MUC1 mRNA Research Articles

Up-regulation of microRNA-136 induces apoptosis and radiosensitivity of esophageal squamous cell carcinoma cells by inhibiting the expression of MUC1

ObjectiveThis present study is performed to figure out the role of microRNA-136 (miR-136) in radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells through the regulation of MUC1. MethodsSeventy-four ESCC patients were divided into radiotherapy sensitive group and radiotherapy resistance group. Colony formation assay and flow cytometry were used to test the radiosensitivity of radiotherapy resistant strain and parent strain. The expression of miR-136 between radiotherapy resistant strain and parent strain was detected by RT-qPCR, and the expression of miR-136 in Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells was detected after different doses of X-ray irradiation. Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells with overexpression of miR-136 or co-overexpression of miR-136 and MUC1 were constructed. Cell proliferation, colony formation and apoptosis was detected by CCK-8 assay, colony formation assay, and flow cytometry, respectively. ResultsThe expression of miR-136 in ESCC tissues was lower and MUC1 mRNA and protein expression was higher than that in adjacent normal tissues. The expression of miR-136 was negatively correlated with the expression of MUC1 mRNA in ESCC. Low expression of miR-136 and high expression of MUC1 were associated with tumor size, lymph node metastasis and distant metastasis. The expression of miR-136 increased while the expression of MUC1 decreased in the radiotherapy sensitive group of ESCC patients relative to the radiotherapy resistant group. The colony formation ability of radiation resistant cell line was stronger than that of parent cell line, and the apoptosis rate showed an opposite trend. Up-regulation of miR-136 reduced the survival rate, suppressed colony formation ability and induced apoptosis of ESCC cells under irradiation, which was reversed by upregulated MUC1. ConclusionThis study demonstrates that up-regulation of miR-136 induces apoptosis and radiosensitivity of ESCC cells by inhibiting the expression of MUC1.

Impact of Maternal Malnutrition on Gut Barrier Defense: Implications for Pregnancy Health and Fetal Development.

Small intestinal Paneth cells, enteric glial cells (EGC), and goblet cells maintain gut mucosal integrity, homeostasis, and influence host physiology locally and through the gut-brain axis. Little is known about their roles during pregnancy, or how maternal malnutrition impacts these cells and their development. Pregnant mice were fed a control diet (CON), undernourished by 30% vs. control (UN), or fed a high fat diet (HF). At day 18.5 (term = 19), gut integrity and function were assessed by immunohistochemistry and qPCR. UN mothers displayed reduced mRNA expression of Paneth cell antimicrobial peptides (AMP; Lyz2, Reg3g) and an accumulation of villi goblet cells, while HF had reduced Reg3g and mucin (Muc2) mRNA and increased lysozyme protein. UN fetuses had increased mRNA expression of gut transcription factor Sox9, associated with reduced expression of maturation markers (Cdx2, Muc2), and increased expression of tight junctions (TJ; Cldn-7). HF fetuses had increased mRNA expression of EGC markers (S100b, Bfabp, Plp1), AMP (Lyz1, Defa1, Reg3g), and TJ (Cldn-3, Cldn-7), and reduced expression of an AMP-activator (Tlr4). Maternal malnutrition altered expression of genes that maintain maternal gut homeostasis, and altered fetal gut permeability, function, and development. This may have long-term implications for host-microbe interactions, immunity, and offspring gut-brain axis function.

Thymoquinone-Induced Tristetraprolin Inhibits Tumor Growth and Metastasis through Destabilization of MUC4 mRNA.

Tristetraprolin (TTP), a well-characterized AU-rich element (ARE) binding protein, functions as a tumor suppressor gene. The purpose of this study was to investigate whether a bioactive substance derived from a natural medicinal plant affects the induction of TTP and to elucidate its mechanism. We examined the effects of natural bioactive materials including Resveratrol (RSV), thymoquinone (TQ) and curcumin on the expression of TTP in cancer cell. TQ derived from a natural plant Nigella sativa increased the expression levels of TTP mRNA and proteins in a dose-dependent manner in gastric and breast cancer cells. TQ-induced TTP increased the instability of MUC4 mRNA by direct binding of TTP to ARE in the 3′UTR of MUC4 mRNA. The induction of TTP by TQ also reduced the proliferation, migration and invasion of cancer cells. The expression of the epithelial-mesenchymal (EMT)-related genes, which were target genes of TTP, was also decreased by the TQ treatment. In the in vivo experiments using mouse melanoma cells, TQ-induced TTP inhibited metastasis of tumor cells. We have found that TQ-induced TTP might inhibit metastasis by reducing tumor cell migration and invasion through destabilization of MUC4 mRNA, which suggest the MUC4 as a novel target to TTP.

Ascites from ovarian cancer patients stimulates MUC16 mucin expression and secretion in human peritoneal mesothelial cells through an Akt-dependent pathway

BackgroundCA125 is a well-established ovarian cancer (OC) serum biomarker. The CA125 heavily glycosylated epitope is carried by the MUC16 mucin, a high molecular weight transmembrane mucin. Upon proteolytic cleavage, the extracellular domain of MUC16 is released from the cell surface into malignant ascites and blood vessels. Previous studies have shown that both tumor and surrounding mesothelial cells may express MUC16. Although little is known about the regulation of MUC16 expression in these cells, recent evidence suggest that inflammatory cytokines may stimulate MUC16 expression. Because malignant ascites is a pro-inflammatory environment, we investigated whether OC ascites stimulate the expression and release of MUC16 by human peritoneal mesothelial cells (HPMCs).MethodsHPMCs were isolated from peritoneal lavages of women operated for conditions other than cancer. MUC16 protein expression was determined by immunoblot, immunofluorescence or immunohistochemistry depending on the experiments. The release of MUC16 from the cell surface was measured using EIA and MUC16 mRNA expression by ddPCR.ResultsWe show that high-grade serous ascites from patients with OC (n = 5) enhance MUC16 expression in HPMCs. Malignant ascites, but not benign peritoneal fluids, stimulate the release of MUC16 in HPMCs in a dose-dependent manner, which is abrogated by heat inactivation. Moreover, we establish that ascites-induced MUC16 expression occurs at the post-transcriptional level and demonstrate that ascites-induced MUC16 expression is mediated, at least partially, through an Akt-dependent pathway. A cytokine array identified upregulation of several cytokines and chemokines in ascites that mediate MUC16 upregulation versus those that do not, including CCL7, CCL8, CCL16, CCL20, CXCL1, IL-6, IL-10, HGF and IL-1 R4. However, when individually tested, none of these factors affected MUC16 expression or secretion. Concentrations of CA125 in the serum of a given patient did not correlate with the ability of its corresponding ascites to stimulate MUC16 release in HPMCs.ConclusionsCollectively, these data indicate that mesothelial cells are an important source of MUC16 in the context of ovarian cancer and malignant ascites is a strong modulator of MUC16 expression in HPMCs and uncover the Akt pathway as a driving factor for upregulation of MUC16. Factors in ascites associated with enhanced MUC16 expression and release remains to be identified.

Expression of inositol-requiring enzyme 1β is downregulated in azoxymethane/dextran sulfate sodium-induced mouse colonic tumors.

Inflammatory bowel disease (IBD) is a risk factor in colon cancer. Endoplasmic reticulum (ER) stress is associated with IBD and cancer. In the current study an azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced mouse colonic tumor model was established to analyze the expression of ER stress chaperone molecules. Female C57BL/6 mice were intraperitoneally injected with 12 mg/kg AOM. On the 7th day following AOM injection, mice were treated with 1% DSS supplemented to the drinking water for 7 days, then followed by 14 days of normal drinking water. The cycle of 7 days DSS plus 14 days normal water was repeated twice and colonic tumors were evaluated for their number and size. Mice in the control group were injected with saline and received normal drinking water for the course of the experiment. mRNA levels of cytokines, inositol-requiring enzyme (IRE)1α and 1β, their downstream targets X-box binding protein (XBP)1u, XBP1s and mucin (MUC) 2 and interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α were detected by reverse transcription-quantitative polymerase chain reaction. IRE1α, IRE1β and MUC2 protein expression was evaluated by immunohistochemistry, and IRE1α and IRE1β levels were further assessed by western blot analysis. It was observed that tumors developed in the distal colon of mice treated with AOM/DSS. IL-6, IL-8 and TNF-α mRNA levels were significantly increased in mice of the tumor group compared with mice of the control group. There were no significant differences in IRE1α mRNA and protein expression between the two groups and XBP1s mRNA levels were increased in the tumor compared with the control group. IRE1β and MUC2 mRNA levels were significantly decreased in the tumor compared with the control group (decreased by 42 and 30%, respectively). IRE1β and MUC2 proteins were predominately expressed in colonic epithelial cells and expression was decreased in the tumor compared with the control group. In conclusion, the downregulation of IRE1β and MUC2 may reduce the ability of colon tissues to resist inflammation, thus promoting the occurrence and development of colonic tumors.

P126 MULTIPLE SIGNALING PATHWAY DEFECTS IN THE INTESTINAL STEM CELL NICHE LIKELY TRIGGER CD-LIKE ILEITIS IN SAMP1/YITFC MICE

Intestinal epithelial barrier dysfunction is considered as a risk factor in the pathogenesis of Crohn’s disease (CD), and since there is no FDA approved therapy available, there is a need for identifying a relevant model to study and test therapies to correct this defect. Previously, we demonstrated that inflammatory environment of CD affects properties of intestinal stem cell (ISC) niche, ablating its ability to form enteroids (EnOs) in our mouse model for CD-like ileitis (SAMP/YitFc, SAMP) (doi.org/10.1016/S0016-5085(17)32213-8). However, EnOs from SAMP mice even prior to disease onset (5-wks, SAMP-5) harbor impaired features, such as smaller size (2.6 ± 0.4 vs 3.9 ± 0.2 fold to ctrl at day 1, P=0.003), number (0.6 ± 0.1 fold to ctrl, P=0.000), reduced viability (63.7%±7.5 vs ctrl P=0.003) and rudimental branching complexity [cyst-like structures (42.7%±2.3 vs 11%±1.2)], compared to controls (ctrls). These EnOs produce exacerbated expression of genes involved in epithelial secretory cell functions when challenged with purified bacterial ligands, such as LPS [higher Muc2 (10.8 ± 3.2 vs 2.5 ± 1.8 P=0.00), Lyz (11.0 ± 5.2 vs 1.1 ± 0.4 P=0.00), Reg3γ (6.7 ± 9.9 vs 1.1 ± 0.5 P=0.04), Crp (7.4 ± 5.4 vs 1.3 ± 0.6 P=0.05), and Defa1 (4006.0 ± 1472.5 vs 2.1 ± 0.7 P=0.00) mRNA vs AKR EnOs]. To further investigate this phenotype, we generated the first high-resolution transcriptional profile of SAMP ISC niche by laser capture microdissection and RNA sequencing. We found that signaling pathways involved in the cellular energy machinery were dramatically reduced in inflamed SAMP (25-wks, SAMP-25) ISC niche compared to that of inflammation-free mice [mitochondrial energy metabolism (P ≤ 0.00, Padj ≤ 0.002, normalized enrichment score NES ≤ -2.4); oxidative phosphorylation (P ≤ 0.00 Padj ≤ 0.001 NES ≤ -2.5)]. Fibrosis (P ≤ 0.04 Padj≤ 0.09 NES ≤ 1.3) and UPR (P ≤ 0.01 Padj ≤ 0.03 NES ≤ 1.5) pathways were enriched in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Bile acid metabolism (P ≤ 0.00, Padj ≤ 0.002 NES ≤ -1.8), ROS (P ≤ 0.001 Padj ≤ 0.003 NES ≤ -1.8), Wnt3a (P ≤ 0.001 Padj ≤ 0.01 NES ≤ -1.8), and Notch1 (P ≤ 0.001 Padj ≤ 0.02 NES ≤ -1.8) pathways were decrease in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Collectively, these data suggest that multiple signaling pathways contribute to ISC niche defect in SAMP mice, and that many key biological functions are already altered prior to ileitis onset and may be responsible for disease initiation. Finally, the evidence that altered gene expression is maintained in ex vivo expanded EnOs indicates that these cultures recapitulate reactive epithelial changes specific of experimental CD. This aspect is of paramount importance since it emphasizes the physiologically relevant value of EnOs generated from SAMP mice, rather than those originated from healthy individuals, to mimic CD-like ileitis.

P126 MULTIPLE SIGNALING PATHWAY DEFECTS IN THE INTESTINAL STEM CELL NICHE LIKELY TRIGGER CD-LIKE ILEITIS IN SAMP1/YITFC MICE

Intestinal epithelial barrier dysfunction is considered as a risk factor in the pathogenesis of Crohn’s disease (CD), and since there is no FDA approved therapy available, there is a need for identifying a relevant model to study and test therapies to correct this defect. Previously, we demonstrated that inflammatory environment of CD affects properties of intestinal stem cell (ISC) niche, ablating its ability to form enteroids (EnOs) in our mouse model for CD-like ileitis (SAMP/YitFc, SAMP) (doi.org/10.1016/S0016-5085(17)32213-8). However, EnOs from SAMP mice even prior to disease onset (5-wks, SAMP-5) harbor impaired features, such as smaller size (2.6 ± 0.4 vs 3.9 ± 0.2 fold to ctrl at day 1, P=0.003), number (0.6 ± 0.1 fold to ctrl, P=0.000), reduced viability (63.7%±7.5 vs ctrl P=0.003) and rudimental branching complexity [cyst-like structures (42.7%±2.3 vs 11%±1.2)], compared to controls (ctrls). These EnOs produce exacerbated expression of genes involved in epithelial secretory cell functions when challenged with purified bacterial ligands, such as LPS [higher Muc2 (10.8 ± 3.2 vs 2.5 ± 1.8 P=0.00), Lyz (11.0 ± 5.2 vs 1.1 ± 0.4 P=0.00), Reg3γ (6.7 ± 9.9 vs 1.1 ± 0.5 P=0.04), Crp (7.4 ± 5.4 vs 1.3 ± 0.6 P=0.05), and Defa1 (4006.0 ± 1472.5 vs 2.1 ± 0.7 P=0.00) mRNA vs AKR EnOs]. To further investigate this phenotype, we generated the first high-resolution transcriptional profile of SAMP ISC niche by laser capture microdissection and RNA sequencing. We found that signaling pathways involved in the cellular energy machinery were dramatically reduced in inflamed SAMP (25-wks, SAMP-25) ISC niche compared to that of inflammation-free mice [mitochondrial energy metabolism (P ≤ 0.00, Padj ≤ 0.002, normalized enrichment score NES ≤ -2.4); oxidative phosphorylation (P ≤ 0.00 Padj ≤ 0.001 NES ≤ -2.5)]. Fibrosis (P ≤ 0.04 Padj≤ 0.09 NES ≤ 1.3) and UPR (P ≤ 0.01 Padj ≤ 0.03 NES ≤ 1.5) pathways were enriched in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Bile acid metabolism (P ≤ 0.00, Padj ≤ 0.002 NES ≤ -1.8), ROS (P ≤ 0.001 Padj ≤ 0.003 NES ≤ -1.8), Wnt3a (P ≤ 0.001 Padj ≤ 0.01 NES ≤ -1.8), and Notch1 (P ≤ 0.001 Padj ≤ 0.02 NES ≤ -1.8) pathways were decrease in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Collectively, these data suggest that multiple signaling pathways contribute to ISC niche defect in SAMP mice, and that many key biological functions are already altered prior to ileitis onset and may be responsible for disease initiation. Finally, the evidence that altered gene expression is maintained in ex vivo expanded EnOs indicates that these cultures recapitulate reactive epithelial changes specific of experimental CD. This aspect is of paramount importance since it emphasizes the physiologically relevant value of EnOs generated from SAMP mice, rather than those originated from healthy individuals, to mimic CD-like ileitis.

53 Pre-implantation exposure to bisphenol A and 4-tert-octylphenol result in disruption of calcium channels

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all miscarriage cases in humans. Calcium (Ca2+) has been shown to involve many cellular signal transduction pathways as well as regulation of cell adhesion, which is necessary for the physiology process of endometrial epithelial cell transformation and stromal cell decidualization during embryo implantation. Exposure to endocrine-disrupting chemicals (ED) can regulate the expression of genes associated with calcium transport in during pregnancy such as TRPV5, TRPV6, PMCA, and NCX1. Additionally, exposure to ED during early gestation results in disrupted intrauterine implantation, uterine receptive, leading to implantation failure. In this study, oestrogen (E2), bisphenol A (BPA), octylphenol (OP), and/or ICI 182,780 (oestrogen receptor antagonist, ICI) were injected subcutaneously from gestation Day 1 to gestation Day 3 post coitus. The number of implantation sites was significantly lower in the OP group, and no implantation site was observed in the E2 and ED+ICI groups. There were differences in the expression of calcium transient transport channel between maternal uterine and implantation. The level of TRPV6 and TRPV5 mRNA and protein was significantly increased by ED and/or ICI treatment in the uterus. The levels of TRPV5 and TRPV6 gene expression were significantly increased by ED with/without ICI treatment in the uterus. However, TRPV5 and TRPV6 gene expression was significantly lower in implantation site samples. The NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Both mRNA and protein levels of MUC1 were markedly higher in all groups, except the BPA group when compared with the vehicle group in the uterus. The LIF and HOXA-10 mRNA were significantly low in E2; BPA+ICI; OP; and/or ICI in both the uterus and implantation site. Expression of the oestrogen receptor (ERa) and progesterone receptor (PR) was significantly lower in all groups except the BPA group when compared with the vehicle group. Taken together, E2, BPA, and OP disrupt the success of implantation through altered expression of calcium transport genes.

Molecular insights into genome-wide association studies of chronic kidney disease-defining traits

Genome-wide association studies (GWAS) have identified >100 loci of chronic kidney disease-defining traits (CKD-dt). Molecular mechanisms underlying these associations remain elusive. Using 280 kidney transcriptomes and 9958 gene expression profiles from 44 non-renal tissues we uncover gene expression partners (eGenes) for 88.9% of CKD-dt GWAS loci. Through epigenomic chromatin segmentation analysis and variant effect prediction we annotate functional consequences to 74% of these loci. Our colocalisation analysis and Mendelian randomisation in >130,000 subjects demonstrate causal effects of three eGenes (NAT8B, CASP9 and MUC1) on estimated glomerular filtration rate. We identify a common alternative splice variant in MUC1 (a gene responsible for rare Mendelian form of kidney disease) and observe increased renal expression of a specific MUC1 mRNA isoform as a plausible molecular mechanism of the GWAS association signal. These data highlight the variants and genes underpinning the associations uncovered in GWAS of CKD-dt.

Expression of Mucins and Claudins in the Colon during Acute and Chronic Experimental Colitis.

We studied changes in the expression of mRNA for mucins and claudins in the medial part of the colon in male C57Bl/6 mice on the model of acute and chronic colitis induced by substitution of drinking water with 1% solution of dextran sodium sulphate for 5 days. In acute colitis, the expression of the main structural component of glycocalyx, mucin Muc3, decreased and expression of pore-forming claudin Cldn2 increased, which reflected enhanced permeability of tight junctions. In the chronic colitis group, in comparison with the normal group, we observed an increase in expression of mRNA of main structural mucus component Muc2, enhanced of expression of Muc1 associated with carcinogenesis, and reduced expression of Muc13, which led to a more severe course of colitis; the expression of pore-forming claudin Cldn2 was elevated. These findings indicate that the imbalance in the expression of mucins and claudins plays an important role in the mechanisms of development of acute and chronic colitis.

Effects of Bisphenol A and 4-tert-Octylphenol on Embryo Implantation Failure in Mouse.

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all human miscarriage cases. Calcium ion has been shown to be involved in many cellular signal transduction pathways as well as in the regulation of cell adhesion, which is necessary for the embryo implantation process. Exposure to endocrine-disrupting chemicals (EDs) during early gestation results in disruption of intrauterine implantation and uterine reception, leading to implantation failure. In this study, ovarian estrogen (E2), bisphenol A (BPA), or 4-tert-octylphenol (OP), with or without ICI 182,780 (ICI) were injected subcutaneously from gestation day 1 to gestation day 3 post-coitus. The expression levels of the calcium transport genes were assessed in maternal uteri and implantation sites. The number of implantation sites was significantly low in the OP group, and implantation sites were absent in the E2, ICI and EDs + ICI groups. There were different calcium transient transport channel expression levels in uterus and implantation site samples. The levels of TRPV5 and TRPV6 gene expression were significantly increased by EDs with/without ICI treatment in utero. Meanwhile, TRPV5 and TRPV6 gene expression were significantly lower in implantation sites samples. NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Compared to vehicle treatment in the uterus, both the MUC1 mRNA and protein levels were markedly high in all but the BPA group. Taken together, these results suggest that both BPA and OP can impair embryo implantation through alteration of calcium transport gene expressions and by affecting uterine receptivity.

Ileum terminal antibiotic infusion affects jejunal and colonic specific microbial population and immune status in growing pigs

BackgroundCompared with oral antibiotics (primarily disrupt foregut microbiota), the present study used antibiotics with ileum terminal infusion to disrupt the hindgut microbiota, and investigated the changes in specific bacterial composition and immune indexes in the jejunum and colon, and serum of growing pigs. Twelve barrows (45 d of age, 12.08 ± 0.28 kg) fitted with a T-cannula at the terminal ileum, were randomly assigned to two groups and infused either saline without antibiotics (Control) or with antibiotics (Antibiotic) at the terminal ileum. After 25 d experiment, all pigs were euthanized for analyzing bacterial composition and immune status.ResultsIleum terminal antibiotic infusion (ITAI) altered dominant bacteria counts, with a decrease in Bifidobacterium, Clostridium cluster IV and Clostridium cluster IV in the colon (P < 0.05), and an increase in Escherichia coli in the jejunum (P < 0.05). ITAI decreased (P < 0.05) short-chain fatty acids concentrations in the colon. ITAI decreased interleukin-8 (IL-8), IL-10 and secretory immunoglobulin A (sIgA) concentrations, and down-regulated IL-10, Mucin-1 (MUC1), Mucin-2 (MUC2) and zonula occludens-1 (ZO-1) mRNA expression in the colonic mucosa (P < 0.05). In the jejunal mucosa, ITAI decreased interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), sIgA and IgG levels together with down-regulation of IFN-γ, TNF-α, MUC2 and ZO-1 mRNA expression (P < 0.05). Furthermore, ITAI decreased IL-10, INF-γ, TNF-α, IgA and IgG concentrations in serum (P < 0.05). Correlation analysis revealed that the change in intestinal microbiota was correlated with alterations of Ig and cytokines.ConclusionsITAI affected jejunal and colonic specific bacteria counts, and altered some immune markers levels in the jejunal and colonic mucosa and serum. These findings implicate the potential contribution of hindgut bacteria to immune response in the intestinal mucosa and serum of growing pigs.

Attenuating effect of melatonin on lipopolysaccharide-induced chicken small intestine inflammation

Enriched melatonin (MEL) has been found in the mammalian intestine and has been recently demonstrated to alleviate rodent colitis. In this study, the effect of MEL on lipopolysaccharide (LPS)-induced intestinal inflammations was investigated in new chicken hatchlings. The chicks were fed with a diet supplemented with MEL (12.5mg/day) from D1 to D10. Meanwhile, the chicks in the LPS or MEL + LPS groups were injected with LPS (10mg/kg BW, i.p.) at D10. LPS treatment for 6h increased the expression of IL-6, IL-4, caspase-3 mRNAs and TUNEL-positive cell populations, but decreased populations of the goblet and PCNA+ cells, IgA production and the expression of MUC2 mRNA in the duodenum. Compared with the LPS group, MEL pre-feeding alleviated duodenal inflammation and decreased the expression of TNF-α mRNAs by 23.6% (P = 0.004), IL-6 mRNAs by 69.4% (P = 0.001), IL-4 mRNAs by 4.1% (P = 0.824) and caspase-3 mRNAs by 45.8% (P < 0.001). Conversely, MEL pre-feeding attenuated the LPS-induced changes of IgA production by 161.6% (P = 0.013) and PCNA+ cell populations by 172.1% (P < 0.001) in the duodenum. TLR4 mRNA was also up-regulated by LPS treatment but down-regulated by MEL pre-feeding. In conclusion, dietary MEL could attenuate LPS-induced chick duodenal inflammation by down-regulating the expression of inflammatory cytokines, promoting epithelial cell proliferation, improving the immunological barrier and inhibiting epithelial apoptosis via the mediation of TLR4.

Detection of miR-21, MUC1mRNA and VEGF Protein Biomarkers Expression Changes in Oral Squamous Cell Carcinomas (OSCC) in Peripheral Blood

Background and purpose: Oral squamous cell carcinoma is a common type of cancer and one of the most important causes of death in the world; therefore, this study investigates the expression of miR-21, MUC1 mRNA and VEGF protein in peripheral blood for early detection and better treatment of this cancer using Real-Time RT-PCR and Elisa.Materials and methods: In this experimental study, 50 OSCC patients and 50 healthy subjects were selected as controls. The total RNA in peripheral blood was extracted by RNA extraction kit and quality evaluation of extracted RNA was measured by NanoDrop.For the synthesis of cDNA, Viva 2-steps RT-PCR kit was used. Expression of miR-21 and MUC1 mRNA were measured by Real-Time RT-PCR and used Elisa for VEGF protein. The relationship between the expressions of these biomarkers was evaluated with tumor staging and progression of cancer.Results: The average age difference is not significant in these two groups by using t-test. The miR-21 was positive in 43 out of 50 OSCC patients (86%), and it was also 14 out of 50 healthy subjects (28%). The MUC1 mRNA was positive in 35 out of 50 OSCC patients, indicating 70% sensitivity, and it was positive in 9 of the 50 healthy subjects(18%). The serum levels of VEGF protein were measured using Elisa; it was positive in 32 out of 50 OSCC patients (64%) and this biomarker in healthy subjects report was not positive, indicating that the specificity is 100%.Conclusions: Overall, the miR-21, MUC1 mRNA and VEGF protein biomarkers could be suggested as a diagnostic screening test for the early detection of diseases. To prove further research is recommended further studies with a larger sample size to be done.

Peroxisome proliferator-activated receptor gamma activation promotes intestinal barrier function by improving mucus and tight junctions in a mouse colitis model

Background and aimsDefects in mucus and intestinal epithelia can lead to intestinal inflammation in colitis. Reduced peroxisome proliferator-activated receptor gamma (PPARγ) in the mucosa may contribute to inflammation. However, the roles of PPARγ in the intestinal barrier remain poorly understood. MethodsChronic colitis was induced in C57BL/6 mice by administration of dextran sulfate sodium (DSS) for 27 days. Three days before DSS treatment, mice were treated with the PPARγ agonist rosiglitazone (Ro) orally at 20 mg kg−1 day−1. ResultsThe colitis based on disease activity index and colonic histopathology was significantly ameliorated in the DSS + Ro group. Additionally, mice in the DSS + Ro group had a thicker mucous layer than those in DSS + NS group, and muc2 mRNA expression was elevated significantly along with the mouse atonal homolog, SAM-pointed domain-containing Ets-like factor, and anterior gradient 2 genes. Moreover, tight junctions were up-regulated, whereas long myosin light chain kinase and phosphorylation of the myosin II light chain were lower in DSS + Ro mice. Similarly, after HT-29 and Caco-2 cells were treated by LPS or LPS + Ro, PPARγ activation by Ro could effectively improve the intestinal barrier, including intestinal mucus and tight junctions. ConclusionsOur results demonstrate that activated PPARγ could effectively promote intestinal mucus integrity by increasing the number of goblet cells, the glycosylation of mucins, and tight junctions via an MLCK-dependent mechanism.

MiR-581-Related Single Nucleotide Polymorphism, rs2641726, Located in MUC4 Gene, is Associated with Gastric Cancer Incidence.

MUC4 is aberrantly expressed in several carcinomas including breast, colon, ovarian, lung, prostate, stomach and pancreatic cancers. MUC4 can regulate cell apoptosis negatively and facilitate stomach tumorigenesis. In this research, we aimed to evaluate the possible association between rs2641726 (C > A) polymorphism of MUC4 and gastric cancer risk in the Iranian population. In this case-control study, we collected blood samples from 168 gastric cancer patients and 66 healthy subjects. Allele-specific primer polymerase chain reaction method was applied to genotype rs2641726 in the obtained DNA samples. This study demonstrated that rs2641726 C allele was significantly associated with the incidence of gastric cancer, odds ratio = 3.382, 95% confidence interval: 1.840-6.217 (P < 0.001). Furthermore, the distribution of this risk allele was highly enriched in the samples with stage III. In silico studies revealed that the C allele of rs2641726, located within MUC4 3'UTR, is potential to attenuate the interaction between miR-581 and MUC4 mRNA. This disturbing effect, which might result in higher expression of MUC4 oncoprotein, was proposed for the mechanism of action of the rs2641726 risk allele. rs2641726 C allele is significantly enriched in gastric cancer specimens. The attenuating effect of this allele on miR-581 and MUC4 interaction might be a potential mechanism of action by which C allele imposes its oncogenic impact.

Quercetin Increases MUC2 and MUC5AC Gene Expression and Secretion in Intestinal Goblet Cell-Like LS174T via PLC/PKCα/ERK1-2 Pathway.

The main dietary flavonoid quercetin, is known to preserve the integrity of gastrointestinal barrier and to have anti-inflammatory, anti-cancer, anti-fibrotic, and other beneficial properties. Many of the biological effects of quercetin appear to be associated to the modulation of cell signaling pathways, rather than to its antioxidant activity. In spite of the large number of data available on the molecular and cellular mechanisms by which quercetin exerts its biological effects, including protection of intestinal barrier function, there is a lack of data about the role of this substance on the expression and/or the secretion of mucins released by intestinal goblet cells. Here we investigated the effects of quercetin on the secretion and the gene expression of the main intestinal gel-forming mucins, MUC2 and MUC5AC, and the signaling mechanisms underlined, in human intestinal goblet cell-like LS174T. We found that quercetin increases intracellular Ca2+ levels and induces MUC2 and MUC5AC secretion in a Ca2+-dependent manner. Quercetin also induces mRNA levels of both secretory mucins. Quercetin stimulation of LS174T cells increases phosphorylation levels of extracellular signal regulated kinase (ERK)1-2 and protein kinase C (PKC) α and the induction of MUC2 and MUC5AC secretion and mRNA relies on phospholipase C (PLC), PKC, and ERK1-2 signaling pathways since the PLC inhibitor U73122, the PKC inhibitor bisindolylmaleimide (BIM) and the ERK1-2 pathway inhibitor PD98059, all revert the stimulatory effects of quercetin. We also demonstrated that the induction of mucin gene expression by quercetin is not limited to goblet cells. Indeed, quercetin induces mRNA levels of MUC2 and MUC5AC via PKCα/ERK1-2 pathway also in the human intestinal epithelial Caco-2 cells. These data highlight a novel mechanism thereby quercetin, regulating the secretory function of intestinal goblet cells and mucin levels in enterocytes may exert its protective effects on intestinal mucosal barrier.

Protease Activated Receptor‐2 Mediates Giardia‐Induced Disruptions of the Intestinal Mucus Barrier

BackgroundGiardia duodenalis has been associated with disruptions of the mucus layers of the small and large intestines, in part via Giardia's cysteine proteases. Disruption of the intestinal mucus barrier has been implicated in a variety of intestinal disorders, but mechanisms currently remain unclear.HypothesisGoblet cell function and mucus disruptions are modulated by activation of protease activated receptor 2 (PAR2), which is highly expressed through the gastrointestinal tract and is known to regulate mucus production in the airway and stomach. Giardia's cysteine proteases cleave and activate PAR2 on intestinal goblet cells.MethodsMUC2 mucin and PAR gene expression were assessed in the human colonic epithelial cell line LS174T using quantitative PCR (qPCR). LS174T were pre‐treated with the PAR2 antagonist p2Pal‐218S pepducin, and incubated with Giardia trophozoites or the PAR1 and PAR2 agonist peptides TFLLRN and 2fLIGRLO, respectively. Ability of Giardia to cleave PAR1 and PAR2 at the N‐terminal domain was determined using CHO cells transformed with N‐luciferase tagged PAR1 or PAR2. Following incubation with Giardia, culture supernatant was collected, reacted with luciferin, and luminescence was measured. Wild‐type and PAR2 deficient mice were infected with Giardia trophozoites. Thickness of the mucus layer was measured by fluorescently staining mucus with fluorescein‐coupled WGA, and expression of Muc2 and Muc5ac genes were determined in the colon and jejunum via qPCR.ResultsLS174T cells express functional and responsive PAR2, but little or no PAR1. Treatment of cells with 2fLIGRLO PAR2 agonist, but not TFLLRN PAR1 agonist, increased MUC2 gene expression. Incubation of cells with Giardia trophozoites increased MUC2 mRNA production, and this increase was abolished by pre‐treatment of cells with a PAR2 antagonist. Giardia also caused decreased PAR2 expression in LS174T. Giardia NF and Giardia GSM are both able to cleave PAR1 and PAR2 at the N terminal domain. Cleavage by NF was more efficient than GSM, and PAR1 was cleaved more extensively than PAR2 by both strains. Giardia infection increased Muc2 and Muc5ac expression in the colon and increased Muc5ac expression in the jejunum of wild‐type mice compared to uninfected controls. In PAR2−/− mice, Giardia infection reduced expression of Muc5ac in the jejunum, and had no impact on either gene in the colon. Giardia infection caused thinning of the mucus layer in wild‐type mice. In contrast, infection induced thickening of the mucus layer in PAR2−/− mice.ConclusionsUsing a model of Giardia infection, the findings demonstrate that PAR2 plays a significant role in mucin gene regulation and function in mice and in a human colonic goblet cell line, and that Giardia is capable of cleaving both PAR1 and PAR2 receptors with strain‐specific efficiencies.Support or Funding InformationCrohn's Colitis Canada (CCC), NSERCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RT-PCR of peritoneal washings predicts peritoneal pancreatic cancer recurrence

BackgroundPeritoneal recurrence of pancreatic cancer is a frequent and lethal outcome after R0 resection. A method to predict peritoneal recurrence could be helpful in its prevention. Materials and methodsPeritoneal washings were prospectively obtained from 29 patients in whom R0 resection was performed. Cytological examination (CY) and real-time reverse transcription polymerase chain reaction (RT-PCR) of the peritoneal washing for the detection of cancer-related genes, CEACAM5, KRT7, KRAS, and MUC1, were performed. Clinicopathological characteristics and real-time RT-PCR results of the peritoneal washing were compared between patients whose pancreatic cancer recurred peritoneally (n = 7) and those patients who it did not recur (n = 22). ResultsOnly one CY-positive (CY+) case was detected, and that patient recurred. MUC1 mRNA expression was significantly higher in the recurrence group (P = 0.015). Cumulative incidence-function analysis demonstrated that peritoneal recurrence rate was significantly higher in MUC1-positive (MUC1+) patients (P = 0.044). MUC1+ patients had significantly decreased disease-free survival (P = 0.009) and disease-specific survival (P = 0.031). MUC1 protein was detected in the primary tumor in 18 of 29 patients. However, no significant difference was observed in the expression of MUC1 protein in peritoneal washings from the primary tumor (P = 0.579). ConclusionsHigh expression of MUC1 mRNA in peritoneal washings is a significant risk factor for peritoneal recurrence of pancreatic cancer after R0 resection along with poor disease-specific survival. RT-PCR of MUC1 mRNA in peritoneal washing may be useful for individualization of adjuvant chemotherapy.

MUC16overexpression induced by gene mutations promotes lung cancer cell growth and invasion

Air pollution is one of the leading causes of lung cancer. Air pollution-related lung cancer is a deteriorating public health problem, particularly in developing countries. The MUC16 gene is one of the most frequently mutated genes in air pollution-related lung cancer. In the present study, MUC16 mRNA expression was increased in ∼50% of air pollution-related lung cancer samples obtained from patients residing in air-polluted regions (Xuanwei and Fuyuan, Yunnan, China), and MUC16 mRNA levels were correlated with the degree of air pollution. Furthermore, sequencing of the captured MUC16 gene identified 561 mutation sites within the MUC16 gene in the air pollution-related lung cancer tissues. Interestingly, some mutations at specific sites and one region were associated with MUC16 mRNA up-regulation. Therefore, we further investigated the impacts of gene mutation on MUC16 expressions and cell behaviors in cultured cells by inducing certain mutations within the MUC16 gene using CRISPER/Cas9 genome editing technology. Certain mutations within the MUC16 gene induced MUC16 overexpression at both the mRNA and the protein level in the cultured cells. Additionally, MUC16 overexpression induced by gene mutations had functional effects on the behavior of lung cancer cells, including increasing their resistance to cisplatin, promoting their growth, and enhancing their migration and invasion capabilities. Based on the data, we suggest that MUC16 mutations potentially associated with air pollution may participate in the development and progression of air pollution-related lung cancer. In addition to ovarian cancer, MUC16 may be a candidate biomarker for lung cancer.