Gastroenterology

Gastroenterology

Volume 155, Issue 1, July 2018, Pages 144-155
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
In Inflamed Intestinal Tissues and Epithelial Cells, Interleukin 22 Signaling Increases Expression of H19 Long Noncoding RNA, Which Promotes Mucosal Regeneration

https://doi.org/10.1053/j.gastro.2018.03.058Get rights and content

Background & Aims

Inflammation affects regeneration of the intestinal epithelia; long noncoding RNAs (lncRNAs) regulate cell functions, such as proliferation, differentiation, and migration. We investigated the mechanisms by which the lncRNA H19, imprinted maternally expressed transcript (H19) regulates regeneration of intestinal epithelium using cell cultures and mouse models of inflammation.

Methods

We performed RNA-sequencing transcriptome analyses of intestinal tissues from mice with lipopolysaccharide (LPS)-induced sepsis to identify lncRNAs associated with inflammation; findings were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization analyses of intestinal tissues from mice with sepsis or dextran sulfate sodium (DSS)–induced mucosal wound healing and patients with ulcerative colitis compared to healthy individuals (controls). We screened cytokines for their ability to induce expression of H19 in HT-29 cells and intestinal epithelial cells (IECs), and confirmed findings in crypt epithelial organoids derived from mouse small intestine. IECs were incubated with different signal transduction inhibitors and effects on H19 lncRNA levels were measured. We assessed intestinal epithelial proliferation or regeneration in H19ΔEx1/+ mice given LPS or DSS vs wild-type littermates (control mice). H19 was overexpressed in IECs using lentiviral vectors and cell proliferation was measured. We performed RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays to study functions of H19 in IECs.

Results

In RNA-sequencing transcriptome analysis of lncRNA expression in intestinal tissues from mice, we found that levels of H19 lncRNA changed significantly with LPS exposure. Levels of H19 lncRNA increased in intestinal tissues of patients with ulcerative colitis, mice with LPS-induced and polymicrobial sepsis, or mice with DSS-induced colitis, compared with controls. Increased H19 lncRNA localized to epithelial cells in the intestine, regardless of Lgr5 messenger RNA expression. Exposure of IECs to interleukin 22 (IL22) increased levels of H19 lncRNA with time and dose, which required STAT3 and protein kinase A activity. IL22 induced expression of H19 in mouse intestinal epithelial organoids within 6 hours. Exposure to IL22 increased growth of intestinal epithelial organoids derived from control mice, but not H19ΔEx1/+ mice. Overexpression of H19 in HT-29 cells increased their proliferation. Intestinal mucosa healed more slowly after withdrawal of DSS from H19ΔEx1/+ mice vs control mice. Crypt epithelial cells from H19ΔEx1/+ mice proliferated more slowly than those from control mice after exposure to LPS. H19 lncRNA bound to p53 and microRNAs that inhibit cell proliferation, including microRNA 34a and let-7; H19 lncRNA binding blocked their function, leading to increased expression of genes that promote regeneration of the epithelium.

Conclusions

The level of lncRNA H19 is increased in inflamed intestinal tissues from mice and patients. The inflammatory cytokine IL22 induces expression of H19 in IECs, which is required for intestinal epithelial proliferation and mucosal healing. H19 lncRNA appears to inhibit p53 protein and microRNA 34a and let-7 to promote proliferation of IECs and epithelial regeneration.

Section snippets

Materials and Methods

Detailed protocols are provided in the Supplementary Materials and Methods.

Inflammation Results in the Induction of Intestinal H19 Long Noncoding RNA That Is Localized to Lgr5+ and Lgr5 Epithelial Cells in the Intestinal Mucosa

Although lncRNAs are thought to be a vast family of functional molecules associated with diverse biological processes in cells, their roles in sustaining tissue homeostasis in vivo remain largely unknown. To fill this knowledge gap, we profiled gene expression in the small intestine of mice with lipopolysaccharide (LPS)-induced sepsis using RNA sequening (RNA-seq) transcriptome analysis. LPS challenge for 24 hours resulted in alterations in the expression of a large number of protein-coding

Discussion

H19 lncRNA is an imprinted and maternally expressed transcript that is found in various tissues during embryogenesis but is silenced after birth.17 Its expression can be reactivated in numerous disease conditions, such as tumors and rheumatoid arthritis.18, 19 H19-deficient mice appear healthy, develop normally, and are fertile,20 suggesting that H19 lncRNA is not essential for embryonic development and viability. However, little is known about the role of H19 lncRNA in inflammation and

Acknowledgments

The authors thank members of the Jun Sun laboratory for advice on preparing mouse intestinal epithelial organoids. Accession number for RNA-sequencing data in the Gene Expression Omnibus database: GSE97371. Author contributions: Study concept and design: H.G. and X.-D.T. were involved in the overall design of experiments and interpretation of results. H.G., H.-F.B., and F.L. performed in vivo experiments. H.G., H.-F.B., F.L., P.W., and X.W. performed in vitro experiments. H.G. and L.W. designed

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    Conflicts of interest The authors disclose no conflicts.

    Funding This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases grant R01DK064240 (X.-D.T), National Institute of General Medical Sciences grants R01GM117628 (X.-D.T.) and R01GM122406 (X.-D.T.), US Department of Veterans Affairs Merit Award I01BX001690 (X.-D.T.), Dorothy M. and Edward E. Burwell Professorship (X.-D.T.), and Ministry of Health of the People’s Republic of China Special Project grant no. 201002020 (J.Q.). The sponsors had no role in study design or collection, analysis, or interpretation of the data.

    Author names in bold designate shared co-first authorship.

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