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. 2021 Oct;161(4):1303-1317.e3.
doi: 10.1053/j.gastro.2021.05.060. Epub 2021 Jun 9.

Circular RNA CircHIPK3 Promotes Homeostasis of the Intestinal Epithelium by Reducing MicroRNA 29b Function

Lan Xiao  1 , Xiang-Xue Ma  2 , Jason Luo  2 , Hee K Chung  2 , Min S Kwon  2 , Ting-Xi Yu  2 , Jaladanki N Rao  1 , Rosemary Kozar  3 , Myriam Gorospe  4 , Jian-Ying Wang  5
Affiliations

Circular RNA CircHIPK3 Promotes Homeostasis of the Intestinal Epithelium by Reducing MicroRNA 29b Function

Lan Xiao et al. Gastroenterology. 2021 Oct.

Abstract

Background & aims: Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that form covalently closed circles. Although circRNAs influence many biological processes, little is known about their role in intestinal epithelium homeostasis. We surveyed circRNAs required to maintain intestinal epithelial integrity and identified circular homeodomain-interacting protein kinase 3 (circHIPK3) as a major regulator of intestinal epithelial repair after acute injury.

Methods: Intestinal mucosal tissues were collected from mice exposed to cecal ligation and puncture for 48 hours and patients with inflammatory bowel diseases and sepsis. We isolated primary enterocytes from the small intestine of mice and derived intestinal organoids. The levels of circHIPK3 were silenced in intestinal epithelial cells (IECs) by transfection with small interfering RNAs targeting the circularization junction of circHIPK3 or elevated using a plasmid vector that overexpressed circHIPK3. Intestinal epithelial repair was examined in an in vitro injury model by removing part of the monolayer. The association of circHIPK3 with microRNA 29b (miR-29b) was determined by biotinylated RNA pull-down assays.

Results: Genome-wide profile analyses identified ∼300 circRNAs, including circHIPK3, differentially expressed in the intestinal mucosa of mice after cecal ligation and puncture relative to sham mice. Intestinal mucosa from patients with inflammatory bowel diseases and sepsis had reduced levels of circHIPK3. Increasing the levels of circHIPK3 enhanced intestinal epithelium repair after wounding, whereas circHIPK3 silencing repressed epithelial recovery. CircHIPK3 silencing also inhibited growth of IECs and intestinal organoids, and circHIPK3 overexpression promoted intestinal epithelium renewal in mice. Mechanistic studies revealed that circHIPK3 directly bound to miR-29b and inhibited miR-29 activity, thus increasing expression of Rac1, Cdc42, and cyclin B1 in IECs after wounding.

Conclusions: In studies of mice, IECs, and human tissues, our results indicate that circHIPK3 improves repair of the intestinal epithelium at least in part by reducing miR-29b availability.

Keywords: CircRNAs; IBD; IEC; MicroRNAs; Mucosal Injury.

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Conflict of interest statement

Conflicts of interest

The authors disclose no conflicts.

Figures

Figure 1.
Figure 1.
Changes in expression of circRNAs in the intestinal mucosa in septic mice and patients with critical illnesses. (A) Heat map (a) and scatter plot (b) depictions of circRNAs differentially expressed in the small intestinal mucosa in mice exposed to CLP for 48 h as measured by circRNA microarray. (B) Differential expression analysis of circRNAs in results described in A. Values are the means from three animals. (C) Levels of mucosal circHIPK3 and other circRNAs in the small intestine as examined by Q-PCR analysis. Values are the means ± SEM (n = 5). * P < 0.05 compared with sham mice. (D) Total levels and quantification of copy numbers of circHIPK3 in the intestinal mucosa from patients with active CD and UC as measured by Q-PCR and droplet digital Q-PCR analyses, respectively. Values are the means ± SEM (n = 6). * P < 0.05 compared with controls. (E) RNA-FISH analysis of circHIPK3 with fluorescent LNA-RNA detection probe in the mucosa from patients with CD, UC, and sepsis, as shown in yellow-green. Scale bars: 25 μm. All these experiments were repeated in tissue samples obtained from 4 control individuals, 4 patients with CD, 4 patients with UC 4 or 4 patients with sepsis and showed similar results.
Figure 2.
Figure 2.
CircHIPK3 enhances intestinal epithelial repair after wounding. (A) Levels of circHIPK3 (left) and HIPK3 mRNA and HIPK3 protein (right) in Caco-2 cells 48 h after transfection with the circHIPK3 expression vector. Values are the means ± SEM (n = 3). * P < 0.05 compared with control. (B) Epithelial repair after wounding in cells described in A. Left: images of epithelial repair; and right: summarized data. Values are the means ± SEM (n = 6). (C) Levels of circHIPK3 (left) and the mRNA and protein (right) of HIPK3 48 h after transfection. Values are the means ± SEM (n = 3). (D) Epithelial repair after wounding in cells described in C. Values are the means ± SEM (n = 6).
Figure 3.
Figure 3.
CircHIPK3 silencing prevents increase in the levels of migration- and growth-associated proteins and alters actin dynamics. (A) Immunoblots of various proteins relevant to cell migration (top) and growth (bottom) after wounding. (B) Immunostaining of F-actin in cells described in A. F-actin: green; nuclei: blue. Scale bar, 25 μm. Three separate experiments were performed and showed similar results.
Figure 4.
Figure 4.
CircHIPK3 is essential for renewal of the intestinal epithelium. (A) Cell growth after circHIPK3 silencing in vitro. Values are means ± SEM (n = 3). * P < 0.05 compared with C-siRNA. (B) Growth of small intestinal organoids after circHIPK3 silencing ex vivo. Left: bright field microscopy analysis of growth of organoids on day 8; and right: confocal analysis of BrdU (red) and DAPI (blue) on day 3 after culture. Scale bars: 100 μm. (C) Quantification of surface area (top) and BrdU positive cells (bottom) of the organoids described in B (n = 6). (D) Immunostainings of lysozyme- (left) and villin-positive (right) cells in intestinal organoids on day 5 after transfection with si-cHIPK3 or C-siRNA. Red, lysozyme; purple, villin. Scale bars: 100 μm. (E) Levels of circHIPK3 in the small intestinal mucosa of mice on day 5 after intraperitoneal injection with a recombinant circHIPK3 lentiviral expression vector (L-cHIPK3) or empty control lentiviral vector (Vector). * P < 0.05 compared with control vector (n = 6). (F) Proliferating cells in small intestinal crypts as measured by BrdU labeling (1 h post-injection, green) in mice described in E. (G) Quantification of BrdU-positive cells in the mucosa described in F. * P < 0.05 compared with control vector (n = 6).
Figure 5.
Figure 5.
CircHIPK3 acts as a sponge of miR-29b in vitro. (A) Schematic of the putative binding sites of miR-29b on circHIPK3 transcript. (B) Levels of miR-29b 24 h after transfection with biotinylated miR-29b. Values are the means ± SEM (n = 3). * P <0.05 compared with control scramble oligomer. (C) Binding of biotinylated miR-29b to various circRNAs. Left, levels of circHIPK3, Cdr1as, and circPABPN1 in the materials pulled down by biotin-miR-29b; and right, levels of total input circRNAs. (D) Levels of luciferase reporter activity after circHIPK3 silencing or its overexpression. Left: Schematic of the chimeric mRNA vector bearing two perfect binding sites (BS) of miR-29b. * P <0.05 compared with cells transfected with C-siRNA or control vector.
Figure 6.
Figure 6.
CircHIPK3 regulates intestinal epithelial repair by interacting with miR-29b. (A) Levels of miR-29b (left) and U6 RNA (right) 24 h after transfection with biotinylated miR-29b. Values are the means ± SEM (n = 3). * P <0.05 compared with control scramble. (B) Levels of mRNAs encoding Rac1, Cdc42, RhoA, cyclin B1 (CycB1), CDK4, and p21 in the materials pulled down by biotin-miR-29b. * P <0.05 compared with control scramble. (C) Immunoblots of various proteins. (D) Immunostaining of F-actin at 6 (top) and 24 (bottom) h after wounding in cells described in C. (E) Summarized data in cells described D (bottom). *, +P < 0.05 compared with control and miR-29b expression alone, respectively. (F) Model proposed to explain the influence of circHIPK3 upon intestinal epithelial repair after wounding.
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