR. Results miRNA profiling: hsa-miR-369-3p, hsa-miR-376a, hsa-miR-376, hsa-miR-411#, hsa-miR-411, and mmu-miR-379 were downregulated in CD patients compared with the controls; hsa-miR-200c, hsa-miR-181-2#, and hsa-miR-125a-5p were upregulated (P < 0.05). Validation cohort: Only hsa-miR-16 was significantly downregulated in patients with CD compared with patients without CD (fold change 0.83, P = 0.02). Receiver operating characteristic analyses showed an area under the curve of 0.65. miRNAs could not discriminate inflammatory from stricturing CD or small bowel CD from CD involving the colon. Conclusion In a clinically relevant cohort of patients, miRNAs in plasma identified in the present and previous studies were inadequate biomarkers for the diagnosis of CD. Eur J Gastroenterol Hepatol 27:1038–1044 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Introduction Crohn’s disease (CD) is a chronic systemic inflammatory disease mainly involving the gastrointestinal tract. Gut inflammation is transmural and with a segmental dis- tribution. All bowel segments may be involved, although CD is most commonly located in the colon and distal ileum [1]. In the majority of patients, the phenotype changes over time from inflammatory lesions to stricturing or penetrat- ing disease [2]. On a cellular level, CD is characterized by an impaired interaction between the gut microbiota and the immune system. Pathogenic mechanisms involve loss of barrier function, increased influx of antigens into the lamina propria, and an exaggerated immune response dominated by proinflammatory T-cells and cytokines [e.g. interferon γ, tumor necrosis factor-α (TNF-α), and interleukins 17 and 23] [3]. Mediators of chronic inflammation stimulate remodeling of the extracellular matrix, leading to fibrosis and eventually stenosis. MicroRNAs (miRNAs) are small noncoding single- stranded RNA molecules containing ∼ 18–24 nucleotides. They act as regulators of gene expression at the post- transcriptional level – either by inducing mRNA degra- dation or by inhibiting the translational process [4], and it is estimated that more than one third of human genes are targets for miRNA regulation [5]. However, although a large number of miRNAs have been identified, little is known about their function (http://www.mirbase.org) [6]. There is growing evidence that miRNAs play a role in the induction of cancer, inflammatory, and autoimmune dis- eases [7]. In the intestinal tract, miRNAs are involved in tissue homeostasis, intestinal cell differentiation, and the maintenance of intestinal barrier function [8]. Recently, several studies have shown a differential expression of miRNAs in tissue samples [9–12] and blood [9,13–15] from patients with inflammatory bowel diseases (IBDs) compared with healthy controls, the latter suggesting that miRNAs may serve as novel biomarkers of these diseases. Furthermore, in a recent study, levels of miRNAs in serum were shown to change during induction therapy with infliximab [16]. The aim of this study was to (a) determine the expres- sion of miRNAs in the plasma of patients with CD com- pared with patients with suspected CD and a normal diagnostic workup, (b) validate the miRNA expression in a second cohort of patients with clinically suspected or known CD, and (c) assess miRNAs performance as biomarkers of CD. a Department of Internal Medicine, Section of Gastroenterology, b Department of Clinical Biochemistry, Lillebaelt Hospital, Vejle and c Department of Medical Gastroenterology, Odense University Hospital, Odense, Denmark Correspondence to Michael D. Jensen, MD, PhD, Department of Internal Medicine, Section of Gastroenterology, Lillebaelt Hospital Vejle, Kabbeltoft 25, 7100 Vejle, Denmark Tel: + 45 7940 6345; fax: + 45 7940 6887; e-mail: michael.dam.jensen@rsyd.dk Received 7 April 2015 Accepted 5 June 2015 European Journal of Gastroenterology & Hepatology 2015, 27:1038–1044 Keywords: Crohn disease, microRNAs, plasma ’ Original article 0954-691X Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/MEG.0000000000000430 1038 Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.