The Colonic Mucosal MicroRNAs, MicroRNA-219a-5p, and MicroRNA-338-3p Are Downregulated in Irritable Bowel Syndrome and Are Associated With Barrier Function and MAPK Signaling

Abstract

Background & aims: Alterations in microRNA (miRNA) and in the intestinal barrier are putative risk factors for irritable bowel syndrome (IBS). We aimed to identify differentially expressed colonic mucosal miRNAs, their targets in IBS compared to healthy controls (HCs), and putative downstream pathways.

Methods: Twenty-nine IBS patients (15 IBS with constipation [IBS-C], 14 IBS with diarrhea [IBS-D]), and 15 age-matched HCs underwent sigmoidoscopy with biopsies. A nCounter array was used to assess biopsy specimen-associated miRNA levels. A false discovery rate (FDR) < 10% was considered significant. Real-time polymerase chain reaction (PCR) was used to validate differentially expressed genes. To assess barrier function, trans-epithelial electrical resistance (TEER) and dextran flux assays were performed on Caco-2 intestinal epithelial cells that were transfected with miRNA-inhibitors or control inhibitors. Protein expression of barrier function associated genes was confirmed using western blots.

Results: Four out of 247 miRNAs tested were differentially expressed in IBS compared to HCs (FDR < 10%). Real-time PCR validation suggested decreased levels of miR-219a-5p and miR-338-3p in IBS (P = .026 and P = .004), and IBS-C (P = .02 and P = .06) vs. HCs as the strongest associations. Inhibition of miR-219a-5p resulted in altered expression of proteasome/barrier function genes. Functionally, miR-219a-5p inhibition enhanced the permeability of intestinal epithelial cells as TEER was reduced (25-50%, P < .05) and dextran flux was increased (P < .01). Additionally, inhibition of miR-338-3p in cells caused alterations in the mitogen-activated protein kinase (MAPK) signaling pathway genes.

Conclusion: Two microRNAs that potentially affect permeability and visceral nociception were identified to be altered in IBS patients. MiR-219a-5p and miR-338-3p potentially alter barrier function and visceral hypersensitivity via neuronal and MAPK signaling and could be therapeutic targets in IBS.

Keywords: Barrier Function; MAPK Signaling; miR-219a-5p; miR-338-3p; miRNA.

Figure 1

shows the heatmap of the normalized expression of 12 differentially expressed miRNAs. Heatmap colors range from dark blue, white, and dark red, representing low, average, and high expression, respectively. Dx, diagnosis; M, Male; F, Female; IBS-C constipation-predominant IBS; IBS-D, diarrhea-predominant IBS. The bottom-panel shows variables associated with the samples.

Figure 2

shows a network of deregulated genes and select gene ontology (GO) terms associated with the inhibition of miR-219a-5p. Nodes and edges are described in the top right panel of the figure.

Figure 3

shows A) western blots of tight junction protein 1 (TJP1 or ZO1) and E-cadherin (CDH1) proteins performed in NCM460 cells, B) Protein levels of ZO1 and CDH1 by densitometry. The error bars represent mean ± standard deviation, and the data represent fold changes relative to control. The experiments were performed in duplicate.

Figure 4

shows, A) a progressive decrease in trans-epithelial electrical resistance (TEER) over 3 days in Caco-2 cells treated with miR-219a-5p inhibitor (Anti_miR_219, red line) compared to cells treated with control miRNA (Anti_miR_Control, blue line), and B) an increased dextran flux over 6 hours (measured every 2 hours) in cells treated with miR-219a-5p inhibitor (Anti_miR_219, red line) compared to cells treated with control miRNA (Anti_miR_Control, blue line). *, p<0.05; **, p<0.01.

Figure 5

shows a network of deregulated genes and select gene ontology (GO) terms associated with the inhibition of hsa-miR-338-3p. Nodes and edges are described in the top right panel of the Figure.