Transgenic Expression of miR-222 Disrupts Intestinal Epithelial Regeneration by Targeting Multiple Genes Including Frizzled-7
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Defects in intestinal epithelial integrity occur commonly in various pathologies. miR-222 is implicated in many aspects of cellular function and plays an important role in several diseases, but its exact biological function in the intestinal epithelium is underexplored. We generated mice with intestinal epithelial tissue-specific overexpression of miR-222 to investigate the function of miR-222 in intestinal physiology and diseases in vivo. Transgenic expression of miR-222 inhibited mucosal growth and increased susceptibility to apoptosis in the small intestine, thus leading to mucosal atrophy. The miR-222-elevated intestinal epithelium was vulnerable to pathological stress, since local overexpression of miR-222 not only delayed mucosal repair after ischemia/reperfusion-induced injury, but also exacerbated gut barrier dysfunction induced by exposure to cecal ligation and puncture. miR-222 overexpression also decreased expression of the Wnt receptor Frizzled-7 (FZD7), cyclin-dependent kinase 4 and tight junctions in the mucosal tissue. Mechanistically, we identified the Fzd7 messenger ribonucleic acid (mRNA) as a novel target of miR-222 and found that [miR-222/Fzd7 mRNA] association repressed Fzd7 mRNA translation. These results implicate miR-222 as a negative regulator of normal intestinal epithelial regeneration and protection by downregulating expression of multiple genes including the Fzd7. Our findings also suggest a novel role of increased miR-222 in the pathogenesis of mucosal growth inhibition, delayed healing and barrier dysfunction.
The mammalian intestinal epithelium is in a constant state of renewal, characterized by active proliferation of stem cells localized near the base of the crypts and progression of these cells up the crypt-villus axis with cessation of proliferation and subsequent differentiation and apoptosis (1,2). The epithelium of the human small intestine undergoes ∼1011 mitoses per day, and this dynamic turnover rate is tightly regulated by numerous factors at multiple levels (1, 2, 3). In response to stressful environments, the successful repair of damaged mucosa and maintenance of the intestinal epithelial integrity require epithelial cell decisions that regulate signaling networks controlling expression of various genes involved in cell survival, apoptosis, migration and proliferation (2,3). Disruption of the intestinal epithelial integrity occurs commonly during critical surgical conditions, leading to the translocation of luminal toxic substances and bacteria to the bloodstream and in some instances multiple organ dysfunction syndrome and death (4,5). However, the exact mechanism by which the intestinal epithelial integrity is preserved under biological and pathological conditions remains largely unknown.
Micro-ribonucleic acids (miRNAs) are small noncoding RNAs that bind to specific mRNAs and inhibit translation and/or promote mRNA degradation (6,7). High-throughput and functional studies show that miRNAs play important roles in physiological and pathological processes (6,8,9). Recently, miRNAs have emerged as master regulators of the gut epithelial homeostasis (3,10,11). We have profiled global miRNA expression in cultured intestinal epithelial cells (IECs) (2) and intestinal mucosa in mice (13) and have found that several miRNAs, including miRNA-222, miRNA-29b, miRNA-322/503 and miR-195, are highly expressed in the intestinal epithelium, and their expression levels are affected rapidly in response to food starvation and polyamine depletion. Further studies show that control of the expression of these miRNAs is crucial for maintenance of normal intestinal epithelial integrity and plays an important role in mucosal pathology. For example, mucosal atrophy in fasted mice is associated with increased miR-29b, whereas miR-29 silencing by systemic delivery of locked nucleic acid-modified anti-miR-29b oligonucleotides promotes the mucosal growth (13). Moreover, miR-322/503 regulates IEC apoptosis (14), and miR-195 represses rapid epithelial restitution after wounding (15).
miR-222 (also referred as miR-222-3p) is highly conserved among different species and is clustered with miR-221 in tandem on the X chromosome (16). miR-222 modulates distinct cellular functions (17, 18, 19), and its role in cancer development can be either tumor suppressive or oncogenic, depending on cellular content and tumor type (20,21). miR-222 in human colorectal cancer cells acts in a positive feedback loop to increase expression levels of RelA and STAT3 (22), but miR-222 is a potential repressor of estrogen receptor (ER)-α expression in 3T3-L1 adipocytes (23). Our previous studies indicate that miR-222 and the RNA-binding protein (RBP) CUGBP1/CELF1 (CUG triplet repeat, RNA binding protein 1/CUGBP Elav-like member 1) synergistically inhibit cyclin-dependent kinase 4 (CDK4) translation in normal IECs by recruiting the Cdk4 mRNA to processing bodies (12). Because most of our knowledge about miR-222 functions comes from studies conducted in cultured cells, the exact in vivo functions of miR-222, particularly in the intestinal epithelium, is not well understood. Here we developed transgenic mice that specially overexpress miR-222 in the intestinal epithelium and demonstrate that tissue-specific overexpression of miR-222 reduces growth and survival of the small intestinal mucosa by targeting multiple genes, including the Wnt receptor Frizzled-7 (Fzd7).
Materials and Methods
Generation of miR222-Tg Mice
To generate miR-222 transgenic (miR222-Tg) mice, a 770-base pair (bp) fragment, including the mouse miR-222 locus on chromosome X (289-bp primary miR-222 sequence) and human β-globin intron (222-bp 5′ upstream sequence and 259-bp 3′ sequence), was cloned into the pIRES-AcGFP1-Nuc vector (Supplementary Figure S1) by using an miR-222 cloning primer set (Supplementary Table S1). The A33 promoter was used to drive intestinal epithelial tissue-specific overexpression of the genomic miR-222 precursor, as reported by others (24,25). Transgenic founders on a pure C57BL/6J background were established by pronuclear injection at the University of Maryland, Baltimore transgenic animal core. Genotyping was performed by polymerase chain reaction (PCR) in deoxyribonucleic acid (DNA) extracted by tail clippings to identify the first generation of recombinant mice with miR222/green flourescence protein (GFP) bicistronic RNA (Supplementary Figure S2). Two founders were obtained, and they were further characterized for the transmission or the expression of the transgene. Transgenic colonies were subsequently established. Male miR-222-Tg mice mated with wild-type (WT) female mice to generate miR-222-Tg mice and their WT littermates for experiments. Representative results from two independent founders are reported here and compared with those obtained from littermate controls.
All experiments were approved according to animal experimental ethics committee guidelines by the University of Maryland Baltimore Institutional Animal Care and Use Committee. Mice were housed and handled in a specific pathogen-free breeding barrier and cared for by trained technicians and veterinarians. To examine gut mucosal growth, bromodeoxyuridine (BrdU) was incorporated in intestinal mucosa by intraperitoneal injection of 2 mg BrdU (Sigma-Aldrich) in phosphate-buffered saline. A 4-cm small intestinal segment taken from 0.5 cm distal to the ligament of Trietz and the segment of middle colon were collected 1 h after injection. To generate intestinal ischemia/reperfusion (I/R)-induced injury, mice were exposed to 30-min superior mesenteric artery ischemia, followed by 2-h reperfusion (26). Sham operation for controls involved laparotomy without mesenteric ischemia. Cecal ligation and puncture (CLP) was induced as described previously (28). The ligated cecum was punctured with a 25-gauge needle and slightly compressed with an applicator until a small amount of stool appeared. In sham-operated animals, the cecum was manipulated but without ligation and puncture. In experiments with apoptosis, mice were injected (intraperitoneally) with tumor necrosis factor (TNF)-α at the dose of 25 µg/kg body weight, and the mucosal tissues were harvested for measurement of apoptotic cell death at 5 h after injection.
Dissected and opened intestines were mounted onto a solid surface and fixed in formalin and paraffin. Sections at 5 µm were stained with hematoxylin and eosin (H&E) for general histology. Slides were examined in a blinded fashion by coding them, and only after examination was complete were they decoded. By using a grade micrometer eyepiece, the overall length of villus and crypts of each section was measured, and the villus:crypt ratio was calculated. Microscopic damages in the intestinal mucosa were measured and semiquantified as described previously (27).
Assays of Gut Permeability In Vivo
Fluorescein isothiocyanate (FITC)-conjugated dextran dissolved in water (Sigma-Aldrich; 4KD, 600 mg/kg) was administered to mice via gavage as described (28,33). Blood was collected 4 h thereafter via cardiac puncture. The serum concentration of the FITC-dextran was determined by using a plate reader with an excitation wavelength at 490 nm and an emission wavelength of 530 nm. The concentration of FITC-dextran in sera was determined by comparison to the FITC-dextran standard curve.
Chemicals and Cell Cultures
Tissue culture medium and dialyzed fetal bovine serum were from Invitrogen, and biochemicals were from Sigma-Aldrich. The antibodies recognizing CDK4, proliferating cell nuclear antigen (PCNA), claudin-1, zona occludens-1 (ZO-1), occludin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology and BD Biosciences, and the secondary antibody conjugated to horseradish peroxidase was from Sigma-Aldrich. Pre-miR™ miRNA precursor and Anti-miR™ miRNA inhibitor of miR-222 were purchased from Ambion. Biotin-labeled miRNA-222 was custom-made by Dharmacon. The IEC-6 cells, stable Wnt5a-transfected fibroblasts (Wnt5a-Fs) and control fibroblasts (Con-Fs) were purchased from ATCC. IEC-6 cells were derived from normal rat intestinal crypt cells, and fibroblasts were originally isolated from mouse subcutaneous connective tissues. To establish the coculture model, stable Wnt5a-Fs or Con-Fs were precultured on the dishes for 24 h and then cocultured with IEC-6 cells for additional 4 d before wounding as described (29).
The fragments of Fzd7 coding region (CR) and 3’-untranslated region (UTR) were subcloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) to generate the pmir-GLO-Luc-Fzd7-CR and pmirGLO-Luc-Fzd7-3′-UTR reporter constructs as described previously (12,33). All of the primer sequences for generating these constructs are provided in Supplementary Table S1. The pMyc-TA-luciferase reporter plasmid was purchased from Addgene. Transient transfections were performed by using the Lipofectamine Reagent as recommended by the manufacturer, and the levels of firefly luciferase activity were normalized to Renilla luciferase activity.
Western Blot Analysis
Whole-cell lysates were prepared by using 2% sodium dodecyl sulfate (SDS), sonicated and centrifuged at 4°C for 15 min. The supernatants were boiled and size-fractionated by SDS-polyacrylamide gel electrophoresis (PAGE). After the blots were incubated with primary antibody and then secondary antibodies, immunocomplexes were developed by using chemiluminescence.
Reverse Transcriptase (RT)-PCR and Real-time Quantitative PCR Analysis
Total RNA was isolated by using an RNeasy mini kit (Qiagen) and used in reverse transcription and PCR amplification reactions as described (15). The levels of the Gapdh PCR product were assessed to monitor the evenness in RNA input in RT-PCR samples. Real-time quantitative PCR (Q-PCR) analysis was performed by using the 7500-Fast RealTime PCR Systems with specific primers, probes and software (Applied Biosystems). For miRNA studies, the levels of miRNA-222 were also quantified by Q-PCR by using Taqman MicroRNA assay; small nuclear RNA (snRNA) U6 was used as the endogenous control.
Biotin Labeled miR-222 Pulldown Assays
Biotin-labeled miR-222 was transfected, and 24 h later, whole-cell lysates were collected, mixed with Dynabead M-280 Streptavidin beads (Invitrogen) and incubated at 4°C with rotation overnight (13). After the beads were washed thoroughly, the bead-bound RNA was isolated and subjected for RT, followed by Q-PCR analysis. Input RNA was extracted and served as control.
Assays of Newly Translated Protein and Polysome Analysis
New synthesis of nascent FZD7 protein was detected by a Click-iT protein analysis detection kit (Life Technologies) and performed following the company’s manual. Briefly, cells were incubated in methionine-free medium and then exposed to L-azidohomoalanine (AHA). After mixing cell lysates with the reaction buffer for 20 min, the biotinalkyne/azide-modified protein complex was pulled down by using paramagnetic streptavidin-conjugated Dynabeads. The pull-down material was resolved by 10% SDS-PAGE and analyzed by Western immunoblotting analysis using antibodies against or GAPDH.
Polysome analysis was performed as described (14). Briefly, cells at ∼70% confluence were incubated in 0.1 mg/mL cycloheximide and then lifted by scraping in polysome extraction buffer (PEB) lysis buffer. Nuclei were pelleted, and the resulting supernatant was centrifuged through a 10–50% linear sucrose gradient to fractionate cytoplasmic components according to their molecular weights. The eluted fractions were prepared with a fraction collector (Brandel), and their quality was monitored at 254 nm by using a UV-6 detector (ISCO). After RNA in each fraction was extracted, the levels of each individual mRNA were quantified by Q-PCR in each of the fractions.
Measurement of Epithelial Repair In Vitro
Epithelial injury model and repair assays were carried out as described previously (29). Cells were plated at 6.25 × 104/cm2 in Dulbecco modified Eagle medium containing fetal bovine serum on 60-mm dishes thinly coated with Matrigel according to the manufacturer’s instructions (BD Biosciences) and were incubated as described for stock cultures. Cells were fed on d 2, and mono-layer was wounded by removing part of the monolayer on d 4; repair was assayed at various times after wounding by using National Institutes of Health image analysis. All experiments were carried out in triplicate, and the results were reported as the percent of wound width covered.
All values were expressed as the means ± standard error of the mean (SEM). An unpaired, two-tailed Student t test was used when indicated with P < 0.05 considered significant. When assessing multiple groups, one-way analysis of variance was used with the Tukey post hoc test. The statistical software used was SPSS17.1.
All supplementary materials are available online at https://doi.org/www.molmed.org .
Transgenic Expression of miR-222 in IECs Represses Growth of Small Intestinal Mucosa
Transgenic expression of miR-222 did not directly induce apoptosis in the intestinal epithelium, since there was no significant cell death observed in miR222-Tg mice as examined by transferase-mediated dUTP nick-end labeling (TUNEL) staining (Supplementary Figure S4Aa). To determine the susceptibility of intestinal epithelium to induced apoptosis, littermates and miR222-Tg mice were exposed to TNFα for 5 h. As shown, typical morphological features characteristic of apoptotic cell death were induced in the intestinal mucosa in both littermate controls and miR222-Tg mice: increased levels of TUNEL-positive cells and active caspase-3 (Supplementary Figures S4Ab, B, C). However, miR222-Tg mice exhibited increased percentages of apoptotic cells and more active caspase-3 compared with those observed in littermates after treatment with TNFα. These results indicate that local overexpression of miR-222 in the epithelium causes small intestinal mucosal atrophy by repressing IEC proliferation and increasing sensitivity to apoptosis.
MiR-222-Overexpressing Intestinal Epithelium Is Vulnerable to Pathological Stress
MiR-222 Interacts with and Represses Fzd7 mRNA Translation
MiR-222-Modulated FZD7 Plays a Critical Role in Intestinal Epithelial Repair
Our previous studies demonstrated that inhibition of IEC-6 cell proliferation by polyamine depletion is associated with increased miR-222 expression, whereas miR-222 silencing enhances cell division (3,12). However, this study was conducted in cultured IECs, and miR-222 has been considered to act as a tumor suppressor or oncogene in cancer development (12,21, 22, 23); therefore, the exact function of miR-222 in the intestinal epithelium in vivo remains to be fully investigated. By using a tissue-specific transgenic expression approach, here we provided powerful genetic evidence that miR-222 plays an important role in the regulation of intestinal mucosal regeneration and protection. Transgene-driven overexpression of miR-222 in IECs caused small intestinal mucosal atrophy, delayed epithelial repair after I/R-induced injury and aggravated the barrier dysfunction in mice exposed to CLP. The miR-222-overexpressing epithelium also exhibited decreased expression of the Wnt receptor FZD7; miR-222 was found to directly interact with and repress Fzd-7 mRNA translation. These findings advance our understanding of the physiological function of miR-222 in maintenance of the intestinal epithelial integrity and highlight the involvement of increased miR-222 in gut mucosal pathology.
The results reported here are the first demonstration that miR-222 functions as a biological repressor of normal mucosal growth in the small intestine. As shown, miR222-Tg mice exhibited a significant inhibition of mucosal growth, as evidenced by a decrease in cell proliferation in the crypts and subsequent shrinkage of crypts and villi in the mucosal tissue. These observations from in vivo studies are consistent with the findings obtained from our previous in vitro cell biology studies that ectopic overexpression of miR-222 precursors in cultured IEC-6 cells inhibits Cdk4 mRNA translation, thus causing G1 phase growth arrest (12). This inhibitory phenotype in mucosal growth by miR-222 occurs only in the small intestine, since there were no differences in the rates of colonic mucosal growth between miR222-Tg mice and littermates, although miR-222 levels in the colonic mucosa were also increased in miR222-Tg mice. The exact reasons for which miR-222 overexpression failed to alter colonic mucosal growth remain unknown, but basal mucosal turnover rate in the colon is lower than that observed in the small intestine (32). Consistent with this finding, we recently reported that targeted deletion of the RBP HuR in IECs causes mucosal growth inhibition in the small intestine but not in the colon (33). The miR-222-elevated epithelium also displayed the impaired mucosal maturation and increased susceptibility to TNFα-induced apoptosis in the small intestine, and these changes may also contribute to disruption of the intestinal epithelial integrity in miR222-Tg mice. On the other hand, inhibition of miR-222 by the simple systemic delivery of locked nucleic acid-modified antimiR-222 was found to block exercise-induced cardiac growth in mice (43). Interestingly, basal rates of cardiac growth and cardiomyocyte proliferation are also slow under biological conditions.
Another significant finding from this study is that the miR-222-overexpressing intestinal epithelium exhibits an increased vulnerability to pathological stress. Increased levels of miR-222 in the intestinal epithelium not only delayed mucosal repair after I/R-induced injury but also exacerbated the gut barrier dysfunction in mice exposed to CLP. The inhibitory effect of miR-222 on mucosal repair is not surprising, because miR-222 modulates expression of several migration-associated (34) and proliferation-promoting proteins (12,35,36) that are essential for rapid epithelial restitution and chronic mucosal healing after injury (37). This notion is further supported by our in vitro studies showing that elevation of miR-222 in cultured IEC-6 cells repressed epithelial repair after wounding. On the other hand, transgenic miR-222 expression deteriorated the gut barrier dysfunction in CLP mice by downregulating expression of TJ proteins, since miR-222-overexpressing epithelium displayed an additional decrease in the levels of claudin-1 and ZO-1 after exposure to CLP. The TJ proteins are the apical-most element of the junctional complex and seal epithelial cells together in a way that prevents even small molecules from leaking between cells (10,38). TJs are highly dynamic, and their constituent protein complexes undergo continuous remodeling and turnover under tight regulation by numerous extracellular and intracellular factors. Maintenance of dynamic levels of TJ proteins is critical for normal function of the epithelial barrier, whereas disruption of TJ expression results in acute gut barrier dysfunction. However, it remains unknown at present whether miR-222 directly targets the TJ mRNAs or regulates TJ expression indirectly.
The results presented here also show that Fzd7 mRNA is a novel target of miR-222 and that [miR-222/Fzd7 mRNA] association represses FZD7 translation. The expression level of FZD7 protein in miR-222-elevated intestinal epithelium decreased dramatically, although there was no change in total Fzd7 mRNA content in miR222-Tg mice. Studies using biotin-labeled miR-222 demonstrated that miR-222 directly bound Fzd7 mRNA but not Cdk2, JunD or Myc mRNAs in IEC-6 cells. Through the use of various ectopic reporters bearing partial transcripts spanning the Fzd7 CR and 3′-UTR with or without miR-222-binding sites, our results further show that miR-222 interacted predominantly with the Fzd7 CR but not with the Fzd7 3′-UTR. These findings are consistent with other results that miR-222 associates with the mRNAs such as MMP1 and SOD2 (34), p27kip1 (35), eNOS (18), LASS2 (19), PPP2R2A (40), TIMP3 (38), ER-α (23) and CD4 (17), thus destabilizing mRNAs and/or repressing their translation. Generally, miR-222 interacts with the 3′-UTRs of target transcripts, although, in some instances, it also associates with the CR or 5′-UTR of target mRNAs for its regulatory actions. In this regard, we have reported that miR-222 represses Cdk4 mRNA translation by interacting with the Cdk4 CR rather than the 3′-UTR (12).
Intestinal epithelium-specific transgenic expression of miR-222 inhibits small intestinal mucosal growth and delays repair of damaged mucosa at least partially by inactivating Wnt signaling as a result of repression of FZD7 expression. Wnt signaling is crucial for gut development and acts as a key regulator of normal epithelial renewal and mucosal repair after injury (30,31). In response to stress, released Wnt proteins in the extracellular milieu bind to serpentine receptors of the FZD family, which leads to an accumulation of dephosphorylated β-catenin and its stabilization (30). Subsequently, the stabilized β-catenin undergoes nuclear translocation and association with TCF transcription factors, enabling transactivation of their target genes. Target deletion of conditional Wnt gene or overexpression of the Wnt natural inhibitor, Dickkopf1, disrupts gut development, represses mucosal growth and delays healing after injury (41,42). Consistent with our previous studies (29), coculture of IECs with Wnt-overexpressing fibroblasts enhanced epithelial repair, but this stimulation was prevented by miR-222 overexpression. Moreover, FZD7 levels decreased dramatically in the intestinal mucosal tissue in miR222-Tg mice and in the miR-222-overexpressing population of IEC-6 cells. FZD7 silencing or decreased levels of FZD7 by miR-222 overexpression inhibited Wnt-dependent transcriptional activity and repressed epithelial repair in the presence of Wnt5a.
These findings indicate that miR-222 functions as a negative regulator of intestinal epithelium homeostasis by altering Wnt signaling activity through posttranscriptional regulation of FZD7 expression.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
Funding was provided by Merit Review Awards (to JY Wang, DJ Turner and JN Rao) from the U.S. Department of Veterans Affairs; grants from the National Institutes of Health (DK57819, DK61972 and DK68491 to JY Wang); and the National Institute on Aging-Intramural Research Program (to M Gorospe). JY Wang is a Senior Research Career Scientist, Biomedical Laboratory Research and Development Service, U.S. Department of Veterans Affairs.
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