To determine whether CD-associated circulating miRNA levels change with treatment, we analyzed the sera of 24 patients with incident CD, similar to our larger cohort in terms of demographics and disease severity, at the time of diagnosis and 6 months later. Between baseline and 6 months, median PCDAI scores decreased significantly (P < 0.001). At the 6-month study visit, the proportions of study subjects who had received the following medication during that interval were systemic steroids 16 (67%), methotrexate 1 (4%), 6-mercaptopurine or azathioprine 10 (42%), and infliximab 2 (8%). Significant reductions were observed in the levels of miR-484 and miR-195 (Fig. 3). All other panel miRNAs showed no significant change following treatment, although most trended downward (Fig. 3B). Changes in panel miRNA levels and PCDAI scores were not significantly correlated. These results suggest that CD-associated circulating miRNAs may be markers of response to therapeutic intervention.
One obstacle to the clinical use of circulating miRNA as a biomarker derives from its acellular nature. In assays of cellular miRNAs, a variety of “housekeeping” RNA species are used commonly to correct for differences in tissue mass, RNA yield, or quality. Because no such internal controls are present for assays of circulating miRNA, in our initial assays, 2 artificial C elegans miRNAs were added at the time of RNA purification and used as surrogate references (see Patients and Methods). However, it was noted in our LDA analyses that miR-150 and miR-342–3p were present at equivalent levels in patients with CD and controls (Supplementary Fig. 3A [http://links.lww.com/MPG/A52]). We therefore determined whether they could be used as internal reference miRNAs. The results obtained using the endogenous reference miRNAs were nearly identical to those obtained using exogenous reference miRNA (Supplementary Fig. 3B [http://links.lww.com/MPG/A52]). These results indicate that it may be possible to eliminate the use of exogenous miRNA in future analyses.
Circulating miRNAs have recently emerged as candidate biomarkers for disease, particularly cancer (36,37,39,58). The present study is the first to demonstrate the potential of circulating miRNAs as noninvasive biomarkers of pediatric CD. An initial screen of patients with CD by microfluidic qRT-PCR array identified a significantly altered serum miRNA profile in comparison with healthy controls. These findings were subsequently validated in a much larger set of cases and controls.
All 24 miRNAs significantly altered in CD sera were elevated. The pathogenesis of IBD is a complex process involving inflammatory signaling, lymphocyte infiltration of the gut, and epithelial cell damage. Each of these may result in increases in the levels of circulating miRNA. For example, exosomes secreted in the course of inflammatory signaling may carry specific miRNAs into the circulation. The intestine is a highly vascular organ, and thus activated lymphocytes in the lamina propria may contribute to circulating miRNA. Furthermore, injury to intestinal epithelia may result in increases in epithelium-specific miRNAs in the circulation, as has been observed for tissue miRNAs in heart or liver injury (39,59). For instance, we found that circulating miR-192 is elevated in CD; miR-192 is also the most greatly expressed miRNA in intestinal epithelia (31).
Serological testing is frequently used in the diagnosis of children with suspected IBD, although evidence suggests current markers are suboptimal as screening tools for disease in this patient population, with reported sensitivities ranging from 55% to 71% (63–65). Thus, although the diagnosis of CD ultimately must be made on histopathologic grounds, the introduction of improved noninvasive testing may help to close the gap between the onset of symptoms and the final diagnosis, allowing for earlier treatment. Conversely, a negative screening test result may help reduce unnecessary endoscopy/colonoscopy.
The serum miRNAs examined here display encouraging diagnostic utility, performing favorably in comparison with some standard serological markers. MiR-484 and let-7b each possessed sensitivities > 80%, and 3 had specificities > 90% in comparison with healthy controls. In addition, each panel miRNA was unchanged in the serum of celiac patients compared with age-, race-and sex-matched controls, suggesting that these miRNAs may be specific for IBD or CD, rather than simply indicators of intestinal inflammation in general. This finding contrasts with current IBD serological markers, which are often present in non-IBD intestinal disease. For instance, ASCA is detected in a large proportion of patients with celiac disease, whereas perinuclear anti-neutrophil cytoplasmic antibodies can also be present in celiac disease or microscopic colitis (9,12,13).
Once the diagnosis of IBD is made, the current serological markers of CD are of limited use because they correlate poorly with disease activity or outcome in both adult and pediatric patients (66,67). Studies of circulating miRNA suggest that it may be a more dynamic biomarker; for example, levels of plasma miR-1, the most abundantly expressed miRNA in the heart (68), are elevated at the time of diagnosis in acute myocardial infarction and return to normal by the time of hospital discharge (38). Likewise, miRNA released from tumor cells can significantly alter circulating miRNAs levels, which normalize following tumor resection (42,69,70). We have found that after 6 months of treatment, serum miR-484 and miR-195 levels were significantly reduced from levels observed at the time of diagnosis. Reductions in panel miRNA levels did not significantly correlate with improved PCDAI scores. However, it remains possible that serum miRNAs accurately represent improvements at the mucosal level, as clinical scoring systems, as well as other surrogate markers, correlate poorly with mucosal healing (71,72).
A number of limitations to this preliminary analysis can be rectified with a larger cohort of cases and controls. The nonsignificant trend toward decreased circulating levels of several miRNAs in the longitudinal analysis may be because of lack of statistical power. In addition, it will be essential to obtain adequate numbers of CD cases reflecting different disease locations and types of complications. Likewise, it will be important to determine whether UC is characterized by a distinct set of circulating miRNAs, because these may be useful in distinguishing UC from colitis caused by CD. It will also be essential to analyze circulating miRNA levels in patients with gastrointestinal complaints not caused by CD (eg, infectious, allergic, or functional disease) because this is the group in whom noninvasive testing can be most useful. The analysis of other inflammatory conditions, such as rheumatoid arthritis, can also help establish the specificity of the CD-associated circulating miRNAs, although in practice, other clinical features can also serve to distinguish CD and nongastrointestinal disease.
In summary, this pilot study has identified a number of miRNAs significantly increased in the serum of patients with pediatric CD These CD-associated miRNAs display encouraging clinical utility that will require confirmation in large validation groups. Our findings suggest that large-scale investigations combining circulating miRNAs, laboratory, and genetic markers of CD may result in composite models with improved sensitivity and specificity for IBD in general and CD in particular.
The authors wish to thank Dr Alessio Fasano and Ms Debby Kryszak for providing sera from patients with celiac disease and associated controls.
We are grateful to all of the members of the Friedman laboratory; the Division of Gastroenterology, Hepatology, and Nutrition; those who have supported the Center for Pediatric IBD at Children's Hospital of Philadelphia, and most of all, the patients who generously agreed to participate in research on IBD.
1. Dotan I. Serologic markers in inflammatory bowel disease: tools for better diagnosis and disease stratification. Exp Rev Gastroenterol Hepatol 2007; 1:265–274.
2. Koutroubakis IE, Petinaki E, Mouzas IA, et al
. Anti-Saccharomyces cerevisiae
mannan antibodies and antineutrophil cytoplasmic autoantibodies in Greek patients with inflammatory bowel disease. Am J Gastroenterol 2001; 96:449–454.
3. Peeters M, Joossens S, Vermeire S, et al
. Diagnostic value of anti-Saccharomyces cerevisiae
and antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease. Am J Gastroenterol 2001; 96:730–734.
4. Quinton JF, Sendid B, Reumaux D, et al
. Anti-Saccharomyces cerevisiae
mannan antibodies combined with antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease: prevalence and diagnostic role. Gut 1998; 42:788–791.
5. Dubinsky M. What is the role of serological markers in IBD? Pediatric and adult data. Dig Dis 2009; 27:259–268.
6. Vermeire S, Joossens S, Peeters M, et al
. Comparative study of ASCA (Anti-Saccharomyces cerevisiae
antibody) assays in inflammatory bowel disease. Gastroenterology 2001; 120:827–833.
7. Reumaux D, Sendid B, Poulain D, et al
. Serological markers in inflammatory bowel diseases. Best Pract Res Clin Gastroenterol 2003; 17:19–35.
8. Anand V, Russell AS, Tsuyuki R, et al
. Perinuclear antineutrophil cytoplasmic autoantibodies and anti-Saccharomyces cerevisiae
antibodies as serological markers are not specific in the identification of Crohn's disease and ulcerative colitis. Can J Gastroenterol 2008; 22:33–36.
9. Desplat-Jego S, Johanet C, Escande A, et al
. Update on Anti-Saccharomyces cerevisiae
antibodies, anti-nuclear associated anti-neutrophil antibodies and antibodies to exocrine pancreas detected by indirect immunofluorescence as biomarkers in chronic inflammatory bowel diseases: results of a multicenter study. World J Gastroenterol 2007; 13:2312–2318.
10. Olives JP, Breton A, Hugot JP, et al
. Antineutrophil cytoplasmic antibodies in children with inflammatory bowel disease: prevalence and diagnostic value. J Pediatr Gastroenterol Nutr 1997; 25:142–148.
11. Proujansky R, Fawcett PT, Gibney KM, et al
. Examination of anti-neutrophil cytoplasmic antibodies in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr 1993; 17:193–197.
12. Damoiseaux JG, Bouten B, Linders AM, et al
. Diagnostic value of anti-Saccharomyces cerevisiae
and antineutrophil cytoplasmic antibodies for inflammatory bowel disease: high prevalence in patients with celiac disease. J Clin Immunol 2002; 22:281–288.
13. Freeman HJ. Perinuclear antineutrophil cytoplasmic antibodies in collagenous or lymphocytic colitis with or without celiac disease. Can J Gastroenterol 1997; 11:417–420.
14. Beattie RM, Walker-Smith JA, Murch SH. Indications for investigation of chronic gastrointestinal symptoms. Arch Dis Child 1995; 73:354–355.
15. Poullis AP, Zar S, Sundaram KK, et al
. A new, highly sensitive assay for C-reactive protein can aid the differentiation of inflammatory bowel disorders from constipation-and diarrhoea-predominant functional bowel disorders. Eur J Gastroenterol Hepatol 2002; 14:409–412.
16. Walker TR, Land ML, Kartashov A, et al
. Fecal lactoferrin is a sensitive and specific marker of disease activity in children and young adults with inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2007; 44:414–422.
17. Pfefferkorn MD, Boone JH, Nguyen JT, et al
. Utility of fecal lactoferrin in identifying Crohn disease activity in children. J Pediatr Gastroenterol Nutr 2010; 51:425–428.
18. Joishy M, Davies I, Ahmed M, et al
. Fecal calprotectin and lactoferrin as noninvasivemarkers of pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2009; 48:48–54.
19. Tibble JA, Sigthorsson G, Foster R, et al
. Use of surrogate markers of inflammation and Rome criteria to distinguish organic from nonorganic intestinal disease. Gastroenterology 2002; 123:450–460.
20. Wang Y, Russell I, Chen C. MicroRNA and stem cell regulation. Curr Opin Mol Ther 2009; 11:292–298.
21. Vasilatou D Papageorgiou S, Pappa V, et al. The role of microRNAs in normal and malignant hematopoiesis. Eur J Haematol
22. Cai B, Pan Z, and Lu Y. The roles of microRNAs in heart diseases: a novel important regulator. Curr Med Chem
23. Fineberg SK, Kosik KS, Davidson BL. MicroRNAs potentiate neural development. Neuron 2009; 64:303–309.
24. Yi R, Fuchs E. MicroRNA-mediated control in the skin. Cell Death Differ
25. Iorio MV, Croce CM. MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol 2009; 27:5848–5856.
26. Pandey AK, Agarwal P, Kaur K, et al
. MicroRNAs in diabetes: tiny players in big disease. Cell Physiol Biochem 2009; 23:221–232.
27. Hebert SS, De Strooper B. Alterations of the microRNA network cause neurodegenerative disease. Trends Neurosci 2009; 32:199–206.
28. Latronico MV, Condorelli G. MicroRNAs and cardiac pathology. Nat Rev Cardiol 2009; 6:419–429.
29. Kato M, Arce L, Natarajan R. MicroRNAs and their role in progressive kidney diseases. Clin J Am Soc Nephrol 2009; 4:1255–1266.
30. Pauley KM, Cha S, Chan EK. MicroRNA in autoimmunity and autoimmune diseases. J Autoimmun 2009; 32:189–194.
31. McKenna LB, Schug J, Vourekas A, et al
. MicroRNAs control intestinal epithelial differentiation, architecture, and barrier function. Gastroenterology 2010; 39:654–1664, 1664 e1651.
32. Ueda T, Volinia S, Okumura H, et al. Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol
33. Slaby O, Svoboda M, Michalek J, et al
. MicroRNAs in colorectal cancer: translation of molecular biology into clinical application. Mol Cancer 2009; 8:102.
34. Wu F, Zikusoka M, Trindade A, et al
. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha. Gastroenterology 2008; 35:624–635, e1624.
35. Wu F, Zhang S, Dassopoulos T, et al
. Identification of microRNAs associated with ileal and colonic Crohn's disease. Inflamm Bowel Dis 2010; 16:1729–1738.
36. Mitchell PS, Parkin RK, Kroh EM, et al
. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008; 105:10513–10518.
37. Chen X, Ba Y, Ma L, et al
. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 2008; 18:997–1006.
38. Ai J, Zhang R, Li Y, et al
. Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biochem Biophys Res Commun 2010; 391:73–77.
39. Wang GK, Zhu JQ, Zhang JT, et al
. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 2010; 31:659–666.
40. Chim SS, Shing TK, Hung EC, et al
. Detection and characterization of placental microRNAs in maternal plasma. Clin Chem 2008; 54:482–490.
41. Wang K, Zhang S, Marzolf B, et al
. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A 2009; 106:4402–4407.
42. Tsujiura M, Ichikawa D, Komatsu S, et al
. Circulating microRNAs in plasma of patients with gastric cancers. Br J Cancer 2010; 102:1174–1179.
43. Kong X, Du Y, Wang G, et al. Detection of differentially expressed microRNAs in serum of pancreatic ductal adenocarcinoma patients: miR-196a could be a potential marker for poor prognosis. Dig Dis Sci
44. Silva J, García V, Zaballos A, et al. Vesicle-related microRNAs in plasma of NSCLC patients and correlation with survival. Eur Respir J
45. Ng EK, Chong WW, Jin H, et al
. Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut 2009; 58:1375–1381.
46. Wu F, Guo NJ, Tian H, et al
. Peripheral blood microRNAs distinguish active ulcerative colitis and Crohn's disease. Inflamm Bowel Dis 2011; 17:241–250.
47. Thayu M, Denson LA, Shults J, et al
. Determinants of changes in linear growth and body composition in incident pediatric Crohn's disease. Gastroenterology 2010; 139:430–438.
48. Thayu M, Shults J, Burnham JM, et al
. Gender differences in body composition deficits at diagnosis in children and adolescents with Crohn's disease. Inflamm Bowel Dis 2007; 13:1121–1128.
49. Dubner SE, Shults J, Baldassano J, et al
. Longitudinal assessment of bone density and structure in an incident cohort of children with Crohn's disease. Gastroenterology 2009; 136:123–130.
50. Satsangi J, Silverberg MS, Vermeire S, et al
. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut 2006; 55:749–753.
51. Hyams JS, Ferry GD, Mandel FS, et al
. Development and validation of a pediatric Crohn's disease activity index. J Pediatr Gastroenterol Nutr 1991; 12:439–447.
52. Suh MR, Lee Y, Kim JY, et al
. Human embryonic stem cells express a unique set of microRNAs. Dev Biol 2004; 270:488–498.
53. Pavlidis P, Noble WS. Matrix2png: a utility for visualizing matrix data. Bioinformatics 2003; 19:295–296.
54. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001; 98:5116–5121.
55. He L, Thomson JM, Hemann MT, et al
. A microRNA polycistron as a potential human oncogene. Nature 2005; 435:828–833.
56. Kan T, Sato F, Ito T, et al
. The miR-106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim. Gastroenterology 2009; 136:1689–1700.
57. Uren AG, Kool J, Matentzoglu K, et al
. Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks. Cell 2008; 133:727–741.
58. Tan KS, Armugam A, Sepramaniam S, et al
. Expression profile of MicroRNAs in young stroke patients. PLoS One 2009; 4:e7689.
59. Wang K, Zhang S, Marzolf B, et al. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci USA
60. Cekaite L, Clancy T, Sioud M. Increased miR-21 expression during human monocyte differentiation into DCs. Front Biosci (Elite Ed) 2010; 2:818–828.
61. Silva MA, Lopez CB, Riverin F, et al
. Characterization and distribution of colonic dendritic cells in Crohn's disease. Inflamm Bowel Dis 2004; 10:504–512.
62. Davidson LA, Wang N, Shah MS, et al
. n-3 Polyunsaturated fatty acids modulate carcinogen-directed non-coding microRNA signatures in rat colon. Carcinogenesis 2009; 30:2077–2084.
63. Ruemmele FM, Targan SR, Levy G, et al
. Diagnostic accuracy of serological assays in pediatric inflammatory bowel disease. Gastroenterology 1998; 115:822–829.
64. Hoffenberg EJ, Fidanza S, Sauaia A. Serologic testing for inflammatory bowel disease. J Pediatr 1999; 134:447–452.
65. Sabery N, Bass D. Use of serologic markers as a screening tool in inflammatory bowel disease compared with elevated erythrocyte sedimentation rate and anemia. Pediatrics 2007; 19:e193–e199.
66. Teml A, Kratzer V, Schneider B, et al
. Anti-Saccharomyces cerevisiae
antibodies: a stable marker for Crohn's disease during steroid and 5-aminosalicylic acid treatment. Am J Gastroenterol 2003; 98:2226–2231.
67. Desir B, Amre DK, Lu SE, et al
. Utility of serum antibodies in determining clinical course in pediatric Crohn's disease. Clin Gastroenterol Hepatol 2004; 2:139–146.
68. Lagos-Quintana M, Rauhut R, Yalcin A, et al
. Identification of tissue-specific microRNAs from mouse. Curr Biol 2002; 12:735–739.
69. Huang Z, Huang D, Ni S, et al
. Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer. Int J Cancer 2010; 127:118–126.
70. Yamamoto Y, Kosaka N, Tanaka M, et al
. MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma. Biomarkers 2009; 14:529–538.
71. Afzal NA, Van Der Zaag-Loonen HJ, Arnaud-Battandier F, et al
. Improvement in quality of life of children with acute Crohn's disease does not parallel mucosal healing after treatment with exclusive enteral nutrition. Aliment Pharmacol Ther 2004; 20:167–172.
72. Efthymiou A, Viazis N, Mantzaris G, et al
. Does clinical response correlate with mucosal healing in patients with Crohn's disease of the small bowel? A prospective, case-series study using wireless capsule endoscopy. Inflamm Bowel Dis 2008; 14:1542–1547.