Increased duodenal expression of miR-146a and -155 in pediatric Crohn's disease

Abstract

Aim: To evaluate the role of microRNA (miR)-146a, -155 and -122 in the duodenal mucosa of pediatric patients with Crohn's disease (CD) and the effect of transforming growth factor-β (TGF-β) on these miRs in duodenal epithelial and fibroblast cells.

Methods: Formalin-fixed, paraffin-embedded biopsies derived from the macroscopically inflamed (CD inflamed: n = 10) and intact (CD intact: n = 10) duodenal mucosa of pediatric CD patients and control children (C: n = 10) were examined. Expression of miR-146a, -155 and -122 was determined by real-time polymerase-chain reaction (PCR). The expression of the above miRs was investigated in recombinant human TGF-β (1 nmol/L, 24 h) or vehicle treated small intestinal epithelial cells (CCL-241) and primary duodenal fibroblast cells derived from healthy children as well.

Results: Expression of miR-146a was significantly higher in the inflamed duodenal mucosa compared to the intact duodenal mucosa of children with CD (CD inflamed: 3.21 ± 0.50 vs CD intact: 0.62 ± 0.26, P ≤ 0.01) and to the control group (CD inflamed: 3.21 ± 0.50 vs C: 1.00 ± 0.33, P ≤ 0.05). The expression of miR-155 was significantly increased in the inflamed region of the duodenum compared to the control group (CD inflamed: 4.87 ± 1.02 vs

Control: 1.00 ± 0.40, P ≤ 0.001). The expression of miR-122 was unchanged in the inflamed or intact mucosa of CD patients compared to controls. TGF-β treatment significantly decreased the expression of miR-155 in small intestinal epithelial cells (TGF-β: 0.7 ± 0.083 vs

Control: 1 ± 0.09, P ≤ 0.05) and also the expression of miR-146a (TGF-β: 0.67 ± 0.04 vs

Control: 1 ± 0.15, P ≤ 0.01) and miR-155 (TGF-β: 0.72 ± 0.09 vs

Control: 1 ± 0.06, P ≤ 0.05) in primary duodenal fibroblasts compared to corresponding vehicle treated controls. TGF-β treatment did not influence the expression of miR-122.

Conclusion: The elevated expression of miR-146a and -155 in the inflamed duodenal mucosa of CD patients suggests the role of these miRs in the pathomechanism of inflammatory bowel disease. Anti-inflammatory TGF-β plays an important role in the regulation of the expression of these miRs.

Keywords: Crohn’s disease; Inflammatory bowel disease; MicroRNAs; Pediatric; Transforming growth factor-β.

Figure 1

Expression of microRNA-146a (A), microRNA-155 (B) and microRNA-122 (C) in the duodenal mucosa of pediatric patients with Crohn’s disease. Expression of microRNA (miR)-146a was significantly higher in the inflamed duodenal mucosa of children with Crohn’s disease (CD) compared to the intact mucosa and controls. miR-155 showed significantly elevated expression in the inflamed region of the duodenum compared to the control group. There was no significant difference in the expression of miR-122 between the groups. ^aP ≤ 0.05 vs Control; ^bP ≤ 0.001 vs Control; ^dP ≤ 0.01 vs CD intact.

Figure 2

Effect of transforming growth factor-β on the expression of microRNA-146a (A), and microRNA-155 (B) in duodenal epithelial cells. Transforming growth factor (TGF)-β did not have any effect on the expression of microRNA (miR)-146a. TGF-β significantly decreased the expression of miR-155. There was no miR-122 expression detected, ^aP ≤ 0.05 vs Control.

Figure 3

Effect of transforming growth factor-β on the expression of microRNA-146a (A), microRNA-155 (B), and microRNA-122 (C) in duodenal fibroblasts. Transforming growth factor (TGF)-β significantly decreased the expression of microRNA (miR)-146a and miR-155. TGF-β had no effect on the expression of miR-122, ^aP ≤ 0.05 vs Control, ^bP ≤ 0.01 vs Control.

Figure 4

Schematic representation of the target interactions between transforming growth factor-β - microRNA-146a, -155 and -122. Transforming growth factor (TGF)-β is considered to be a major anti-inflammatory cytokine playing an important role in the pathogenesis of inflammatory bowel disease. MicroRNA (miR)-146a, -155 and -122 act as possible regulators of the TGF-β signal transduction, with capacity to induce apoptosis, cell migration, invasion and proliferation. Moreover, TGF-β induces and represses the transcription of various genes. Data shown in the figure are based on MiRTarBase Database. SMAD: Mothers against decapentaplegic homolog; RhoA: Ras homolog gene family, member A.