MicroRNA profiling in Muc2 knockout mice of colitis-associated cancer model reveals epigenetic alterations during chronic colitis malignant transformation.
ABSTRACT: Our previous studies have demonstrated that genetic deletion of the Muc2 gene causes colorectal cancers in mice. The current study further showed that at the early stage (<3 months) the Muc2 knockout mice spontaneously developed chronic inflammation in colon and rectum, similar pathological features as human colitis; and at the late stage (>3 months) the mice exhibited colorectal cancer, including a unique phenotype of rectal prolapsed (rectal severe inflammation and adenocarcinoma). Thus, the age of 3 months might be the key point of the transition from chronic inflammation to cancer. To determine the mechanisms of the malignant transformation, we conducted miRNA array on the colonic epithelial cells from the 3-month Muc2-/- and +/+ mice. MicroRNA profiling showed differential expression of miRNAs (i.e. lower or higher expression enrichments) in Muc2-/- mice. 15 of them were validated by quantitative PCR. Based on relevance to cytokine and cancer, 4 miRNAs (miR-138, miR-145, miR-146a, and miR-150) were validate and were found significantly downregulated in human colitis and colorectal cancer tissues. The network of the targets of these miRNAs was characterized, and interestedly, miRNA-associated cytokines were significantly increased in Muc2-/-mice. This is the first to reveal the importance of aberrant expression of miRNAs in dynamically transformation from chronic colitis to colitis-associated cancer. These findings shed light on revealing the mechanisms of chronic colitis malignant transformation.
Project description:Gut dysbiosis is associated with colitis-associated colorectal carcinogenesis, and the genetic deficiency of the Muc2 gene causes spontaneous development of colitis and colorectal cancer. Whether there are changes of gut microbiota and a linkage between the changes of microbiota and intestinal pathology in Muc2-/- mice are unclear. Muc2-/- and Muc2+/+ mice were generated by backcrossing from Muc2+/- mice, and the fecal samples were collected at different dates (48th, 98th, 118th, 138th, and 178th day). Gut microbiota were analyzed by high-throughput sequencing with the universal 16S rRNA primers (V3?V5 region). All mice were sacrificed at day 178 to collect colonic tissue and epithelial cells for the analysis of histopathology and inflammatory cytokines. On the 178th day, Muc2-/- mice developed colorectal chronic colitis, hyperplasia, adenomas and adenocarcinomas, and inflammatory cytokines (e.g., cyclooxygenase 2 (COX-2), interleukin 6 (IL-6), tumor necrosis factor-? (TNF-?), interleukin 1 ? (IL-1?), i-kappa-B-kinase ? (IKK?)) were significantly increased in colonic epithelial cells of Muc2-/- mice. In general, structural segregation of gut microbiota was observed throughout the experimental time points between the Muc2-/- and Muc2+/+ mice. Impressively, in Muc2-/- mice, Alpha diversities reflected by Shannon and Chao indexes were higher, the phylum of Firmicutes was enriched and Bacteroidetes was decreased, and Desulfovibrio, Escherichia, Akkermansia, Turicibacter, and Erysipelotrichaceae were significantly increased, but Lactobacilli and Lachnospiraceae were significantly decreased. Moreover, the abundance of Ruminococcaceae and butyrate-producing bacteria was significantly higher in the Muc2-/- mice. There were significant differences of gut microbiota between Muc2-/- and Muc2+/+ mice. The dynamic changes of microbiota might contribute to the development of colitis and colitis-associated colorectal carcinogenesis. Therefore, this study revealed specific functional bacteria in the development of colitis and colitis-associated colorectal carcinogenesis, which will benefit the development of preventive and therapeutic strategies for chronic inflammation and its malignant transformation.
Project description:Intestinal epithelia are protected by a layer of mucin secreted by goblet cells against mechanical and chemical injuries, potent causes of inflammation, and the most abundant secreted intestinal mucin is encoded by the Muc2 gene. Genetic deletion of Muc2 causes intestinal inflammation in early stage and tumors after 3 months. The underlying mechanisms are not clear, but epigenetic alterations, particularly, up- and down-regulated microRNAs are involved in the malignant transformation from colitis to cancer. We used miRNA array to profile the differential expression of the miRNAs in Muc2-/- mouse colonic epithelial lin comparison with those in wild-type mice. Total RNA were extracted from mouse colonic epithelial cells and Muc2-/- and +/+, and the RNA were hybridized on Affymetrix miRNA microarray to determine the alterations of miRNAs during colitis development and its malignant transformation from colitis to cancer. To the end, we found miRNA were differential expressed in the Muc2-/- mice, among them 20 miRNAs were significantly downregulated and 71 miRNAs were significantly upregulated in Muc2-/- mice, in comparison with Muc2+/+ mice (change fold >2 or <0.5; T<0.01, p value< 0.05, q value< 0.05).
Project description:<h4>Background</h4>Chronic inflammation associated with ulcerative colitis predisposes individuals to increased colon cancer risk. The aim of these studies was to identify microRNAs that are aberrantly regulated during inflammation and may participate in transformation of colonic epithelial cells in the inflammatory setting.<h4>Methodology/principal findings</h4>We have use quantitative PCR arrays to compare microRNA (miRNA) expression in tumors and control colonic epithelial cells isolated from distal colons of chronically inflamed mice and APC(Min/+) mice. Rank order statistics was utilized to identify differentially regulated miRNAs in tumors that arose due to chronic inflammation and/or to germline APC mutation. Eight high priority miRNAs were identified: miR-215, miR-137, miR-708, miR-31, and miR-135b were differentially expressed in APC tumors and miR-215, miR-133a, miR-467d, miR-218, miR-708, miR-31, and miR-135b in colitis-associated tumors. Four of these (miR-215, miR-708, miR-31, and miR-135b) were common to both tumors types, and dysregulation of these miRNAs was confirmed in an independent sample set. Target prediction and pathway analysis suggests that these microRNAs, in the aggregate, regulate signaling pathways related to MAPK, PI3K, WNT, and TGF-?, all of which are known to be involved in transformation.<h4>Conclusions/significance</h4>We conclude that these four miRNAs are dysregulated at some very early stage in transformation of colonic epithelial cells. This response is not dependent on the mechanism of initiation of transformation (inflammation versus germline mutation), suggesting that the miRNAs that we have identified are likely to regulate critical signaling pathways that are central to early events in transformation of colonic epithelial cells.
Project description:Mutation of tumor suppressor adenomatous polyposis coli (APC) initiates most colorectal cancers and chronic colitis increases risk. APC is a nucleo-cytoplasmic shuttling protein, best known for antagonizing Wnt signaling by forming a cytoplasmic complex that marks ?-catenin for degradation. Using our unique mouse model with compromised nuclear Apc import (Apc(mNLS)), we show that Apc(mNLS/mNLS) mice have increased susceptibility to tumorigenesis induced with azoxymethane (AOM) and dextran sodium sulfate (DSS). The AOM-DSS-induced colon adenoma histopathology, proliferation, apoptosis, stem cell number and ?-catenin and Kras mutation spectra were similar in Apc(mNLS/mNLS) and Apc(+/+) mice. However, AOM-DSS-treated Apc(mNLS/mNLS) mice showed more weight loss, more lymphoid follicles and edema, and increased colon shortening than treated Apc(+/+) mice, indicating a colitis predisposition. To test this directly, we induced acute colitis with a 7 day DSS treatment followed by 5 days of recovery. Compared with Apc(+/+) mice, DSS-treated Apc(mNLS/mNLS) mice developed more severe colitis based on clinical grade and histopathology. Apc(mNLS/mNLS) mice also had higher lymphocytic infiltration and reduced expression of stem cell markers, suggesting an increased propensity for chronic inflammation. Moreover, colons from DSS-treated Apc(mNLS/mNLS) mice showed fewer goblet cells and reduced Muc2 expression. Even in untreated Apc(mNLS/mNLS) mice, there were significantly fewer goblet cells in jejuna, and a modest decrease in colonocyte Muc2 expression compared with Apc(+/+) mice. Colonocytes from untreated Apc(mNLS/mNLS) mice also showed increased expression of inflammatory mediators cyclooxygenase-2 (Cox-2) and macrophage inflammatory protein-2 (MIP-2). These findings reveal novel functions for nuclear Apc in goblet cell differentiation and protection against inflammation-induced colon tumorigenesis.
Project description:miR-148a has been shown to regulate inflammation, immunity and the growth of certain tumors, but its roles in colitis and colorectal tumorigenesis remain largely undetermined. Here we found miR-148a-deficient mice to be more susceptible to colitis and colitis-associated tumorigenesis. Both were associated with increased nuclear factor ?B (NF-?B) and signal transducer and activator of transcription 3 (STAT3) signaling. Bone marrow- and non-bone marrow-derived miR-148a contributed to colitis and colitis-associated tumorigenesis. miR-148a loss of heterozygosity exacerbated Apc<sup>min/+</sup> colon and small intestinal spontaneous tumor development. Restoring miR-148a expression prevented both spontaneous and carcinogen-induced colon tumor development. miR-148a was downregulated in human inflammatory bowel disease (IBD) and colorectal cancer patient tissues. This correlated with a high degree of miR-148a promoter methylation mediated by a complex comprised of P65 and DNA methyltransferase 3 alpha (DNMT3A). miR-148a directly targets several well-accepted upstream regulators of NF-?B and STAT3 signaling, including GP130, IKK?, IKK?, IL1R1 and TNFR2, which leads to decreased NF-?B and STAT3 activation in macrophages and colon tissues. Our findings reveal that miR-148a is an indirect tumor suppressor that modulates colitis and colitis-associated tumorigenesis by suppressing the expression of signaling by NF-?B and STAT3 and their pro-inflammatory consequences.
Project description:MicroRNAs (miRNAs) are epigenetically involved in regulating gene expression. They may be of importance in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to determine the role of miRNAs by their specific blocking in the CD4+CB45RBhi T-cell transfer model of chronic experimental colitis.Colitis caused by transfer of WT CD4+CD45RBhi T cells in severe combined immunodeficiency (SCID) mice shares many features with human IBD. Colonic miRNA expression levels were measured at three time points in colitic mice, where a time-dependent upregulation of multiple miRNAs was seen. To inhibit these miRNAs, specific locked-nucleic-acid-modified (LNA) oligonucleotides were administered in further experiments at the moment the mice demonstrated the first signs of colitis. As controls, PBS and a scrambled sequence of anti-miRNA were used. Genome-wide expression analyses were also performed in order to detect candidate target genes of miR-142-5p, of which inhibition resulted in most effective amelioration of colitis.Anti-miR-142-5p reduced colitis and related wasting disease when administered in the T-cell transfer model, reflected in reduced weight loss and a lower disease activity index (DAI). In further validation experiments we also observed a higher survival rate and less colonic histological inflammation in the antagomir-treated mice. Moreover, by genome-wide expression analyses, we found downstream activation of the anti-inflammatory IL10RA pathway, including three genes also found in the top-20 candidate target genes of miR-142-5p.In conclusion, CD4+CD45RBhi-transfer colitis induces miR-142-5p. Blocking miR-142-5p reduced colitis and prevented wasting disease, possibly by activation of the IL10RA pathway.
Project description:MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.
Project description:<h4>Background</h4>The colonic mucus layer plays a critical role in intestinal homeostasis by limiting contact between luminal bacteria and the mucosal immune system. A defective mucus barrier in animal models allows bacterial contact with the intestinal epithelium and results in spontaneous colitis. A defective mucus barrier is also a key feature of active ulcerative colitis (UC). Alterations in the immune compartment due to intestinal bacterial breach in mice lacking the colon mucus barrier have not been characterized and correlated to active UC.<h4>Aims</h4>To characterize alterations in the immune compartment due to intestinal bacterial breach in Muc2-/- mice, which lack the colon mucus barrier, and correlate the findings to active UC.<h4>Methods</h4>Bacterial contact with colon epithelium and penetration into colon tissue was examined in Muc2-/- mice and colon biopsies from patients with active UC using fluorescence microscopy and qPCR. Neutrophils, lymphocytes, CD103+ dendritic cell subsets and macrophages in colon from Muc2-/- mice and biopsies from UC patients were quantitated by flow cytometry.<h4>Results</h4>Inflamed UC patients and Muc2-/- mice had bacteria in contact with the colon epithelium. Bacterial rRNA was present in colonic mucosa in humans and Muc2-/- mice and in the draining lymph nodes of mice. Inflamed Muc2-/- mice and UC patients had elevated colon neutrophils, T cells and macrophages while a reduced frequency of CD103+ DCs was present in the inflamed colon of both mice and humans.<h4>Conclusions</h4>The parallel features of the colon immune cell compartment in Muc2-/- mice and UC patients supports the usefulness of this model to understand the early phase of spontaneous colitis and will provide insight into novel strategies to treat UC.
Project description:Altered intestinal O-glycan expression has been observed in patients with ulcerative colitis and colorectal cancer, but the role of this alteration in the etiology of these diseases is unknown. O-glycans in mucin core proteins are the predominant components of the intestinal mucus, which comprises part of the intestinal mucosal barrier. Core 3-derived O-glycans, which are one of the major types of O-glycans, are primarily expressed in the colon. To investigate the biological function of core 3-derived O-glycans, we engineered mice lacking core 3 beta1,3-N-acetylglucosaminyltransferase (C3GnT), an enzyme predicted to be important in the synthesis of core 3-derived O-glycans. Disruption of the C3GnT gene eliminated core 3-derived O-glycans. C3GnT-deficient mice displayed a discrete, colon-specific reduction in Muc2 protein and increased permeability of the intestinal barrier. Moreover, these mice were highly susceptible to experimental triggers of colitis and colorectal adenocarcinoma. These data reveal a requirement for core 3-derived O-glycans in resistance to colonic disease.