1. Volume 154, Issue 3, Pages 599-611 (February 2018)
Long Noncoding RNA uc.173 Promotes Renewal of the Intestinal Mucosa by Inducing Degradation of MicroRNA 195 
Lan Xiao, Jing Wu, Jun-Yao Wang, Hee Kyoung Chung, Sudhakar Kalakonda, Jaladanki N. Rao, Myriam Gorospe, Jian-Ying Wang 
Gastroenterology 
Volume 154, Issue 3, Pages (February 2018)
DOI: /j.gastro
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2. Gastroenterology 2018 154, 599-611DOI: (10.1053/j.gastro.2017.10.009)
Copyright © 2018 AGA Institute Terms and Conditions

3. Figure 1
Changes in T-UCR expression in the intestinal mucosa after food starvation. (A) Scatter plot (top) and heat map depiction (bottom) of T-UCRs differentially expressed in the small intestinal mucosa in mice fasted for 48 hours as examined by T-UCR microarray. (B) Differential expression analysis of T-UCRs (>2-fold down or up) observed in results described in A. Values are means ± SEM of data from 4 animals. *P < .05 compared with controls. (C) Basal levels of various T-UCRs relative to uc.173 in the mucosa as measured by RT-Q-PCR analysis. (D) Levels of mucosal uc.173 in the small intestine as measured by RT-Q-PCR analysis. Values are means ± SEM of data from 6 animals. *P < .05 compared with controls. (E) Levels of uc.173 in cells growth-arrested in G1 phase by polyamine depletion. Caco-2 cells were exposed to DFMO (5 mmol/L) alone or DFMO plus putrescine (Put, 10 μmol/L) for 4 days. *P < .05 compared with controls or cells treated with DFMO plus Put. (F) Levels of cytoplasmic (cyto) and nuclear uc.173, host gene UBE2B mRNA, and lncRNA HULC in Caco-2 cells.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 2
Ectopically expressed uc.173 promotes intestinal epithelial renewal. (A) Levels of uc.173 in cells transfected with the uc.173 expression vector (left) for 48 hours as measured by RT-Q-PCR. Values are means ± SEM from 3 separate experiments. *P < .05 compared with cells control vector. (B) Cell growth after uc.173 overexpression (O/E). Twenty-four hours after the transfection, equal number of cells was seeded in 24-well plates. *P < .05 compared with control vector. (C) Sizes of small intestinal organoids after uc.173 overexpression. Organoids were isolated from proximal small intestine and grown in the indicated conditions: i) confocal analysis of BrdU (red) and F-actin (green) on day 3 after culture; and ii) bright field microscopy analysis of growth of organoids on day 8. Scale bars: 100 μm. (D) Quantification of BrdU (left) in organoids described in Ci and surface area (right) of organoids described in Cii (n = 6). *P < .05 compared with control vector.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 3
LNA-mediated uc.173 silencing inhibits intestinal mucosal renewal. (A) Levels of cellular uc hours after transfection with anti-uc.173_1 or anti-uc.173_2 in Caco-2 cells. Values are means ± SEM from 3 separate experiments. *P < .05 compared with control oligonucleotides (Con-oligo). (B) Cell growth after uc.173 silencing. *P < .05 compared with Con-oligo. (C) Levels of uc.173 (left) and uc.346 (right) in the small intestinal mucosa in mice injected intraperitoneally with LNA-anti-uc.173_1 or Con-oligo for 4 consecutive days. On the day 5, total RNA was isolated for RT-Q-PCR analysis. Values are means ± SEM of data from 5 animals. *P < .05 compared with Con-oligo. (D) Photomicrographs of hematoxylin/eosin (H/E) staining of the small intestinal mucosa taken 0.5 cm distal to the ligament of Trietz in mice described in C. Scale bar: 100 μm. (E) Changes in the length of villi (left) and crypt (right) of the mucosa described in C. (F) Expression levels of PCNA in the mucosa described in C. (G) Association of decreased uc.173 with reduction in cell proliferation in patients with Crohn’s disease. Left: in situ hybridization of uc.173 with fluorescent LNA-RNA detection probe in the intestinal mucosa, as shown in yellow-green; middle: immunostaining of Ki67, as shown in red; right: immunostaining of occludin, as shown in green. Experiments were repeated in samples obtained from 5 patients with Crohn’s disease or controls and showed similar results. Scale bars: 100 μm.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 4
uc.173 specifically down-regulates miRNA195 in IECs. (A) The levels of mature miRNAs after uc.173 silencing. Caco-2 cells were transfected with anti-uc.173_1 or Con-oligo, and different miRNAs were examined 48 hours thereafter. Values are means ± SEM from 3 separate experiments. *P < .05 compared with Con-oligo. (B) Levels of mature miRNAs 48 hours after transfection with the uc.173 expression vector. *P < .05 compared with control vector. (C and D) Levels of primary miRNA195 (pri-miR-195) and pri-miRNA222 (pri-miR-222) in cells described in A and B.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 5
uc.173 does not alter transcription of the miRNA195 gene. (A) Levels of miRNA195 (left) and miRNA29b (right) in the small intestinal mucosa after injection with Anti-uc.173_1 for 4 consecutive days. Values are means ± SEM of data from 5 mice. *P < .05 compared with Con-oligo. (B) Immunoblots of p53, JunD and RNA-binding proteins 48 hours after transfection with anti-uc.173_1 in Caco-2 cells. (C) Schematic of miRNA195 promoter luciferase (Luc) reporter constructs. p53 and AP1 indicate the relative location of p53- and AP1-binding sites within the miRNA195 promoter, respectively. (D) Levels of miRNA195 promoter activity in cells described in B: i) uc.173 levels; and ii) activity of the Luc reporter. Values are means ± SEM from 3 separate experiments. (E) Levels of miRNA195 promoter activity in cells overexpressing uc.173: i) uc.173 levels; and ii) activity of the Luc reporter. *P < .05 compared with control vector.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 6
uc.173 interacts with and destabilizes pri-miR-195. (A) Schematic of pri-miR-195 depicting the stem-loop sequence and its complementarity with uc.173. (B) uc.173 association with pri-miR-195 as measured by using biotinylated pri-miR-195. Values are means ± SEM from 3 separate experiments. *P < .05 compared with biotin-labeled mature miRNA195. (C) Stability of the pri-miR-195 after uc.173 overexpression. Levels of pri-miRNAs were examined at different times after administration with actinomycin D. (D) Cell growth in uc.173-silenced cells co-transfected with anti-miR-195. *P < .05 compared with anti-uc.173 alone. (E) Cell growth in uc.173 overexpressing cells co-transfected with pre-miR-195. *P < .05 compared with control vector or uc.173 expression vector alone. (F) Model proposed to explain the influence of uc.173 upon the gut epithelial homeostasis. Uc.173 directly interacts with and destabilizes the pri-miR-195 transcript, thus decreasing the levels of cellular miRNA195.
Gastroenterology  , DOI: ( /j.gastro )
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