MicroRNA signatures differentiate Crohn's disease from ulcerative colitis

Abstract

Background: Excessive and inappropriate immune responses are the hallmark of several autoimmune disorders, including the inflammatory bowel diseases (IBD): Crohn's disease (CD) and ulcerative colitis (UC). A complex etiology involving both environmental and genetic factors influences IBD pathogenesis. The role of microRNAs (miRNAs), noncoding RNAs involved in regulating numerous biological processes, to IBD pathology, in terms of initiation and progression, remains ill-defined. In the present study, we evaluated the relationship between colon, peripheral blood, and saliva whole miRNome expression in IBD patients and non-inflammatory bowel disease (non-IBD) controls to identify miRNAs that could discriminate CD from UC. Quantitative real-time PCR (qRT-PCR) was used to validate and assess miRNA expression.

Results: Microarray analysis demonstrated that upwards of twenty six miRNAs were changed in CD and UC colon biopsies relative to the non-IBD controls. CD was associated with the differential expression of 10 miRNAs while UC was associated with 6 miRNAs in matched colon tissues. CD was associated with altered expression of 6 miRNAs while UC was associated with 9 miRNAs in whole blood. Expression of miR-101 in CD patients and miR-21, miR-31, miR-142-3p, and miR-142-5p in UC patients were altered in saliva.

Conclusions: Our results suggest that there is specific miRNA expression patterns associated with UC versus CD in three separate tissue/body fluids (colon, blood, and saliva). Further, the aberrant miRNA expression profiles indicate that miRNAs may be contributory to IBD pathogenesis, or at least reflect the underlying inflammation. Scrutinizing miRNA expression in saliva and blood samples may be beneficial in monitoring or diagnosing disease in IBD patients. A panel of miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-146a, and miR-375) may be used as markers to identify and discriminate between CD and UC.

Figure 1

Heat map and unsupervised hierarchical clustering of miRNAs in IBD colon biopsies indicate dysregulation of miRNA expression. The miRCURY LNA microarray microRNA profiling service was used to examine miRNA expression in colon biopsies from Normal, CD, and UC subjects. (A) Heat map of fold changes > 0.5. (B) Heat map of fold changes > 3.0. Red color represents an expression level above mean, blue color represents expression lower than the mean. A Delta Log Median ratio of +/−1.0 is equal to a fold change of +/− 2.0.

Figure 2

miRNA expression is altered in colon, blood, and saliva from Crohn’s disease and ulcerative colitis subjects. Total RNA from NL, CD, and UC colon biopsies was pooled and used for TaqMan qRT-PCR analysis for the indicated miRNAs. miRNA expression was normalized to U6 expression. Statistical significance was calculated using the Student’s t-test relative to the healthy NL control. At least 23 distinct specimens were included in each pooled group of colon biopsies. 30 and 5 distinct specimens were included in each pooled group of blood and saliva samples, respectively.

Figure 3

miRNA expression is differentially expressed in matched colon biopsies from Crohn’s disease subjects. Total RNA was isolated from matched endoscopically uninvolved (EU) and endoscopically involved (EI) colon biopsies from CD subjects. The RNA samples were used for TaqMan qRT-PCR analysis for the indicated miRNAs. miRNA expression was normalized to U6 expression. Pairings were subdivided according to miRNA expression trends. Statistical significance was calculated using the Student’s paired t-test relative to the endoscopically uninvolved CD tissue.

Figure 4

miRNA expression is differentially expressed in matched colon biopsies from ulcerative colitis subjects. Total RNA was isolated from matched endoscopically uninvolved (EU) and endoscopically involved (EI) colon biopsies from UC subjects. The RNA samples were used for TaqMan qRT-PCR analysis for the indicated miRNAs. miRNA expression was normalized to U6 expression. Pairings were subdivided according to miRNA expression trends. Statistical significance was calculated using the Student’s paired t-test relative to the endoscopically uninvolved UC tissue.

Figure 5

Roquin-1 and ATG16L1 expression is differentially expressed in a subset of CD and UC subjects. Total RNA from matched endoscopically uninvolved (EU) and endoscopically involved (EI) colon biopsies from (A, C) CD and (B, D) UC subjects was used to analyze Roquin-1 or ATG16L1 expression via qRT-PCR. Gene expression was normalized to GAPDH. Pairings were subdivided according to Roquin-1 or ATG16L1 expression trends. Statistical significance was calculated using the Student’s paired t-test relative to the endoscopically uninvolved IBD tissue.