Altered intestinal epithelial integrity is an important susceptibility trait in inflammatory bowel disease (IBD), and early life stressors are reported to contribute to this disease susceptibility in adulthood. To identify disease mechanisms associated with early-life trauma that exacerbate IBD in adulthood, we used a “double-hit” neonatal inflammation (NI) and adult inflammation (AI) model that exhibits more severe mucosal injury in the colon later in life. In this study, we explore the underlying mechanisms of this aggravated injury. In rats exposed to both NI and AI, we found sustained increases in colonic permeability accompanied by significantly attenuated expression of the epithelial junction protein E-cadherin. Quantitative RT-PCR revealed a decreased Cdh1 (gene of E-cadherin) mRNA expression in NI + AI rats compared with NI or AI rats. Next, we performed microRNA microarrays to identify potential regulators of E-cadherin in NI + AI rats. We confirmed the overexpression of miR-155, a predicted regulator of E-cadherin, and selected it for further analysis based on reported significance in human IBD. Using ingenuity pathway analysis software, the targets and related canonical pathway of miR-155 were analyzed. Mechanistic studies identified histone hyperacetylation at the Mir155 promoter in NI + AI rats, concomitant with elevated RNA polymerase II binding. In vitro, E-cadherin knockdown markedly increased epithelial cell permeability, as did overexpression of miR-155 mimics, which significantly suppressed E-cadherin protein. In vivo, NI + AI colonic permeability was significantly reversed with administration of miR-155 inhibitor rectally. Our collective findings indicate that early-life inflammatory stressors trigger a significant and sustained epithelial injury by suppressing E-cadherin through epigenetic mechanisms.

The integrity of the epithelial cell junction barrier of the intestines is thought to play an essential role in the mediation of inflammation and disease pathogenesis (1). This has been well studied in inflammatory bowel disease (IBD), a group of conditions associated with chronic inflammation of the gastrointestinal tract. Tight and adherens junction proteins have been implicated in the pathogenesis of IBD. This includes E-cadherin, an essential adherens junction protein that is also required for tight junction formation (2). Alterations of E-cadherin structure and downregulation of production promote a proinflammatory state, in part, by facilitating the transmigration of neutrophils (3, 4) and by serving as the binding ligand for the intestinal epithelial lymphocyte homing receptor αE, which is a critical regulator of immune homeostasis in the intestine (5, 6).

Besides structural changes, the prolonged, vacillating immune response characteristic of IBD is thought to be mediated by varied mechanisms. One area of investigation has been perinatal stress and the predisposition to diseases later in life, including IBD. We have previously demonstrated in rats that episodes of neonatal inflammation (NI) create epigenetic changes, leading to an exaggerated immune response when exposed to a second stress later in life (7). This response was associated with an increase in IL-1β production after an episode of adult inflammation (AI) but was dependent on the precedent-setting NI. The exaggerated immune response after NI appeared to be mediated in part by epigenetic changes, specifically, the acetylation around NF-κB–binding motifs leading to exaggerated IL-1β activation following an episode of AI. Importantly, adult rats given multiple inflammatory insults did not generate this aggravated IL-1β response, reinforcing the neonatal period as a critical determinant of the aggravated chronic inflammatory changes.

Within this dual inflammatory event model, the specific role of microRNA (miRNA) remains unclear. miRNAs play an important role in a variety of developmental processes and diseases by modulating gene expression through their interaction with mRNA targets, which inhibits translation and/or degrades mRNA. Despite the inherent limitations of in silico prediction tools and the difficulty in determining direct versus indirect consequences of miRNA interactions, promising discoveries have come from such approaches in the study of IBD (8). Circulating miRNA with differential expression in ulcerative colitis (UC) and Crohn's disease (CD) have been proposed as biomarkers to distinguish between the two IBD subtypes (9). Changes in miRNA expression and the interaction with canonical pathways associated with IBD have previously been noted (10). Specifically, bioinformatics analysis has demonstrated or predicted multiple miRNAs that target E-cadherin, including miR-155. miR-155 plays an important role in various aspects of normal immune function; alterations in miR-155 expression and its association with IBD have been well established (11). Compared with healthy controls (Ctls), patients with UC have a significant increase in miR-155 levels both in the circulation (9) and in colonic mucosa (12). miR-155 is an important mediator of T cell–regulated immunity and is required to induce Th17-driven chronic colitis in mice (13, 14). This is noteworthy because E-cadherin–sensitized dendritic cells also have been shown to increase Th17-driven immune responses and colitis (15).

We hypothesized that, to our knowledge, our novel model of exaggerated immune response, triggered by NI, was mediated by a change in intestinal permeability. We postulated that changes in epithelial integrity would be accompanied by a decrease in the expression of junction proteins. In addition, we hypothesized that the changes, in part, would be mediated by specific miRNAs that interact with target mRNAs of critical junction proteins in the gut. The current work reinforces our previous findings of NI increasing susceptibility to aggravated inflammation when a second stressor is provided. These changes were initiated by increased colonic permeability, corresponding to a significant decrease in the expression of E-cadherin and mediated, at least in part, by miR-155.

FITC-dextran (MW 4000), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Human TNF-α and IL-1β were purchased from Genscript Biotech (Piscataway, NJ).

Caco-2, SW620, CCD 841 CoN, and HEK293 cells purchased from American Type Culture Collection (Manassas, VA) were maintained in DMEM supplemented with 10% FBS and 0.1% penicillin-streptomycin solution. Transient transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA). To measure epithelial monolayer permeability, confluent epithelial cell monolayers, cultured on a permeable surface, were placed in a cell culture plate to form donor-receiver compartments (16). FITC-dextran test compounds are added to the apical compartment with fresh medium in the basolateral compartment. The transport of FITC-dextran across the epithelial cell monolayer was monitored.

Male Sprague Dawley rat littermates were used in the preclinical studies. Five-day-old pups were purchased from Envigo (Houston, TX). The procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch at Galveston.

Rat neonates were randomly divided into four groups: 1) vehicle treatment in both neonatal and adult-life stages (Ctl + Ctl), 2) sham treatment as neonates followed by an inflammatory insult as adults (Ctl + AI), 3) NI and then sham treatment as adults (NI + Ctl), and 4) NI plus AI in combination (NI + AI) (Fig. 1A). Experimenters were blinded to treatment assignment. To induce NI, TNBS (130 mg/kg dissolved in 200 μl of saline containing 10% ethanol, a concentration previously established to ensure neonatal viability) was injected intraluminally 2 cm into the colon of pups on postnatal day 10 (17, 18). The animals were kept in a head-down position while the anus was held closed for 1 min to prevent leakage. Rats in the sham treatment groups received 200 μl of saline. In an interval necessary to induce significant change, 6–8 wk later, animals were subjected to a secondary TNBS (68 mg/kg) insult as AI. Under light anesthesia, 250 μl of TNBS in PBS containing 40% ethanol was injected intrarectally via a catheter, advanced to 8 cm into the colon. Control rats were given 250 μl of saline. Epithelial permeability in the colon was assessed 7, 14, or 28 d after the second TNBS treatment. To determine whether a delayed first-time AI also aggravates the immune response, comparable to NI treatment, we also induced AI with TNBS (130 mg/kg) in 6- to 8-wk-old rats followed by a second AI (68 mg/kg TNBS) 6 wk later (four groups: Ctl + Ctl, Ctl + AI, AI + Ctl, and AI + AI). One week after the second TNBS administration, epithelial permeability in the colon was determined.

To assess colonic permeability, animals were fasted overnight with free access to GoLitely (Braintree Laboratories, Braintree, MA). Under light anesthesia with isoflurane, FITC-dextran dissolved in PBS (40 mg/ml, 200 μl) was administered intraluminally via catheter into the colon. Two hours later, animals were euthanized, and blood was collected for serum fluorescein measurement. For histologic examination, a full-thickness colon specimen was obtained from the proximal, middle, and distal colon; fixed in 10% formalin; embedded in paraffin; sectioned; and stained with H&E (19). Immune cells (neutrophils, leukocytes, B and T lymphocytes, and mast cells) were stained and evaluated by two independent pathologists in a blinded manner. The mucosa/submucosa layers were dissected from the rest of the colon, snap-frozen in liquid nitrogen, pulverized, and stored at −80°C for molecular studies.

Ctl + Ctl and NI + AI rats were used for miR-155 inhibitor treatment. Both groups of rats were treated with either miRCURY LNA miRNA Inhibitor Control (catalog no. YI00199006) or rno-miR-155-5p miRCURY LNA miRNA Inhibitor (catalog no. YI0401318) via enema (10 nm in 200 μl of PBS per injection), once daily from 1 d before through 6 d after the AI (20, 21). Colonic permeability was assessed 24 h after the last dose of treatment. The mucosa/submucosa layers were dissected from the distal colon and used for detection of mRNA and protein by quantitative RT-PCR (RT-qPCR) and Western blot, respectively.

Total RNA containing miRNA was extracted using the miRNeasy Mini Kit (QIAGEN, Valencia, CA), followed by cDNA synthesis using SuperScript III First-Strand Synthesis System or TaqMan Advanced miRNA cDNA Synthesis Kit (Invitrogen). The mRNA and miRNA levels were quantitated using TaqMan-based quantitative PCR (qPCR), with 18S rRNA and U6 small nuclear RNA as internal Ctls, respectively.

Genomic DNA was extracted using DNeasy Blood and Tissue Kit (QIAGEN), followed by sodium bisulfite conversion using EpiMark Bisulfite Conversion Kit (New England Biolabs, Ipswich, MA). SYBR green-based quantitative methylation-specific PCR (qMSP) with primers specific to the methylated rat Mir155 promoter (Rt-Mir155-M-F: 5′-GTC TTG GGA GCT TTT ACA GTG G-3′; Rt-Mir155-M-R: 5′-GGC CAC CAC CTC CTA GCA A-3′) was performed in a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific). Primers specific to the rat Mmp9 promoter (Mmp9-MSP-F: 5′-TGA GGT TGG GAA ATG GTG GAC-3′; Mmp9-MSP-R: 5′-TGT ACA GAC CTG TGA GAC GG-3′) were included as internal Ctl.

Western blotting was performed as described previously (7). Primary Abs were as follows: anti–E-cadherin (catalog no. AF648, 1:1000; R&D Systems), anti–β-actin mouse mAb (catalog no. A5441, 1:5000; Sigma-Aldrich), anti–β-catenin (A11343), claudin (Cldn) 1 (A2196), Cldn2 (A14085), Cldn8 (A8174) and occludin (Ocln) (A2601) rabbit polyclonal Abs (ABclonal Technology, Woburn, MA), and anti–Cldn1 (bs-8482R) and Cldn15 (bs-13753) rabbit polyclonal Abs (Bioss Abs, Woburn, MA). All blots were scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Band density was determined using LI-COR Image Studio Software.

Full-thickness colon tissue was fixed in 10% formalin and embedded in paraffin. Ten-micrometer sections were baked in a 50–55°C oven for 1 h. After Ag retrieval and 1 h blocking with 10% serum, the slides were treated with anti–E-cadherin mouse mAb (sc-71007, 1:50 dilution; Santa Cruz Biotechnology) overnight, followed by washing three times 15 min each with 1× PBS. The sections were then incubated for 1 h at room temperature with ALEXA-conjugated Ab (Invitrogen) diluted 1:400 in PBS. Images were captured using a LEICA DMI 6000 B Microscope.

miRNA microarrays were performed by LC Sciences (Houston, TX) using Microarray Version 21 (MRA-1003). Data were analyzed by subtracting the background and then normalizing the signals using a Locally Weighted Regression Filter. The miRNA transcript was considered as reliably detectable only if the signal intensity was >3 times the background SD; the spot coefficient of variation was <0.5, and at least 50% of the repeated probe signals were above the detection level.

Using ingenuity pathway analysis (IPA) (Redwood, CA) software, miRNAs of interest were imputed and cross-referenced with described TargetScan human targets. Using the biomarker dataset upload, the ingenuity identifier was selected, and an overlay of the tight junction canonical pathway was implemented.

Chromatin immunoprecipitation (ChIP) assays were performed as described previously using ChIP-IT Express Kit (Active Motif, Carlsbad, CA) (18). Abs for the immunoprecipitation are as follows: histone H3 acetyl lysine 9 (H3K9ac, catalog no. 39137), histone H4 acetyl lysine 12 (H4K12ac, catalog no. 39165), and RNA polymerase II (catalog no. 39097; Active Motif). Precipitated DNA, PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA), and primers specific to the rat Mir155 gene promoter (forward: 5′-GGC TTG CTG AAG GCT GTA TG-3′, reverse: 5′-ACA GGT AGG AGT CAG TCA GAG G-3′) were used for real-time PCR. Fold differences in precipitated DNA were normalized against input.

All data were expressed as mean ± SD. We used one-way ANOVA followed by Tukey post hoc analysis for comparison of more than two means and Student t test to compare between two means and considered p < 0.05 to be statistically significant.

First, to evaluate the effect of NI and AI on colonic epithelial integrity, we assessed the intestinal permeability in all four groups of male rats at an interval of 7, 14, and 28 d from the adult inflammatory insult (TNBS) administered at 8 wk (Fig. 1A). Permeability was significantly increased in NI + AI rats compared with Ctl + AI at all intervals (i.e., days 7, 14, and 28) (Fig. 1B–D, p < 0.05). These changes in permeability accompanied increased architectural distortion and epithelial damage, as viewed in H&E-stained colonic specimens of NI + AI rats compared with other groups (Fig. 1E). There was a significant, pan-colonic infiltration of neutrophils in the NI + AI group versus the other three groups (Fig. 1F–H, p < 0.01). These findings were mirrored in an NI + AI–dependent manner in other immune cells known to influence epithelial barrier function: leukocytes, B and T lymphocytes, and mast cells (Supplemental Fig. 1). A small but statistically significant increase in permeability occurred in Ctl + AI rats at day 7 but was not sustained at later time points. This corresponded with an increase in colonic neutrophils. When comparing neutrophil infiltration between groups, the NI + AI group was significantly increased compared with the Ctl + AI group in all areas of the colon (Fig. 1F–H, p < 0.01). There was no significant difference in permeability among NI + Ctl rats compared with the Ctl + Ctl group (Fig. 1B–D). These findings suggest that the combination of NI and AI is necessary to create a significant and sustained increase in permeability and that a single insult later in life will not reproduce the exacerbated response triggered by an initial NI episode.

FIGURE 1.

Aggravated mucosal injury in the rats subjected to both NI and AI in the colon. (A) Diagram displaying the animal protocol, including administration timing of TNBS and normal saline (NlSal). Colonic permeability in rats exposed to NI, AI, or both was measured using FITC-dextran at day 7 (B), 14 (C), and 28 (D). n = 15. (E) H&E staining of the colon. Original magnification ×40. Neutrophil infiltration at day 7 in proximal (F), middle (G), and distal (H) colon was evaluated by two independent pathologists. n = 12. For comparison, four groups of adult rats were exposed to either one or two rounds of AI, and the colonic permeability was assessed 7 d later (I). Neutrophils in the proximal (J), middle (K), and distal (L) colon were also examined by two independent pathologists. ANOVA. n = 12. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus Ctl + AI.

FIGURE 1.

Aggravated mucosal injury in the rats subjected to both NI and AI in the colon. (A) Diagram displaying the animal protocol, including administration timing of TNBS and normal saline (NlSal). Colonic permeability in rats exposed to NI, AI, or both was measured using FITC-dextran at day 7 (B), 14 (C), and 28 (D). n = 15. (E) H&E staining of the colon. Original magnification ×40. Neutrophil infiltration at day 7 in proximal (F), middle (G), and distal (H) colon was evaluated by two independent pathologists. n = 12. For comparison, four groups of adult rats were exposed to either one or two rounds of AI, and the colonic permeability was assessed 7 d later (I). Neutrophils in the proximal (J), middle (K), and distal (L) colon were also examined by two independent pathologists. ANOVA. n = 12. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus Ctl + AI.

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At 7 d, we also evaluated adult rats that were exposed to two rounds of AI, and there was a significant increase in permeability noted in Ctl + AI and AI + AI as compared with Ctl + Ctl (p < 0.05) but no significant change between Ctl + AI and AI + AI (p > 0.05) (Fig. 1I). These findings were accompanied by a significant increase in neutrophil concentration in Ctl + AI and AI + AI groups compared with Ctl + Ctl (p < 0.05) with, again, no significant change between the increased groups (p > 0.05) (Fig. 1J–L). Neither the change in permeability nor neutrophil concentration was as sizable as that seen after a neonatal insult. These findings support our previous findings that multiple AI episodes, later in life, fail to recapitulate the NI + AI paradigm and do not significantly change intestinal permeability in the absence of NI (7).

To elucidate the molecular mechanisms underlying the increase of colonic permeability in NI + AI rats, we compared the expression levels of junction proteins in colonic mucosa/submucosa. Protein levels of β-catenin, Cldn1, Cldn2, Cldn7, Cldn8, Cldn15, and Ocln remained the same in all four groups of rats (Supplemental Fig. 2). However, E-cadherin protein levels were markedly attenuated in the colonic mucosa/submucosa of NI + AI compared with the other three groups of rats (p < 0.05) (Fig. 2A). Immunofluorescence staining corroborated the decreased intensity of E-cadherin expression in NI + AI rats (Fig. 2B). When evaluating E-cadherin protein expression in adult rats subjected to two rounds of AI, there was no significant difference between Ctl + Ctl, Ctl + AI, AI + Ctl, and AI + AI groups (Fig. 2C).

FIGURE 2.

Significant suppression of E-cadherin protein in the colonic mucosa/submucosa of NI + AI rats. (A) E-cadherin protein levels were markedly downregulated in the colonic mucosa/submucosa of NI + AI rats. Top, Representative images of Western blots. Bottom, Arbitrary OD units of E-cadherin (E-cad) were normalized against β-actin and expressed as fold change. ANOVA. n = 12. (B) Immunofluorescence staining of E-cadherin demonstrating decreased relative intensity in the colon of NI + AI rats. Original magnification ×40. (C) Two rounds of AI had no effect on E-cadherin protein expression in the colonic mucosa/submucosa. Top, Representative images of Western blots. Bottom, Arbitrary OD units of the targeting protein were normalized versus β-actin and presented as fold change. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus Ctl + AI.

FIGURE 2.

Significant suppression of E-cadherin protein in the colonic mucosa/submucosa of NI + AI rats. (A) E-cadherin protein levels were markedly downregulated in the colonic mucosa/submucosa of NI + AI rats. Top, Representative images of Western blots. Bottom, Arbitrary OD units of E-cadherin (E-cad) were normalized against β-actin and expressed as fold change. ANOVA. n = 12. (B) Immunofluorescence staining of E-cadherin demonstrating decreased relative intensity in the colon of NI + AI rats. Original magnification ×40. (C) Two rounds of AI had no effect on E-cadherin protein expression in the colonic mucosa/submucosa. Top, Representative images of Western blots. Bottom, Arbitrary OD units of the targeting protein were normalized versus β-actin and presented as fold change. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus Ctl + AI.

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To determine if E-cadherin is transcriptionally downregulated in NI + AI rats, we assessed mRNA levels of Cdh1, which encodes E-cadherin, as well as other junction proteins in the mucosa/submucosa. Respective mRNA levels relative to 18S rRNA were calculated for Cdh1, Ctnnb1, Cldn2, Cldn7, Cldn8, and Ocln. Cdh1 mRNA was significantly reduced in NI + AI rats compared with the other three groups (p < 0.05) (Fig. 3A). Ctnnb1, Cldn2, Cldn7, and Cldn8 were not significantly reduced in any of the treatment groups (Fig. 3B–E). Ocln mRNA levels in colonic mucosa/submucosa were significantly reduced in both Ctl + AI and NI + AI rats (p < 0.05), and there was a significant decrease in Ocln expression in NI + AI rats compared with NI + Ctl (p < 0.05) (Fig. 3F). In contrast, the combination of AI + AI was not able to induce a significant change in Cdh1 mRNA levels (Fig. 3G). In addition, there was no significant change in Ctnnb1 (Fig. 3H) or other tight junction proteins (data not shown) when exposed to AI + AI. Similar findings were demonstrated in Ocln expression in groups exposed to the AI, with Ocln mRNA levels reduced in both Ctl + AI and AI + AI rats, compared with the Ctl + Ctl group (p < 0.05) (p < 0.05) (Fig. 3I). These findings demonstrate that, in contrast to other junctional proteins, Cdh1 is the only junction protein that was significantly reduced when exposed to both NI and AI. We elected to focus on Cdh1 rather than Ocln because the latter was reduced by AI alone.

FIGURE 3.

mRNA levels of junction proteins in the colonic mucosa/submucosa of eight groups of rats. Total RNA was isolated from the colonic mucosa/submucosa. mRNA expression levels of Cdh1 (A), Ctnnb1 (B), Cldn2 (C), Cldn7 (D), Cldn8 (E), and Ocln (F) in Ctl + Ctl, Ctl + AI, NI + Ctl, and NI + AI rats were quantified by TaqMan-based RT-qPCR. n = 12. ANOVA. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus NI + Ctl. mRNA levels of Cdh1 (G), Ctnnb1 (H), and Ocln (I) in Ctl + Ctl, Ctl + AI, AI + Ctl, and AI + AI rats were also determined by RT-qPCR. n = 12. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus AI + Ctl.

FIGURE 3.

mRNA levels of junction proteins in the colonic mucosa/submucosa of eight groups of rats. Total RNA was isolated from the colonic mucosa/submucosa. mRNA expression levels of Cdh1 (A), Ctnnb1 (B), Cldn2 (C), Cldn7 (D), Cldn8 (E), and Ocln (F) in Ctl + Ctl, Ctl + AI, NI + Ctl, and NI + AI rats were quantified by TaqMan-based RT-qPCR. n = 12. ANOVA. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus NI + Ctl. mRNA levels of Cdh1 (G), Ctnnb1 (H), and Ocln (I) in Ctl + Ctl, Ctl + AI, AI + Ctl, and AI + AI rats were also determined by RT-qPCR. n = 12. *p < 0.05 versus Ctl + Ctl, #p < 0.05 versus AI + Ctl.

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To explore the molecular mechanisms underlying the reduction of Cdh1 mRNA levels, we used miRNA microarrays to evaluate global changes in miRNA production in the colon mucosa/submucosa of four groups of rats: Ctl, Ctl + AI (C-T68), NI + Ctl, and NI + AI (NI-T68). The data were deposited in the Gene Expression Omnibus Repository (accession no. GSE138770. Available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138770). A hierarchical cluster analysis was created for all miRNAs with a signal intensity >32 (Supplemental Fig. 3). A t test was then performed comparing the Ctl group to the NI + AI rats. Forty-five miRNA had significant changes in expression levels (p < 0.05) and high signal intensity (>250). When compared with Ctls, 24 miRNAs were increased in colon mucosa/submucosa (Table I) and 21 were decreased (Supplemental Table I, p < 0.05). After the initial analysis, miR-155 was prioritized because of its previously noted modulation of Cdh1 (22).

Table I.
miRNAs increased (p < 0.05) in the colon mucosa/submucosa of NI + AI rats compared with Ctl + Ctl rats
Ctl + Ctl
NI + AI
miRNA Namep ValueMeanMeanLog2(NI + AI/Ctl + Ctl)
rno-miR-139-5p 2.10 × 10−3 1600 3050 0.93 
rno-miR-3596c 7.45 × 10−3 1255 3760 1.58 
rno-miR-205 8.11 × 10−3 4638 6093 0.39 
rno-miR-151-3p 8.86 × 10−3 409 604 0.56 
rno-miR-3596a 9.31 × 10−3 6807 15,051 1.14 
rno-miR-378a-5p 1.46 × 10−2 380 700 0.88 
rno-miR-196c-3p 1.59 × 10−2 14,898 23,674 0.67 
rno-miR-185-5p 1.87 × 10−2 798 972 0.29 
rno-miR-652-3p 1.88 × 10−2 1181 1784 0.59 
rno-miR-18a-5p 2.45 × 10−2 256 446 0.80 
rno-miR-375-3p 2.80 × 10−2 11,682 16,083 0.46 
rno-miR-345-5p 3.22 × 10−2 202 321 0.67 
rno-miR-671 3.48 × 10−2 396 910 1.20 
rno-miR-1843-3p 3.86 × 10−2 353 1090 1.63 
rno-miR-18a-5p 4.02 × 10−2 310 632 1.03 
rno-miR-152-3p 4.23 × 10−2 3368 4503 0.42 
rno-miR-3596d 4.53 × 10−2 3552 7521 1.08 
rno-miR-378b 4.66 × 10−2 2633 4013 0.61 
rno-miR-155-5p 5.02 × 10−2 273 373 0.45 
rno-miR-324-5p 6.17 × 10−2 252 429 0.77 
rno-miR-1839-5p 6.85 × 10−2 236 363 0.62 
rno-miR-140-5p 8.46 × 10−2 333 400 0.27 
rno-miR-33-3p  8.63 × 10−2 203 286 0.50 
rno-miR-324-3p 8.90 × 10−2 290 421 0.54 
Ctl + Ctl
NI + AI
miRNA Namep ValueMeanMeanLog2(NI + AI/Ctl + Ctl)
rno-miR-139-5p 2.10 × 10−3 1600 3050 0.93 
rno-miR-3596c 7.45 × 10−3 1255 3760 1.58 
rno-miR-205 8.11 × 10−3 4638 6093 0.39 
rno-miR-151-3p 8.86 × 10−3 409 604 0.56 
rno-miR-3596a 9.31 × 10−3 6807 15,051 1.14 
rno-miR-378a-5p 1.46 × 10−2 380 700 0.88 
rno-miR-196c-3p 1.59 × 10−2 14,898 23,674 0.67 
rno-miR-185-5p 1.87 × 10−2 798 972 0.29 
rno-miR-652-3p 1.88 × 10−2 1181 1784 0.59 
rno-miR-18a-5p 2.45 × 10−2 256 446 0.80 
rno-miR-375-3p 2.80 × 10−2 11,682 16,083 0.46 
rno-miR-345-5p 3.22 × 10−2 202 321 0.67 
rno-miR-671 3.48 × 10−2 396 910 1.20 
rno-miR-1843-3p 3.86 × 10−2 353 1090 1.63 
rno-miR-18a-5p 4.02 × 10−2 310 632 1.03 
rno-miR-152-3p 4.23 × 10−2 3368 4503 0.42 
rno-miR-3596d 4.53 × 10−2 3552 7521 1.08 
rno-miR-378b 4.66 × 10−2 2633 4013 0.61 
rno-miR-155-5p 5.02 × 10−2 273 373 0.45 
rno-miR-324-5p 6.17 × 10−2 252 429 0.77 
rno-miR-1839-5p 6.85 × 10−2 236 363 0.62 
rno-miR-140-5p 8.46 × 10−2 333 400 0.27 
rno-miR-33-3p  8.63 × 10−2 203 286 0.50 
rno-miR-324-3p 8.90 × 10−2 290 421 0.54 

Using IPA, miR-155 was cross-referenced with TargetScan human targets. Using the biomarker dataset upload, the ingenuity identifier was selected, and an overlay of the tight junction canonical pathway was implemented (data not presented). Protein kinase C iota (PRKCI), a target of miR-155, was also common to tight junction signaling via its inhibition of atypical protein kinase C (aPKC).

To validate the miRNA array data prediction studies, RT-qPCR was performed on miR-155. In the presence of NI + AI, a significant increase in miR-155 expression in colonic mucosa was noted compared with the other three groups (p < 0.05) (Fig. 4A). No significant increase was detected for miR-155 in Ctl + AI versus Ctl + Ctl or NI + Ctl rats. For comparison, we also assessed miR-155 levels in the colonic mucosa of the rats subjected to one or two rounds of AIs. There was a slight elevation in miR-155 levels in Ctl + AI or AI + AI versus Ctl + Ctl or AI + Ctl rats (p > 0.1, Fig. 4B). TNF-α mRNA expression was significantly upregulated in both Ctl + AI and AI + AI rats compared with Ctl + Ctl and AI + Ctl rats (p < 0.05, Fig. 4C). No significant difference was observed in AI + AI versus Ctl + AI rats.

FIGURE 4.

Epigenetic mechanisms underlying miR-155 elevation in NI + AI rats. (A) RT-qPCR validation of miRNA microarray data confirming a significant increase in miR-155 expression in the colonic mucosa/submucosa of NI + AI rats. n = 12. ANOVA. *p < 0.05 versus Ctl + Ctl, Ctl + AI, and NI + Ctl groups. In the colonic mucosa/submucosa of Ctl + Ctl, Ctl + AI, AI + Ctl, and AI + AI rats, miR-155 (B) and TNF-α (C) levels were also quantified by RT-qPCR. n = 8. *p < 0.05 versus Ctl + Ctl and AI + Ctl groups. H3K9ac (D), H4K12ac (E), and RNAPII (F) bound to the rat Mir155 promoter in the colonic mucosa/submucosa of NI + AI rats were quantified by ChIP-qPCR. Chromatin immunoprecipitated with specific Abs, as indicated, was quantified by real-time PCR using primers specific to the core promoter of the rat Mir155 gene and normalized to inputs. DNA methylation (G) on the rat Mir155 promoter was quantified by SYBR green-based qMSP. n = 3 independent experiments. ANOVA. *p < 0.05 versus Ctl + Ctl, Ctl + AI, and NI + Ctl groups.

FIGURE 4.

Epigenetic mechanisms underlying miR-155 elevation in NI + AI rats. (A) RT-qPCR validation of miRNA microarray data confirming a significant increase in miR-155 expression in the colonic mucosa/submucosa of NI + AI rats. n = 12. ANOVA. *p < 0.05 versus Ctl + Ctl, Ctl + AI, and NI + Ctl groups. In the colonic mucosa/submucosa of Ctl + Ctl, Ctl + AI, AI + Ctl, and AI + AI rats, miR-155 (B) and TNF-α (C) levels were also quantified by RT-qPCR. n = 8. *p < 0.05 versus Ctl + Ctl and AI + Ctl groups. H3K9ac (D), H4K12ac (E), and RNAPII (F) bound to the rat Mir155 promoter in the colonic mucosa/submucosa of NI + AI rats were quantified by ChIP-qPCR. Chromatin immunoprecipitated with specific Abs, as indicated, was quantified by real-time PCR using primers specific to the core promoter of the rat Mir155 gene and normalized to inputs. DNA methylation (G) on the rat Mir155 promoter was quantified by SYBR green-based qMSP. n = 3 independent experiments. ANOVA. *p < 0.05 versus Ctl + Ctl, Ctl + AI, and NI + Ctl groups.

Close modal

A direct correlation often exists between the level of histone hyperacetylation and promoter activation for a given gene of interest. To elucidate the epigenetic mechanisms underlying the miR-155 upregulation in NI + AI rats, we evaluated the histone acetylation status surrounding the core promoter region of Mir155. ChIP-qPCR analysis was performed using Abs against either H3K9ac or H4K12ac, and primers specific to the rat Mir155 gene promotor (−87/+6). This revealed that both H3K9ac (Fig. 4D) and H4K12ac (Fig. 4E) were increased significantly at the Mir155 promoter motif in NI + AI rats compared with Ctl + Ctl, Ctl + AI, and NI + Ctl groups (p < 0.05), indicating histone hyperacetylation and chromatin relaxation. In addition, ChIP-qPCR analysis with anti–RNA polymerase II Ab confirmed that the elevation in histone acetylation was accompanied by increased RNA polymerase II at the Mir155 promoter (p < 0.01) (Fig. 4F), indicative of transcriptional activation. NI alone caused slight increases in H3K9ac (Fig. 4D, p = 0.09), H4K12ac (Fig. 4E, p = 0.17), H3K27ac, and H3K4me3 (data not shown, p > 0.05) (23). DNA methylation on the rat Mir155 promoter was also quantified by qMSP with no significant difference among all four groups (Fig. 4G). This further supports the role of acetylation-driven epigenetic modifications in the NI + AI rats.

High levels of H2O2, IL-1β, TNF-α, and LPS have been found in patients with IBD, and we speculated that some of these factors might upregulate miR-155 by enhancing histone acetylation (2426). To test the hypothesis, we incubated SW620 and HEK293 cells with H2O2 (200 μM), IL-1β (10 ng/ml), TNF-α (10 ng/ml), or LPS (10 and 100 ng/ml) for 24 h and detected miR-155 expression by RT-qPCR. We found that only TNF-α significantly elevated miR-155 expression in SW620 cells (Fig. 5A). These findings suggest that proinflammatory cytokines, but not reactive oxygen species or TLR-4 agonists, are responsible for miR-155 overexpression in colonic epithelial cells. These changes in miR-155 expression were sustained from hour 3 to 24 in both cell lines when exposed to a fixed dose of TNF-α in both SW620 (Fig. 5B) and CCD 841 CoN (Fig. 5D) cell lines. In addition, there was a dose-dependent response achieved in both cell lines when exposed to TNF-α (Fig. 5C, 5E). No changes in miR-155 were observed in HEK293 cells (data not shown), indicating that the upregulation of miR-155 by proinflammatory cytokines is cell type specific. To determine whether TNF-α and IL-1β activate miR-155 by inducing histone acetylation surrounding the Mir155 promoter, SW620 cells treated with TNF-α or IL-1β were fixed with 1% formaldehyde and subjected to ChIP-qPCR analysis. Both H3K9ac and H4K12ac at the Mir155 promoter were markedly enhanced by TNF-α compared with the Ctls. No significant increase was detected for H3K9ac and H4K12ac by IL-1β (Fig. 5F, p > 0.05). This data suggested that TNF-α [which is significantly elevated in NI + AI rats (7)] rather than IL-1β primarily activates Mir155 transcription by inducing histone hyperacetylation.

FIGURE 5.

Epigenetic upregulation of miR-155 by TNF-α in SW620 and CCD 841 CoN colon epithelial cells. miR-155 expression levels were quantified by TaqMan Advanced miRNA Assay. (A) TNF-α significantly increased miR-155 expression compared with other known proinflammatory molecules and Ctl. *p < 0.05 versus vehicle-treated cells. In SW620 (B) and CCD 841 CoN (D) cells, a significant increase in miR-155 expression was achieved at 3, 6, 18, and 24 h intervals when exposed to 100 ng/ml TNF-α. In addition, both SW620 (C) and CCD 841 CoN (E) cell lines achieved a dose-dependent increase in miR-155 expression at concentrations of 1 ng/ml of TNF-α and above. *p < 0.05 compared with time zero (time course) or concentration zero (dose response). (F) H3K9ac and H4K12ac surrounding the human MIR155 promoter were evaluated by ChIP-qPCR. SW620 cells were treated with 100 ng/ml IL-1β or TNF-α for 24 h. n = 3 independent experiments. t test. *p < 0.05 versus vehicle-treated cells.

FIGURE 5.

Epigenetic upregulation of miR-155 by TNF-α in SW620 and CCD 841 CoN colon epithelial cells. miR-155 expression levels were quantified by TaqMan Advanced miRNA Assay. (A) TNF-α significantly increased miR-155 expression compared with other known proinflammatory molecules and Ctl. *p < 0.05 versus vehicle-treated cells. In SW620 (B) and CCD 841 CoN (D) cells, a significant increase in miR-155 expression was achieved at 3, 6, 18, and 24 h intervals when exposed to 100 ng/ml TNF-α. In addition, both SW620 (C) and CCD 841 CoN (E) cell lines achieved a dose-dependent increase in miR-155 expression at concentrations of 1 ng/ml of TNF-α and above. *p < 0.05 compared with time zero (time course) or concentration zero (dose response). (F) H3K9ac and H4K12ac surrounding the human MIR155 promoter were evaluated by ChIP-qPCR. SW620 cells were treated with 100 ng/ml IL-1β or TNF-α for 24 h. n = 3 independent experiments. t test. *p < 0.05 versus vehicle-treated cells.

Close modal

To confirm that E-cadherin ablation disrupts cell junction and that miR-155 targets E-cadherin, we depleted E-cadherin in SW620 cells using CDH1 small interfering RNA (siRNA) and overexpressed miR-155 by transient transfection. Intercellular junctions were evaluated by monitoring the SW620 monolayer permeability. As expected, a significant increase in monolayer permeability was noted in the CDH1 siRNA transfection group (Fig. 6A, bar graph). This was accompanied by a significant decrease in E-cadherin protein expression compared with the Ctl siRNA group (Fig. 6A, Western blots). A similar decrease in E-cadherin protein was replicated when the cells were transfected with miR-155 and included a concurrent, significant increase in SW620 monolayer permeability (quantified by comparing fluorescence units counted per second) compared with an miRNA negative Ctl (p < 0.05) (Fig. 6B). Similar results were obtained from Caco-2 cells (data not shown). To augment our findings, in vivo studies were performed. Ctl + Ctl and NI + AI rats were given either miR-155 inhibitor treatment or Ctl via enema daily for 1 d before and 6 d after exposure to AI. Colonic permeability 24 h after the last dose was assessed, and the marked increase of colonic permeability in NI + AI rats (versus Ctl + Ctl rats) was significantly mitigated by the miR-155 inhibitor (Fig. 6C, bar graph). No significant difference was noted in the Ctl + Ctl group. Western blots showed that the decrease in E-cadherin protein expression was also abrogated by the miR-155 inhibitor (Fig. 6C). These findings confirm miR-155 targeting of E-cadherin, mechanistically explaining the miRNA’s association with increased epithelial permeability, and the junction protein’s dependence on NI + AI for diminished expression.

FIGURE 6.

miR-155 disrupts epithelial integrity by suppressing E-cadherin. (A) E-cadherin depletion increased SW620 monolayer permeability. (B) miR-155 suppressed E-cadherin protein expression and increased SW620 monolayer permeability. CDH1 siRNA and miR-155 mimics were overexpressed in SW620 cells by transient transfection. In vitro permeability was assessed by measuring the transport of FITC-dextran across the epithelial cell monolayer. n = 5 independent experiments. t test. *p < 0.05. (C) In vivo inhibition of miR-155 mitigated the increase of colonic permeability and the decrease of E-cadherin in NI + AI rats. Animals were treated with either Ctl or miR-155 inhibitor via daily enema for 7 d. Colonic permeability was evaluated by measuring the transport of FITC-dextran across the colon epithelium. ANOVA. n = 8. E-cadherin (E-cad) and β-actin protein levels were detected by Western blots.

FIGURE 6.

miR-155 disrupts epithelial integrity by suppressing E-cadherin. (A) E-cadherin depletion increased SW620 monolayer permeability. (B) miR-155 suppressed E-cadherin protein expression and increased SW620 monolayer permeability. CDH1 siRNA and miR-155 mimics were overexpressed in SW620 cells by transient transfection. In vitro permeability was assessed by measuring the transport of FITC-dextran across the epithelial cell monolayer. n = 5 independent experiments. t test. *p < 0.05. (C) In vivo inhibition of miR-155 mitigated the increase of colonic permeability and the decrease of E-cadherin in NI + AI rats. Animals were treated with either Ctl or miR-155 inhibitor via daily enema for 7 d. Colonic permeability was evaluated by measuring the transport of FITC-dextran across the colon epithelium. ANOVA. n = 8. E-cadherin (E-cad) and β-actin protein levels were detected by Western blots.

Close modal

Early life adversity mediated through infections and other systemic stressors are independent risk factors in IBD. Investigators have previously illustrated that term birth, breast feeding for more than 12 mo, and avoidance of antibiotic exposure in early life convey some protective effect in the development of IBD (27, 28). In animal models, the early life disruption of normal gut microbiota and the subsequent loss of mucosal homeostasis is thought to confer IBD risk (29). We previously investigated durable mechanisms mediating these pathogenic changes. Using a “two-hit” chemical injury model, we demonstrated that NI induces epigenetic changes, priming the rat for an exaggerated immune response in the setting of a second, adult stressor. Through hyperacetylation of histone H4K12, NI + AI rats had sustained upregulation of IL-1β expression in colonic epithelium. This change was dependent on the presence of NI (7). Key questions remained regarding the colonic structural alterations mediated by these epigenetic changes.

A hallmark feature of IBD is the presence of clinical flares, which in part are mediated by alterations in epithelial integrity. Changes in intestinal permeability cause alterations in the normal immune response (30). In this study, using epithelial permeability as a marker for a loss of junctional integrity, we demonstrated that significant and sustained changes in barrier integrity were dependent on a combination of NI and a subsequent AI response. We also showed that these changes in epithelial permeability were accompanied by a marked decrease in the junction protein E-cadherin, in both rat colonic mucosa/submucosa as well as SW620 and CCD 841 CoN epithelial monolayers.

E-cadherin mediates cell–cell interactions at adherens junctions, but is also recognized as an important immune modulator. This is particularly true in IBD, in which E-cadherin–driven changes in innate immunity have been implicated in disease pathogenesis. Altered localization of E-cadherin has been shown to increase susceptibility to CD (3). The proposed mechanisms are complex but are thought to revolve around increased intestinal permeability. This includes the weakening of tight junctions leading to direct sampling of luminal enteric bacteria by dendritic cells promoting a T cell–mediated immune response (15, 31, 32). In UC, genomic analysis has identified a susceptibility locus including E-cadherin (33). Mice with a tamoxifen-induced E-cadherin knockout exhibited architectural changes and increased mucosal inflammation consistent with UC. These mice also had increased hematochezia and mortality compared with Ctls. Both outcomes were exacerbated by subsequent episodes of systemic inflammation (34).

Interestingly, we did not note a significant reduction in other junction proteins (including β-catenin, Cldn1, Cldn2, Cldn7, Cldn8, or Cldn15) despite NI or AI exposures. Ocln was decreased only when exposed to AI, independent of the presence or absence of NI. There have been varied reports of Ocln expression in IBD. In a study comparing Ocln and E-cadherin expression, Ocln was significantly decreased in the colonic mucosa of patients with UC in areas of active disease and in histologically quiescent areas compared with Ctls. E-cadherin was downregulated only in areas of active inflammation (35). In a subsequent study of Ocln expression in UC and CD, there was no significant change in the luminal epithelium of IBD patients compared with Ctls (36). These findings suggest that further work is warranted on the mechanisms influencing Ocln expression.

Although there were significant decreases in Cdh1 encoding E-cadherin in NI + AI rats, it was relatively smaller than the subsequent decrease in NI + AI protein expression. Although there was a decrease in E-cadherin transcription, this discrepancy suggests there is likely concurrent posttranslational activity mediating total intact E-cadherin levels as well. One example of this is proteolysis mediated in part by matrix metalloproteinases (MMPs). MMP-1 and MMP-3 mRNA were significantly increased in actively inflamed versus quiescent UC and CD mucosa (37), and MMP-2 has been implicated in E-cadherin cleavage (38).

Overall, our data reinforces the role of E-cadherin as a key mediator of epithelial integrity in IBD and suggests that E-cadherin is unique among its cohort of junction proteins in its susceptibility to NI-induced expression changes. Importantly, we then demonstrated several mechanisms of inflammation-dependent changes that mediate alterations in E-cadherin expression and intestinal permeability.

miRNA and their role in the modulation of gene expression represent an active area of research interest in IBD. Initially, the role of miR-155 in tight junction signaling was investigated by bioinformatics analysis using IPA software. miR-155 has previously been demonstrated to influence expression of E-cadherin by targeting RhoA (22). We investigated pathways using predicted interactions of miR-155 and tight junction signaling. Consistent with previously described results, miR-155 was predicted to interfere with tight junction integrity through increasing expression of PRKCI (8). PRKCI overexpression has been shown to functionally inhibit aPKC, a molecule which assists in formation and maintenance of tight junctions (39). The impact of PRKCI expression and its effect on E-cadherin in colonic cells is unclear at this time.

In the present investigation, miR-155 was significantly increased in NI + AI rats compared with Ctl + Ctl, Ctl + AI, and NI + Ctl groups. This augmentation of miR-155 expression was confirmed by RT-qPCR. Using ChIP-qPCR, we interrogated epigenetic alterations mediating the increase in miR-155, and noted increased acetylation of H3K9 and H4K12, dependent on the presence of both NI and AI. Acetylation of lysine residues of histones H3 and H4 allowed for increased RNA polymerase II binding to the Mir155 gene promoter site, resulting in transcriptional activation. We were unable to identify any single histone modification that is responsible for the Mir155 promoter sensitization by NI. We postulate that NI sensitizes the Mir155 promoter by modifying a histone code, which could be a massively complex, rather than a single, lysine residue.

Although increased miR-155 expression has previously been shown to correspond to increased levels of TNF-α, the mechanism of this was previously unknown (40, 41). Recently, we showed that the combination of NI + AI induces an augmented immune response corresponding with a marked increase in TNF-α in the colonic mucosa/submucosa (7). In the current study, we found that AI alone also increased TNF-α, but two rounds of AI failed to exacerbate TNF-α expression. We demonstrated that hyperacetylation at the H3K9 and H4K12 could be induced by exposure to high concentration of TNF-α (≥1 ng/ml, Fig. 5C, 5E), again resulting in a significant increase in miR-155 expression. This was unique to TNF-α, and not IL-1β, and was not observed with other proinflammatory molecules associated with IBD: H2O2, and LPS. In addition, the epigenetic changes in miR-155 expression by TNF-α, were recapitulated in SW620 and CCD 841 CoN colonic epithelial cells but not in HEK 293 kidney epithelial cells.

Increased TNF-α levels have been noted in the mucosa of both UC and CD patients (2426) and play a central role in IBD pathogenesis. Anti–TNF-α Ab therapy has been shown to improve rates of remission in active disease in both UC and CD (42). As a result, anti–TNF-α Ab therapies have been increasingly used in IBD (19). Significant changes in miR-155 expression in the setting of NI + AI, mediated at least in part by TNF-α, and the specificity of miR-155 expression in colonic epithelium provide a compelling mechanism in which miR-155 confers an important and persistent role in the pathogenesis of IBD. Corroboration of this hypothesis might come from future preclinical studies in which miR-155 has been deleted selectively in the colon.

In the present research, we expanded on miR-155 mechanisms for direct alteration in epithelial integrity, confirming that miR-155 interferes with adherens/tight junctions by targeting and decreasing E-cadherin expression. By transfecting SW620 and CCD 841 CoN cells with miR-155, a significant decrease in E-cadherin expression occurred, mimicking transfection with CDH1 siRNA. Although this relationship has been previously described in severe pancreatitis, this is, to our knowledge, the first demonstration of this interaction in an IBD model (22). This was validated by an in vivo model in which colonic permeability was significantly decreased in NI + AI rats receiving a rectally administered miR-155 inhibitor. These findings provide an explanation for our previously noted NI + AI–dependent changes in E-cadherin levels.

In summary, our findings suggest that the combination of NI and AI in rats creates a significant and sustained change in gut permeability, and that a single insult later in life cannot recapitulate the exaggerated response triggered by an initial NI episode. This supports our previous findings that multiple AI episodes do not significantly change colonic permeability in the absence of NI. We demonstrated that, unlike other junction proteins, E-cadherin levels were selectively and significantly reduced by NI + AI exposure. The changes in E-cadherin expression were mediated, at least in part, by miR-155. In addition, miR-155 expression was uniquely increased by TNF-α compared with other proinflammatory molecules. This in total suggests that miR-155 and its interplay with colonic permeability via E-cadherin changes plays a disease-specific role in IBD pathogenesis and is an interesting and pertinent therapeutic target for future clinical evaluation.

LC Sciences (Houston, TX) performed the miRNA microarrays and helped with data deposit.

This work was supported in part by National Institutes of Health Grant R21 AI126097 (to Q.L.) and American Heart Association Grant 17GRNT33460395 (to Q.L.).

The microarray data presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138770) under accession number GSE138770.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • AI

    adult inflammation

  •  
  • CD

    Crohn's disease

  •  
  • ChIP

    chromatin immunoprecipitation

  •  
  • Cldn

    claudin

  •  
  • Ctl

    control

  •  
  • IBD

    inflammatory bowel disease

  •  
  • IPA

    ingenuity pathway analysis

  •  
  • miRNA

    microRNA

  •  
  • MMP

    matrix metalloproteinase

  •  
  • NI

    neonatal inflammation

  •  
  • Ocln

    occludin

  •  
  • PRKCI

    protein kinase C iota

  •  
  • qMSP

    quantitative methylation-specific PCR

  •  
  • qPCR

    quantitative PCR

  •  
  • RT-qPCR

    quantitative RT-PCR

  •  
  • siRNA

    small interfering RNA

  •  
  • TNBS

    2,4,6-trinitrobenzene sulfonic acid

  •  
  • UC

    ulcerative colitis.

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The authors have no financial conflicts of interest.

Supplementary data