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 7

GAS5 elevates the abundance of vtRNAs by increasing gene transcription.

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GAS5 elevates the abundance of vtRNAs by increasing gene transcription....
(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) Changes in the levels of vtRNA1-1 (vtR1-1), vtR1-2, vtR1-3, and vtR2-1 in cells treated as described in A (n = 3). *P < 0.05 compared with control vector. (C) Levels of GAS5 in Caco-2 cells 48 hours after transfection with si-GAS5 or C-siRNA. *P < 0.05 compared with C-siRNA (n = 3). (D) Levels of vtRNAs in cells treated as described in C. *P < 0.05 compared with C-siRNA (n = 3). (E) Levels of mouse vtRNA in the small intestinal mucosa of control and Gas5-knockdown (Gas5-KD) mice. *P < 0.05 compared with controls (n = 5). (F) Structure (top) and activity (bottom) of luciferase (Luc) reporter of the vtRNA1 promoter. Cells were transfected with control pGL3 vector or Luc-vtRNA1 (vtR1) promoter, and the luciferase activity was examined 48 hours later. *P < 0.05 compared with pGL3 (n = 4). (G) Changes in the levels of luciferase activity of the vtR1 promoter in cells overexpressing GAS5 (left) or GAS5-silent cells (right). The luciferase activity was examined 48 hours after cotransfection with GAS5 expression vector and Luc-vtR1-promoter or cotransfection with si-GAS5 and Luc-vtR1-promoter. *P < 0.05 compared with vector or C-siRNA (n = 4). In all studies, 2-tailed Student’s t test was used for statistical analysis.