MUC1 mRNA Expression Research Articles

Dietary flaxseed modulates the colonic microenvironment in healthy C57Bl/6 male mice which may alter susceptibility to gut-associated diseases

Understanding how dietary components alter the healthy baseline colonic microenvironment is important in determining their roles in influencing gut health and gut-associated diseases. Dietary flaxseed (FS) has demonstrated anti-colon cancer effects in numerous rodent models, however, exacerbated acute colonic mucosal injury and inflammation in a colitis model. This study investigates whether FS alters critical aspects of gut health in healthy unchallenged mice, which may help explain some of the divergent effects observed following different gut-associated disease challenges. Four-week-old C57Bl/6 male mice were fed an AIN-93G basal diet (BD) or an isocaloric BD+10% ground FS diet for 3 weeks. FS enhanced colon goblet cell density, mucus production, MUC2 mRNA expression, and cecal short chain fatty acid levels, indicative of beneficial intestinal barrier integrity responses. Additionally, FS enhanced colonic regenerating islet-derived protein 3 gamma (RegIIIγ) and reduced MUC1 and resistin-like molecule beta (RELMβ) mRNA expression which may indicate altered responses in regulating microbial defense and injury repair responses. FS diet altered the fecal microbial community structure (16S rRNA gene profiling), including a 20-fold increase in Prevotella spp. and a 30-fold reduction in Akkermansia muciniphila abundance. A 10-fold reduction in A. muciniphila abundance by FS was also demonstrated in the colon tissue-associated microbiota (quantitative PCR). Furthermore, fecal branched chain fatty acids were increased by FS, indicative of increased microbial-derived putrefactive compounds. In conclusion, consumption of a FS-supplemented diet alters the baseline colonic microenvironment of healthy mice which may modify subsequent mucosal microbial defense and injury-repair responses leading to altered susceptibility to different gut-associated diseases.

Therapeutic efficacy of fibroblast growth factor 10 in a rabbit model of dry eye.

The aim of the present study was to investigate the therapeutic efficacy of fibroblast growth factor 10 (FGF10) in the promotion of healing, survival and expression of mucin in corneal epithelial cells in a rabbit dry eye model. A total of 12 healthy female New Zealand white rabbits were divided randomly into three groups. The lacrimal glands were injected with saline either alone (normal control group) or with concanavalin A (Con A), with either topical phosphate-buffered saline (PBS; PBS control group) or 25 µg/ml FGF10 (FGF10 treatment group). Lacrimal gland inflammation, tear function, corneal epithelial cell integrity, cell apoptosis and mucin expression were subsequently assessed. Lacrimal gland tissue biopsies were performed in conjunction with histology and electron microscopy observations. Tear meniscus height (TMH) and tear meniscus area (TMA) were measured using Fourier domain-optical coherence tomography. Tear membrane break-up time (TBUT) was also assessed and corneal fluorescein staining was performed. The percentages of apoptotic corneal and conjunctival (Cj) epithelial cells (ECs) were counted using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. The mRNA expression levels of Muc1 were determined using reverse transcription-quantitative polymerase chain reaction analyses. The TMH and TMA values of the PBS and treatment groups were found to be significantly reduced, compared with those of the normal control group 3 days after Con A injection. However, the TMH and TMA of the FGF10 treatment group were higher, compared with those of the PBS group 3 and 7 days after treatment, respectively. Furthermore, the FGF10 treatment group exhibited prolonged TBUT, reduced corneal fluorescein staining and repaired epithelial cell ultra-structure7 days after treatment. The percentages of apoptotic corneal- and Cj-ECs in the FGF10 treatment group were significantly reduced, compared with those in the PBS group. FGF10 significantly induced the mRNA expression of Muc1 in the corneal epithelial cells, compared with the normal control group, and induced higher mRNA expression levels of Muc1 in the Cj-ECs, compared with the PBS control group. In the present study, the rabbit dry eye model was successfully established 3 days after lacrimal gland Con A injection. FGF10 eye drops increased TMH and TMA, promoted corneal epithelial healing, reduced apoptosis of the corneal- and Cj-ECs and led to increased expression of Muc1.

Cytokine stimulation of MUC4 expression in human female reproductive tissue carcinoma cell lines and endometrial cancer.

MUC4, a transmembrane glycoprotein, interferes with cell adhesion, and promotes EGFR signaling in cancer. Studies in rat models have demonstrated steroid hormonal regulation of endometrial MUC4 expression. In this study, qRT-PCR screening of mouse tissues determined that Muc4 mRNA also was robustly expressed in mouse uteri. Previous studies from our labs have demonstrated MUC4 mRNA was expressed at levels <1% of MUC1 mRNA in human endometrium and endometriotic tissue. Multiple human endometrial adenocarcinoma cell lines were assayed for MUC4 mRNA expression revealing extremely low basal expression in the Ishikawa, RL-95-2, AN3CA, and KLE lines. Moderate to high expression was observed in HEC50 and HEC-1A cells. MUC4 mRNA expression was not affected by progesterone and/or estrogen treatment, but was greatly stimulated at both mRNA and protein levels by proinflammatory cytokines (IFN-γ and TNF-α), particularly when used in combination. In endometrial tissue, MUC4 mRNA levels did not change significantly between normal or cancerous samples; although, a subset of patients with grade 1 and 2 tumors displayed substantially higher expression. Likewise, immunostaining of human endometrial adenocarcinoma tissues revealed little to no staining in many patients (low MUC4), but strong staining in some patients (high MUC4) independent of cancer grade. In cases where staining was observed, it was heterogeneous with some cells displaying robust MUC4 expression and others displaying little or no staining. Collectively, these observations demonstrate that while MUC4 is highly expressed in the mouse uterus, it is not a major mucin in normal human endometrium. Rather, MUC4 is a potential marker of endometrial adenocarcinoma in a subset of patients.

Chronic cigarette smoke exposure induces microbial and inflammatory shifts and mucin changes in the murine gut.

Inflammatory bowel diseases (IBD) are complex multifactorial diseases characterized by an inappropriate host response to an altered commensal microbiome and dysfunctional mucus barrier. Cigarette smoking is the best known environmental risk factor in IBD. Here, we studied the influence of chronic smoke exposure on the gut microbiome, mucus layer composition and immune factors in conventional mice. We compared smoke-exposed with air-exposed mice (n = 12) after a smoke exposure of 24 weeks. Both Illumina sequencing (n = 6) and denaturing gradient gel electrophoresis (n = 12) showed that bacterial activity and community structure were significantly altered in the colon due to smoke exposure. Interestingly, an increase of Lachnospiraceae sp. activity in the colon was observed. Also, the mRNA expression of Muc2 and Muc3 increased in the ileum, whereas Muc4 increased in the distal colon of smoke-exposed mice (n = 6). Furthermore, we observed increased Cxcl2 and decreased Ifn-γ in the ileum, and increased Il-6 and decreased Tgf-β in the proximal colon. Tight junction gene expression remained unchanged. We infer that the modulating role of chronic smoke exposure as a latently present risk factor in the gut may be driven by the altered epithelial mucus profiles and changes in microbiome composition and immune factors.

Effect of β-glucan on MUC4 and MUC5B expression in human airway epithelial cells.

β-Glucan is found in the cell walls of fungi, bacteria, and some plant tissues, and is detected by the innate immune system. Furthermore, this recognition is known to worsen respiratory symptoms in patients with allergic and inflammatory airway diseases. However, the means by which β-glucan affects the secretion of major mucins by human airway epithelial cells has not been elucidated. Therefore, in this study, the effect and signaling pathway of β-glucan on mucins MUC4 and MUC5B were investigated in human airway epithelial cells. In NCI-H292 cells and human normal nasal epithelial cells, the effect and signaling pathway of β-glucan on MUC4 and MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA). β-Glucan increased MUC4 and MUC5B expression and activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). SB203580 (a p38 MAPK inhibitor) and pyrrolidine dithiocarbamate (PDTC; a NF-κB inhibitor) inhibited β-glucan-induced MUC4 and MUC5B expression. In addition, siRNA knockdown of p38 MAPK blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of NF-κB. Furthermore, Toll-like receptor 4 (TLR4) mRNA expression was increased by β-glucan, and siRNA knockdown of TLR4 blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of p38 MAPK and NF-κB. These results demonstrate that in human airway epithelial cells β-glucan induces MUC4 and MUC5B expression via the TLR4-p38 MAPK-NF-κB signaling pathway.

MUC1 Promoter-Driven DTA as a Targeted Therapeutic Strategy against Pancreatic Cancer.

Mucin1 (MUC1) is overexpressed in pancreatic ductal adenocarcinoma (PDA) and is associated with tumor aggressiveness, suggesting that MUC1 is a promising therapeutic target for promoter-driven diphtheria toxin A (DTA). Endogenous MUC1 transcript levels were analyzed by quantitative PCR (qPCR) in multiple PDA cells (Capan1, HPAFII, Su.86.86, Capan2, Hs766T, MiaPaCa2, and Panc1). Expression levels were correlated with luciferase activity and cell death after transfection with MUC1 promoter-driven luciferase and DTA constructs. MUC1-positive (+) cells had significantly elevated MUC1 mRNA expression compared with MUC1-negative (-) cells. Luciferase activity was significantly higher in MUC1(+) cells when transfected with MUC1 promoter-driven luciferase and MUC1(+) cells underwent enhanced cell death after transfection with a single dose of MUC1 promoter-driven DTA. IFNγ pretreatment enhanced MUC1 expression in MUC1(-) cells and induced sensitivity to MUC1-DTA therapy. Matched primary and metastatic tumor lesions from clinical specimens revealed similar MUC1 IHC labeling patterns, and a tissue microarray of human PDA biopsies revealed increased immunolabeling with a combination of MUC1 and mesothelin (MSLN) antibodies, compared with either antibody alone. Combining MUC1 with MSLN-targeted DTA enhanced drug efficacy in an in vitro model of heterogeneous PDA. These data demonstrate that MUC1 promoter-driven DTA preferentially kills MUC1-expressing PDA cells and drugs that enhance MUC1 expression sensitize PDA cells with low MUC1 expression. MUC1 expression in primary and metastatic lesions provides a rationale for the development of a systemic MUC1 promoter-driven DTA therapy that may be further enhanced by combination with other promoter-driven DTA constructs.

Bile acid induces MUC2 expression and inhibits tumor invasion in gastric carcinomas.

Bile acids might induce mucin expression and regulate tumor behavior in esophageal and colon cancers. However, little is known about the effect of bile acids on tumor invasiveness of gastric carcinoma (GC). The aim of the current study was to elucidate the mechanisms by which bile acids regulate tumor invasion in GC. We investigated bile acid-induced MUC2 expression and cell invasion and migration in the cultured GC cell lines, SNU-216, and MKN45. In addition, immunohistochemical analysis of MUC2 and Snail was performed on 389 archival paraffin-embedded tissues of GC to evaluate the correlation of their expression with prognosis. Deoxycholic acid (DCA), a secondary bile acid, had no effect on the viability of SNU-216 and MKN45 GC cells at low concentrations (0-100 μM), but decreased viability at a higher concentration (200 μM). MKN45 cells showed higher MUC2 expression than SNU-216 cells under basal conditions. DCA treatment upregulated MUC2 mRNA expression in both SNU-216 and MKN45 cells. Expression of Snail and MMP9 was markedly decreased by DCA treatment, and E-cadherin expression was subsequently increased. DCA significantly inhibited invasion and migration of SNU-216 and MKN45 cells. In human GC, MUC2 expression showed a negative correlation with Snail expression (P = 0.021) and a significantly positive correlation with better prognosis (P = 0.023). Taken together, our data suggest that DCA induced MUC2 expression in GC cells and inhibited tumor invasion and migration. Additionally, MUC2-expressing GCs showed low rates of Snail expression and were associated with a favorable prognosis.

Expressions of MUC1 and vascular endothelial growth factor mRNA in blood are biomarkers for predicting efficacy of gefitinib treatment in non-small cell lung cancer.

BackgroundGefitinib, an EGFR-tyrosine kinase inhibitor, significantly improve prognosis in patients with advanced non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the usefulness of MUC1 and vascular endothelial growth factor (VEGF) mRNA expression in peripheral blood as means of predicting benefit from gefitinib therapy in NSCLC patients.MethodsMUC1 and VEGF mRNA expressions were detected in peripheral blood of 66 patients with advanced NSCLC before (B0) and 4 weeks after treatment (B4w) with gefitinib, using real-time quantitative-PCR assay. Correlations between blood MUC1 and VEGF mRNA expression at B0 and B4w and the response to gefitinib treatment and survival were analyzed.ResultsBlood levels of MUC1 and VEGF mRNA at B0 and at B4w were significantly higher in patients with progressive disease than in those with partial response and stable disease. Furthermore, blood MUC1 and VEGF mRNA positivity at two time points were strongly associated with shorter progression-free survival (PFS) and overall survival (OS) (P = 0.005 and P = 0.008 at B0, and P < 0.001 and P = 0.001 at B4w, respectively, for MUC1; P = 0.004 and P = 0.009 at B0, and P = 0.001 and P < 0.001 at B4w, respectively, for VEGF). Multivariate analyses demonstrated that blood MUC1 and VEGF mRNA positivity at B0 and B4w were independent factors for predicting worse PFS and OS.ConclusionsMUC1 and VEGF mRNA positivity in blood seem to be indicators of unfavorable response and poor PFS and OS in patients with advanced NSCLC treated with gefitinib and may be promising noninvasive and repeatable markers for predicting efficacy of gefitinib treatment.

Sex-determined susceptibility and differential MUC2 mRNA expression during the course of murine intestinal eimeriosis.

Parasitic diseases differ in prevalence, course, and severity between males and females. The study was designed to compare males with females for the susceptibility to Eimeria papillata infection as well as the expression of the mucin gene, MUC2. Oocysts output was detected to be more in the feces of male mice (3.5 × 10(4) ± 4000 oocysts/g feces) than in females (2 × 10(4) ± 2000 oocysts/g feces). In addition, infected males showed a significant higher number of meronts, gamonts, and developing oocysts compared to infected female mice. Moreover, E. papillata induced a marked goblet cell hypoplasia where the jejuna of infected male mice contained lower numbers of goblet cells per ten villus-crypt units compared to infected females. Also, the expression of MUC2 mRNA is found to be more expressed in infected females than males. In addition, testosterone, nitric oxide, and inducible nitric oxide synthase activities were found to be higher in infected male mice than in infected females. In general, male Swiss albino mice have been shown to be relatively more susceptible to infection with E. papilaata when compared with female mice.

Effects of monochromatic light stimuli on the development and Muc2 expression of goblet cells in broiler small intestines during embryogenesis

The effects of monochromatic light on the ontogeny, differentiation, and Muc2 expression level in goblet cells were studied in the small intestines of late-stage broiler embryos. The embryos were exposed to blue light (B group), green light (G group), red light (R group), or darkness (D group) throughout the incubation period. On d 15 of incubation (E15), a few acidic goblet cells (only the sulfated subtype) were observed, and Muc2 mRNA expression was detected. On E18, however, neutral, acidic, and intermediate types, as well as the sulfated subtype, were observed in the small intestine, and a decreasing gradient of goblet cell density was found along the duodenum to ileum axis. Up to E21, 3 types of goblet cells and 3 acidic cell subtypes were found in all the small intestines. The goblet cell density increased along the duodenum to ileum axis. Monochromatic light stimulation resulted in no significant differences in the density and types of goblet cells between the different treatment groups on E15 and E18, but an increased Muc2 mRNA expression level was detected on E18 in the G group compared with the other treatment groups. On E21, the goblet cell density, proportion of acidic goblet cells, and Muc2 mRNA expression level increased in the G group compared with other treatment groups. These results suggest that the ontogeny and differentiation of goblet cells in broiler embryos display temporal and spatial differences. Green monochromatic light may have the potential to promote the proliferation and maturation of as well as the expression of Muc2 mRNA in goblet cells of broiler embryos.

MUC1 expression in Fallopian tubes of women with hydrosalpinx

ObjectiveTo analyze the expression of MUC1 in Fallopian tubes with or without hydrosalpinx, using four different types of antibody. Study designIn a case-control study, immunohistochemical expression of MUC1 was examined in Fallopian tubes derived from women with hydrosalpinx (n=10) and normal controls (n=10). Four different antibodies were used for the detection of both extracellular (214D4, HMFG1, VPM654) and intracellular (EPR1023) MUC1 epitopes. Staining intensity was measured with ImageJ software. Expression of MUC1 mRNA was quantified by quantitative RT-PCR. Statistical analysis was performed with Student t-test (mean±SD) and Mann–Whitney U-test (median [range]). ResultsThe mean (±SD) and median [range] intensity of MUC1 in controls vs. hydrosalpinx were: 214D4-67.5±11.3 vs. 74.8±14.69 (P=0.22); HMFG1-95.3 [642–1079] vs. 97.0 [502–1550] (P=0.91); VPM654-41.1 [314–914] vs. 46.0 [390–1424] (P=0.1); EPR1023-24.7±7.3 vs. 57.4±31.3 (P=0.01). MUC1 mRNA was 0.16 [008–05] vs. 0.09 [005–019] (P=0.06). Ectodomains and mRNA of MUC1 are unchanged in tubes from hydrosalpinx patients. In contrast, immunodetection of the MUC1 cytoplasmic tail is enhanced in tubes from hydrosalpinx. ConclusionFallopian tubes with hydrosalpinx have a selective accumulation of MUC1 cytoplasmic tail, but not difference in the ectodomain.

Tu1228 Effect of Phosphatidylcholine (PC)-Aspirin on Small Bowel Mucosal Injury

Background: Aspirin can induce small bowel injury. There is no proven treatment of aspirin enteropathy other than withdrawal. Recently, a novel aspirin associated with phosphatidylcholine(PC) is designed to be gastric protective due to hydrophobic property of PC. Lysophosphatidic acid (LPA), a metabolite from PC and mucin were found to play roles in integrity of gastrointestinal tract epithelium. This study hypothesized that PC-aspirin would reduce aspirin-induced small bowel injury. Methods: Sprague-Dawley rats were intra-duodenally administrated with either PC-aspirin (100mg, n=12-14), Plain aspirin (100mg, n=12-14) or vehicle (1ml 2%methtcellylulose, n=12), and euthanized after 90 minutes. Small bowel injury was assessed by lesion area, vascular permeability (i.v. 1% Evan's blue solution), intestinal hemoglobin concentration and histological change (H&E stained slides were scored with a scoring system). Apoptotic index was estimated with western blot and TUNEL staining (apoptotic cell in 5 villi would be counted). The expressions of lysophosphatidic acid receptor 2 (LPA2), MUC2 the mainly expressed secretory mucin in intestine, and TLR4, MyD88, NF-κΒp50 were test with Western blot or real-time PCR. Mucins were stained with Periodic Acid Schiff (PAS). Prostaglandin E2 (PGE2) concentration were detect with ELISA. Results: Comparing to plain aspirin-treated rats, PC-aspirin caused fewer injuries in the small bowel as assessed in: lesion area (4.82±1.61mm2 vs 196.00±84.89mm2 P=0.003), vascular permeability (42.81±7.69ug/ml vs 74.73±18.61ug/ml P=0.003), intestinal hemoglobin concentration (66.67±16.81ng/ml vs 91.83±14.80ng/ml P=0.020) and histological change (2.12±0.56 vs 3.06±1.07 P=0.044)(Figure 1). Also a decreased apoptotic index was found in PC-aspirin group (16.43±4.28 vs 38.71±5.59 P<0.001 for TUNEL, 0.49±0.14 vs 1.06±0.33 P=0.007 for cleaved caspase-3, 0.81±0.29 vs 1.40±0.56 P=0.043 for cleaved caspase-7, Figure 1). Immunohistochemical staining and western blot (0.21±0.06 vs 0.14±0.06 P=0.044 for PCaspirin vs Plain aspirin) showed that LPA2-positive cells were strongly expressed in surfacelining of small bowel mucosa in the control and PC-aspirin groups but not in the plain aspirin group (Figure 2). Moreover, PAS staining reveals a thicker mucin layer in PC-aspirin rats, with a higher MUC2 mRNA level than plain aspirin rats (1.70±0.75 vs 0.98±0.45 P= 0.039)(Figure 2). The tissue expressions of TLR4, MyD88, NF-κΒp50 and COX-2 were increased while PGE2was decreased in both plain aspirin and PC-aspirin groups. Conclusion: PC-aspirin significantly decreased aspirin induced small bowel injury, this effect may associated with (1) increased LPA2 expression, which protect mucosa from apoptotic damages, and (2) increased MUC2 mRNA expression and decreased mucin layer consumption caused by aspirin, which help to suppress bacterial invasion.

Mucin 1 and cytokines mRNA in endometrium of dairy cows with postpartum uterine disease or repeat breeding

Mucin (MUC) 1 is an inducible innate immune effector, an important component of defense against bacterial invasion, and is linked with infertility in humans. The objectives were to evaluate messenger RNA (mRNA) expression of MUC1 and cytokine genes in the endometrium of cows with various postpartum uterine inflammatory conditions or with a history of repeat breeding. Endometrial samples were collected from lactating dairy cows diagnosed with metritis (n = 4), endometritis (n = 4), subclinical endometritis (n = 4), or no uterine pathology (normal; n = 4). In addition, endometrial samples were collected from repeat breeder cows with (n = 4) or without (n = 4) subclinical endometritis, and unaffected cows (n = 4). Quantitative polymerase chain reaction was used to determine mRNA abundances of MUC1, Toll-like receptor (TLR) 4, interleukin (IL) 1β, IL6, IL8, tumor necrosis factor (TNF) α, insulin-like growth factor (IGF) 1, and IGF-binding protein (BP) 2. The mRNA expressions were significantly greater for cows with metritis and clinical endometritis compared with cows with no uterine inflammation, except for IL6. However, mRNA expressions for these target genes were not different for cows with subclinical endometritis, compared with cows without uterine inflammation, except for IL1β and TNFα mRNA (P < 0.01). All mRNA expressions were greater (P < 0.001) for repeat breeder cows with subclinical endometritis compared with normal cows. However, in repeat breeder cows without subclinical endometritis, only expressions of MUC1, IGF1, and IGF BP2 were greater compared with normal cows (P < 0.01). Based on functional protein networks, there were significant associations between these transcripts. In conclusion, endometrial expressions of MUC1 and cytokine genes differed among normal, fertile versus diseased, and subfertile dairy cows. Perhaps, these altered gene expressions contribute to endometrial insufficiency and consequently pregnancy wastage.

Abstract P5-03-14: Low dose ionising radiation effects in normal breast progenitor sub-populations

Abstract The human breast is a radiosensitive tissue and radiation exposure is proposed to contribute to mammary gland carcinogenesis through changes to stem and progenitor cells that result in aberrant proliferation, differentiation and genome instability. However, little is known about the low dose radiation response (LD IR) under conditions of relevance to environmental and occupational and medical imaging exposures. This study utilises 2-D and 3-D breast models derived from two normal breast cell lines (MCF10A and HME1) and primary human mammary epithelial cells from reduction mammoplasty surgery, to investigate the mechanisms underpinning sensitivity of normal breast to LD IR and elucidate its role in breast cancer initiation and progression. We have shown that DNA damage can be detected in both cell lines after doses &amp;lt;50 mGy and that an increased level of DNA damage foci at 1 h post LD IR is observed in a subpopulation of cells. Using antibodies to cell surface markers suggested to be putative breast stem cell markers, we have performed Fluoresence Activated Cell Sorting (FACS) to isolate subpopulations from the MCF10A cell line. CD49fhi, EpCAM-, MUC1- cells thought to be progenitor like cells were found to be more resistant to LD IR than the EpCAM+, MUC1+ population. As the pathways that govern survival/proliferation of stem and progenitor cells versus differentiation are interconnected, we have investigated if radiation resistance is also correlated with changes to breast cell differentiation. It was observed that LD IR modulated breast cell lineage markers in 2D cultures of both cell lines. We subsequently found that radiation induces a decrease in MUC1 mRNA expression levels which coincides with increased Lef1 expression. Lef1 has been shown to promote Mre11 expression thereby conferring elevated DNA repair and radiation resistance in tumour cells. Based on our findings, we hypothesize that progenitor cell populations within normal breast cell lines may be more efficient at detecting DNA DSBs resulting from LD IR leading to a pro-survival response. We are currently investigating these effects in primary mammary epithelial cell samples, using FACS to isolate progenitor subpopulations. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-14.

Effects of Fungi and Eosinophils on Mucin Gene Expression in Rhinovirus-Infected Nasal Epithelial Cells

PurposeFungi, rhinoviruses (RVs), and eosinophils are associated with upper respiratory diseases. We evaluated the effects of fungal stimulation and eosinophil co-culture on the expression of mucin genes in RV-infected nasal polyp epithelial cells.MethodsNasal polyp epithelial cells were obtained from chronic rhinosinusitis patients. Cultured epithelial cells were stimulated with Alternaria and Aspergillus with or without RV-16 infection. The epithelial cells were co-cultured with eosinophils for 16 h. MUC4, MUC5AC, MUC5B, and MUC8 mRNA expressions in the epithelial cells were quantified using real-time RT-PCR. To determine the underlying mechanism, nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and mitogen-activated protein kinase (MAPK) inhibitors were used to inhibit mucin gene expression.ResultsFungi and RV-16 induced mucin gene expression in nasal polyp epithelial cells. However, there was no synergistic increase in mucin gene expression, with the exception of MUC4 mRNA expression stimulated by 25 µg/mL Aspergillus. When RV-16-infected epithelial cells were stimulated with fungi and then co-cultured with eosinophils, MUC4, MUC5B, and MUC8 mRNA expressions increased. Mucin gene expression was inhibited by NF-κB inhibitors.ConclusionsRV-16, airborne fungi, and eosinophils may exacerbate the inflammatory process in nasal mucosal diseases by enhancing mucin gene expression.

Tanshinone IIA inhibits lipopolysaccharide-induced MUC1 overexpression in alveolar epithelial cells

The anti-inflammatory function of tanshinone IIA (TIIA), an active natural compound from Chinese herbal medicine Danshen, has been well recognized, and therefore TIIA has been widely used to treat various inflammatory conditions associated with cardiac and lung diseases. Mucin 1 (Muc1) plays important anti-inflammatory roles in resolution of acute lung inflammation. In this study, we investigated the effects of TIIA on LPS-induced acute lung inflammation, as well as its relationship to Muc1 expression in mouse lung and MUC1 in human alveolar epithelial cells. TIIA pretreatment significantly inhibited LPS-induced pulmonary inflammation in both Muc1 wild-type (Muc1(+/+)) and knockout (Muc1(-/-)) mice, as manifested by reduced neutrophil infiltration and reduced TNF-α and keratinocyte chemoattractant levels in bronchoalveolar lavage fluid. The inhibitory effects of TIIA on airway inflammation were associated with reduced expression of Muc1 in Muc1(+/+) mouse lung. Moreover, pretreatment with TIIA significantly inhibited LPS-induced MUC1 expression and TNF-α release in A549 alveolar epithelial cells. TNF-α upregulated MUC1 mRNA and protein expression in A549 cells, which was inhibited by pretreatment with TIIA. The LPS-induced MUC1 expression was blocked when A549 cells were transfected with siRNA targeting for TNF-α receptor 1. Furthermore, TIIA inhibited LPS-induced nuclear translocation of NF-κB and upregulation of Toll-like receptor 4 in A549 cells. Taken together, these results demonstrate that TIIA suppressed LPS-induced acute lung inflammation regardless of the presence of Muc1, and TIIA inhibited LPS- and TNF-α-induced MUC1/Muc1 expression in airway epithelial cells, suggesting that MUC1/Muc1 does not account for the mechanisms of the anti-inflammatory effects of TIIA in the airway.

Evaluation of Mucin-1 protein and mRNA expression as prognostic and predictive markers after neoadjuvant chemotherapy for breast cancer

BackgroundMucin-1 (MUC1) is a promising antigen for the development of tumor vaccines. We evaluated the frequency of MUC1 expression and its impact on therapy response and survival after neoadjuvant chemotherapy for breast cancer. Patients and methodsPre-treatment core biopsies of patients from the GeparTrio neoadjuvant trial (NCT 00544765) were evaluated for MUC1 by immunohistochemistry (IHC; N = 691) and quantitative RT-PCR (qRT-PCR; N = 286) from formalin-fixed paraffin-embedded (FFPE) samples. ResultsMUC1 protein and mRNA was detectable in the majority of cases and was associated with hormone-receptor-positive status (P < 0.001). High MUC1 protein and mRNA expression were associated with lower probability of pathologic complete response (P = 0.017 and P < 0.001) and with longer patient survival (P = 0.03 and P < 0.001). In multivariable analysis, MUC1 protein and mRNA expression were independently predictive (P = 0.001 and P < 0.001). MUC1 protein and mRNA expression were independently prognostic for overall survival (P = 0.029 and P = 0.015). ConclusionsMUC1 is frequently expressed in breast cancer and detectable on mRNA and protein level from FFPE tissue. It provides independent predictive information for therapy response and survival after neoadjuvant chemotherapy. In clinical immunotherapy trials, MUC1 expression may serve as a predictive marker.

PGE2 induces MUC2 and MUC5AC expression in human intrahepatic biliary epithelial cells via EP4/p38MAPK activation

Background. MUC2 and MUC5AC overproduction is considered to be associated with hepatolithiasis and related to inflammation. However, mechanisms underlying MUC upregulation under inflammatory stimulation in human intrahepatic biliary epithelial cells (HIBECs) are not completely understood.Material and Methods. Expression of MUC2 and MUC5AC mRNA in HIBECs was detected by real-time PCR. Expression of COX-2, EP4, and phosphorylated ERK, JNK and p38MAPK protein was detected by Western blot. Concentrations of PGE2, IL-1ß and TNF-α in cell culture supernatants were measured using the Quantikine Elisa kit.Results. COX-2 expression as well as PGE2 production in HIBECs was upregulated significantly by LPS, which was completely blocked by either TLR4 antagonist or NFkB inhibitor. Selective COX-2 inhibitor suppressed LPS-induced MUC2 and MUC5AC mRNA expression remarkably. Exogenous PGE2 increased MUC2 and MUC5AC mRNA expression in a dosage-dependent manner independent of IL-1ß and TNF-α. PGE2 receptor EP4 agonist elevated MUC2 and MUC5AC expression, whereas EP4 antagonist had the opposite effect. Expression of phosphorylated p38MAPK was upregulated by exogenous PGE2, and p38MAPK inhibitor reduced MUC2 and MUC5AC expression in HIBECs. In addition, it was found that levels of PGE2, MUC2 and MUC5AC in bile samples from the hepatic ducts affected by intrahepatic stones were significantly higher than those from the unaffected hepatic ducts of patients with hepatolithiasis.Conclusions. Our findings indicate that PGE2 induces MUC2 and MUC5AC expression in HIBECs via EP4-p38MAPK signaling.

The silence of MUC2 mRNA induced by promoter hypermethylation associated with HBV in Hepatocellular Carcinoma

BackgroundTo evaluate the promoter methylation status of MUC2 gene and mRNA expression in patients with hepatocellular carcinoma.MethodsWe analyzed MUC2 methylation by MSP, and MUC2 mRNA by real-time PCR in 74 HCC.ResultsMUC2 mRNA were lower in HCC tissues (Mean -ΔCt = −4.70) than that in Non-HCC tissues (Mean -ΔCt = −2.98). Expression of MUC2 was elevated in only 23 (31.08%) of the 74 HCC patients. MUC2 promoter was hypermethylated in 62.2% (46/74) of HCCs, and in only 18.9% (14/74) of non-tumor samples. MUC2 mRNA were lower in HCC patients with hypermethylation (Mean -ΔΔCt = −2.25) than those with demethylation (Mean -ΔΔCt = −0.22), and there is a decreased tendency for MUC2 mRNA in HCC patients with promoter hypermethylation (p = 0.011). There was a significantly correlation found between MUC2 mRNA and HBV and AFP in HCC. The loss of MUC2 mRNA and hypermethylation could be poor prognostic factors. After treated by 5-Aza-CdR and TSA, we found that MUC2 mRNA induced significantly in 7721, Huh7 and HepG2 cells.ConclusionThe results suggested that MUC2 mRNA silenced by promoter hypermethylation is associated with high levels HBV in HCC.

Mucins 1‐shRNA inhibit the proliferation and HIF‐1alpha protein expression on human cholangiocarcinoma cells

To explore the effects of Mucins (MUC)1-shRNA on the proliferation and hypoxia inducible factor (HIF)-1alpha expression of human cholangiocarcinoma (CCA) QBC939 cells in vitro. MUC1-shRNA was constructed and transfected with Lipofectamine™ 2000 into cultured CCA cells. MUC1 mRNA and protein expression levels were determined by RT-PCR and Western blot, respectively. The cellular proliferation and HIF-1alpha expression of QBC939 cells were evaluated by the MTT assay and Western blot, respectively. After transfection, the expression levels of MUC1 mRNA and protein in the experimental group decreased significantly in QBC939 (P < 0.01). The proliferation of MUC1 shRNA-transfected group was 0.30 ± 0.05, 38.32 ± 1.43%, 15.18 ± 1.32%, and there were remarkable differences when compared with the control groups (P < 0.05). Significant inhibition of HIF-1alpha protein expression in MUC1 shRNA-transfected group was also discovered (P < 0.05). MUC1-shRNA could inhibit proliferation and significantly weaken HIF-1alpha protein expression of QBC939 cells, suggesting its potential as a therapeutic target of CCA.