IL1B Increases Intestinal Tight Junction Permeability by Upregulation of MIR200C-3p, Which Degrades Occludin mRNA. Journal Article

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Authors: Rawat, M; Nighot, M; Al-Sadi, R; Gupta, Y; Viszwapriya, D; Yochum, G; Koltun, W; Ma, TY
Article Title: IL1B Increases Intestinal Tight Junction Permeability by Upregulation of MIR200C-3p, Which Degrades Occludin mRNA.
Abstract: BACKGROUND AIMS: Defects in the epithelial tight junction (TJ) barrier contribute to development of intestinal inflammation associated with diseases. Interleukin 1 beta (IL1B) increases intestinal permeability in mice. We investigated microRNAs that are regulated by IL1B and their effects on expression of TJ proteins and intestinal permeability. METHODS: We used TargetScan to identify microRNAs that would bind the 3'UTR of occludin mRNA; regions that interacted with microRNAs were predicted using the V-fold server and Assemble2, and 3-dimensional models were created using UCSF Chimera linked with Assemble2. Caco-2 cells were transfected with vectors that express microRNAs, analyzed by immunoblots and real-time PCR, and grown as monolayers; permeability in response to IL1B was assessed with the marker inulin. Male C57BL/6 mice were given intraperitoneal injections of IL1B and intestinal recycling perfusion was measured; some mice were given dextran sodium sulfate to induce colitis and/or gavage with an antagonist to MIR200C-3p (antagomiR-200C) or the non-specific antagomiR (control). Intestinal tissues were collected from mice and analyzed by histology and real-time PCR; enterocytes were isolated by laser capture microdissection. We also analyzed colon tissues and organoids from patients with and without ulcerative colitis. RESULTS: Incubation of Caco-2 monolayers with IL1B increased TJ permeability and reduced levels of occludin protein and mRNA without affecting the expression of other transmembrane TJ proteins. Targetscan identified MIR122, MIR200B-3p, and MIR200C-3p, as miRNAs that might bind to the occludin 3'UTR. MIR200C-3p was rapidly increased in Caco-2 cells incubated with IL1B; the antagomiR-200c prevented the IL1B-induced decrease in occludin mRNA and protein and reduced TJ permeability. Administration of IL1B to mice increased small intestinal TJ permeability, compared with mice given vehicle; enterocytes isolated from mice given IL1B had increased expression of MIR200C-3p and decreased levels of occludin mRNA and protein. Intestinal tissues from mice with colitis had increased levels of IL1B mRNA and MIR200C-3p and decreased levels of occludin mRNA; gavage of mice with antagomiR-200C reduced levels of MIR200C-3p and prevented the decrease in occludin mRNA and the increase in colonic permeability. Colon tissues and organoids from patients with ulcerative colitis had increased levels of IL1B mRNA and MIR200C-3p compared with healthy controls. Using 3-dimensional molecular modeling and mutational analyses, we identified the nucleotide bases in the occluding mRNA 3'UTR that interact with MIR200C-3p. CONCLUSIONS: Intestine tissues from patients with ulcerative colitis and mice with colitis have increased levels of IL1B mRNA and MIR200C-3p, which reduces expression of occludin by enterocytes and thereby increases TJ permeability. Three-dimensional modeling of the interaction between MIR200C-3p and the occludin mRNA 3'UTR identified sites of interaction. The antagomiR-200C prevents the decrease in occludin in enterocytes and intestine tissues of mice with colitis, maintaining the TJ barrier.
Journal Title: Gastroenterology
ISSN: 1528-0012; 0016-5085
Publisher: Unknown  
Date Published: 2020