Long noncoding RNA GAS5 disrupts intestinal epithelial barrier function by increasing small vault RNA levels
Ting-Xi Yu, Hee Kyoung Chung, Amy VanderStoep, Bridgette Warner, Hongxia Chen, Haonan Zhao, Ana S.G. Cunningham, Rosemary Kozar, Myriam Gorospe, Lan Xiao, Jian-Ying Wang
Ting-Xi Yu, Hee Kyoung Chung, Amy VanderStoep, Bridgette Warner, Hongxia Chen, Haonan Zhao, Ana S.G. Cunningham, Rosemary Kozar, Myriam Gorospe, Lan Xiao, Jian-Ying Wang
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Research Article Cell biology Gastroenterology

Long noncoding RNA GAS5 disrupts intestinal epithelial barrier function by increasing small vault RNA levels

Abstract

Disruptions in the integrity of the intestinal epithelium occur commonly in inflammatory bowel disease (IBD) and critical surgical disorders, but the underlying mechanisms remain largely unknown. Here we identified long noncoding RNA GAS5 as a repressor of intestinal mucosal growth and the function of the gut epithelial barrier. The levels of tissue GAS5/Gas5 increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with IBD. Transient and tissue-specific knockdown of Gas5 in mice using CRISPR/Cas9 enhanced the renewal of the mucosa of the small intestine, increased the levels of tight junction (TJ) proteins ZO-1, ZO-2, claudin-1, and claudin-2, and improved gut barrier function. Conversely, ectopic overexpression of GAS5 in intestinal organoids and in cultured intestinal epithelium cells decreased the levels of these TJ proteins and caused epithelial barrier dysfunction. Mechanistic studies revealed that GAS5 acted as a transcriptional enhancer of the gene encoding small noncoding vault RNAs (vtRNAs) and that GAS5 repressed TJ expression by increasing the levels of vtRNAs. Together, our results indicate that GAS5 disrupts the integrity of the intestinal epithelium by impairing mucosal growth and epithelial barrier function and that it represses TJ expression, at least in part, via vtRNAs.

Authors

Ting-Xi Yu, Hee Kyoung Chung, Amy VanderStoep, Bridgette Warner, Hongxia Chen, Haonan Zhao, Ana S.G. Cunningham, Rosemary Kozar, Myriam Gorospe, Lan Xiao, Jian-Ying Wang

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Figure 5

Ectopically expressed GAS5 disrupts intestinal epithelial barrier function in cultured IECs.

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Ectopically expressed GAS5 disrupts intestinal epithelial barrier functi...
(A) Levels of GAS5 in Caco-2 cells 48 hours after transfection with GAS5 expression vector. Values are the mean ± SEM (n = 3). *P < 0.05 compared with control vector. (B) Expression levels of intercellular junction proteins in cells treated described in A, as assessed by Western blot analysis. GAPDH was included as a loading control. (C) Distribution of tight junction proteins in cells treated as described in A. Forty-eight hours after transfection, cells were fixed, permeabilized, and incubated first with antibodies against different intercellular junction proteins and then with TRITC-conjugated anti-IgG. Original magnification, ×500. (D) Changes in TEER (left) and FITC-dextran paracellular permeability (right) in cells treated as described in A. TEER assays were performed on 12-mm Transwell filters; paracellular permeability was assayed by adding the membrane-impermeable trace molecule FITC-dextran to the insert medium. Values are the mean ± SEM (n = 6). *P < 0.05 compared with control vector. In A and D, statistical significance was analyzed using unpaired, 2-tailed Student’s t test. In B and C, 3 separate experiments were performed and showed similar results.