Long noncoding RNA uc.230/CUG-binding protein 1 axis sustains intestinal epithelial homeostasis and response to tissue injury
Ting-Xi Yu, Sudhakar Kalakonda, Xiangzheng Liu, Naomi Han, Hee K. Chung, Lan Xiao, Jaladanki N. Rao, Tong-Chuan He, Jean-Pierre Raufman, Jian-Ying Wang
Ting-Xi Yu, Sudhakar Kalakonda, Xiangzheng Liu, Naomi Han, Hee K. Chung, Lan Xiao, Jaladanki N. Rao, Tong-Chuan He, Jean-Pierre Raufman, Jian-Ying Wang
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Research Article Gastroenterology

Long noncoding RNA uc.230/CUG-binding protein 1 axis sustains intestinal epithelial homeostasis and response to tissue injury

Abstract

Intestinal epithelial integrity is commonly disrupted in patients with critical disorders, but the exact underlying mechanisms are unclear. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control different cell functions and are involved in pathologies. Here, we investigated the role of T-UCRs in intestinal epithelial homeostasis and identified T-UCR uc.230 as a major regulator of epithelial renewal, apoptosis, and barrier function. Compared with controls, intestinal mucosal tissues from patients with ulcerative colitis and from mice with colitis or fasted for 48 hours had increased levels of uc.230. Silencing uc.230 inhibited the growth of intestinal epithelial cells (IECs) and organoids and caused epithelial barrier dysfunction. Silencing uc.230 also increased IEC vulnerability to apoptosis, whereas increasing uc.230 levels protected IECs against cell death. In mice with colitis, reduced uc.230 levels enhanced mucosal inflammatory injury and delayed recovery. Mechanistic studies revealed that uc.230 increased CUG-binding protein 1 (CUGBP1) by acting as a natural decoy RNA for miR-503, which interacts with Cugbp1 mRNA and represses its translation. These findings indicate that uc.230 sustains intestinal mucosal homeostasis by promoting epithelial renewal and barrier function and that it protects IECs against apoptosis by serving as a natural sponge for miR-503, thereby preserving CUGBP1 expression.

Authors

Ting-Xi Yu, Sudhakar Kalakonda, Xiangzheng Liu, Naomi Han, Hee K. Chung, Lan Xiao, Jaladanki N. Rao, Tong-Chuan He, Jean-Pierre Raufman, Jian-Ying Wang

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Figure 8

Uc.230 prevents apoptosis by increasing CUGBP1 via interaction with miR-503.

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Uc.230 prevents apoptosis by increasing CUGBP1 via interaction with miR...
(A) Immunoblots of RBPs and anti-apoptotic proteins after silencing or overexpressing uc.230. Caco-2 cells were transfected with anti-uc.230 or uc.230 expression vector, and levels of various proteins were examined 48 hours after the transfections. (B) Levels of biotinylated miR-miR-503 (left) and U6 RNA (right) 24 hours after transfection with biotinylated miR-503. Values are the means ± SEM (n = 3). *P < 0.05 compared with cells transfected with scrambled oligomer. (C) Binding of biotinylated miR-503 to uc.230 and mRNAs Cugbp1 and Cdk2 in cells described in B. *P < 0.05 compared with scrambled oligomer (n = 3). (D) Levels of miR-503 48 hours after cells were transfected with pre–miR-503 alone or cotransfected with pre–miR-503 and uc.230 expression vector. *P < 0.05 compared with cells transfected with control vector or uc.230 expression vector alone (n = 3). (E) Effect of increasing uc.230 on miR-503/Cugbp1 mRNA association. Cells were transfected with bio-miR-503 or scrambled oligomer 24 hours after cells were transfected with uc.230 expression vector. The levels of Cugbp1 mRNA in the materials pulled down by miR-503 were examined 24 hours thereafter. *P < 0.05 compared with cells transfected with scrambled oligomer and +P < 0.05 compared with cells transfected with bio-miR-503 with control vector. (F) Representative immunoblots of CUGBP1 48 hours after cells were transfected with pre–miR-503 alone or cotransfected with pre–miR-503 and uc.230 expression vector. Experiments were repeated 3 separate times and showed similar results. (G and H) Percentages of TNFα/CHX–induced apoptosis in cells cotransfected with uc.230 expression vector and siCUGBP1 or cotransfected with anti-uc.230 and CUGBP1 expression vector. Cells were treated with TNFα/CHX 48 hours after cotransfection, and apoptosis was measured 4 hours thereafter. Values are means ± SEM (n = 5). *P < 0.05 compared with untreated group; +P < 0.05 compared with cells transfected with vector or control oligo; and #P < 0.05 compared with cells transfected with uc.230 expression vector or anti-uc.230 alone. In B, C, and E, statistical significance was analyzed using unpaired, 2-tailed Student’s t tests, while comparisons of means between more than 2 groups were performed by 1-way ANOVA with Tukey’s multiple-comparison test for D, G, and H.