Downregulation of occludin protein in necrotizing enterocolitis is associated with altered expression of microRNA-874

Author(s):
Christie Buonpane; Guillermo Ares; John Sincavage; Carrie Yuan; Douglas Wood; Catherine Hunter

Background:

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates. NEC is associated with increased intestinal permeability due to changes in tight junction proteins in the intestinal epithelium (i.e. occludin). MicroRNAs (miRNAs) are small non-coding RNAs that serve as key regulators of tight junction proteins by inducing translational inhibition or transcript degradation. Aberrant expression of miRNAs is found in the intestinal epithelium of patients with inflammatory bowel disease and other intestinal disorders; however, no studies have examined the role of miRNAs in NEC.

Hypothesis:

We hypothesize that decreased occludin is regulated by miRNA expression in the intestinal epithelium during NEC.

Methods:

To test our hypothesis, we analyzed patterns of 3 occludin-regulating miRNAs (miR-21, miR-122, and miR-874). We examined human intestinal NEC (N=6) vs control samples (N=6) as well as an in vitro enteroid model. The enteroids (intestinal epithelial organoids) were exposed to lipopolysaccharide (LPS) for 24 hours to create an in vitro model of experimental NEC compared to untreated controls. Changes in miRNA, mRNA and protein were analyzed by RT-PCR, western blot and immunofluorescence. Data was analyzed with Student’s t-test or ANOVA.

Results:

Significant changes of miR-21 or 122 were not identified. Decreased expression of miR-874 (p=0.002) in human intestinal NEC samples was associated with decreased occludin mRNA (p=0.006) and protein expression (p=0.009). Similarly, enteroids treated with LPS expressed lower levels of miR-874 than the controls (p=0.008). This finding was accompanied by a 2.7 fold decrease in occludin mRNA (p=0.005).

Conclusions:

There is a downregulation of occludin RNA and protein in NEC preceded by decreased miR-874 expression. Thus, miR-874 may be an important regulator and biomarker of NEC.