TRPV4 activation in human corneal epithelial cells promotes membrane mucin production.

Given that the corneal epithelium is situated on the outermost part of the eye, its functions can be influenced by external temperatures and chemical substances. This study aimed to elucidate the expression profile of chemosensory receptors in corneal epithelial cells and analyze their role in eye function regulation. A comprehensive analysis of 425 chemosensory receptors in human corneal epithelial cells-transformed (HCE-T) revealed the functional expression of TRPV4. The activation of TRPV4 in HCE-T cells significantly increased the expression of membrane-associated mucins MUC1, MUC4, and MUC16, which are crucial for stabilizing tear films, with efficacy comparable to the active components of dry eye medications. The present study suggests that TRPV4, which is activated by body temperature, regulates mucin expression and proposes it as a novel target for dry eye treatment.

Circadian disruption reduces MUC4 expression via the clock molecule BMAL1 during dry eye development.

Circadian disruption, as a result of shiftwork, jet lag, and other lifestyle factors, is a common public health problem associated with a wide range of diseases, such as metabolic disorders, neurodegenerative diseases, and cancer. In the present study, we established a chronic jet lag model using a time shift method every 3 days and assessed the effects of circadian disruption on ocular surface homeostasis. Our results indicated that jet lag increased corneal epithelial defects, cell apoptosis, and proinflammatory cytokine expression. However, the volume of tear secretion and the number of conjunctival goblet cells did not significantly change after 30 days of jet lag. Moreover, further analysis of the pathogenic mechanism using RNA sequencing revealed that jet lag caused corneal transmembrane mucin deficiency, specifically MUC4 deficiency. The crucial role of MUC4 in pathogenic progression was demonstrated by the protection of corneal epithelial cells and the inhibition of inflammatory activation following MUC4 replenishment. Unexpectedly, genetic ablation of BMAL1 in mice caused MUC4 deficiency and dry eye disease. The underlying mechanism was revealed in cultured human corneal epithelial cells in vitro, where BMAL1 silencing reduced MUC4 expression, and BMAL1 overexpression increased MUC4 expression. Furthermore, melatonin, a circadian rhythm restorer, had a therapeutic effect on jet lag-induced dry eye by restoring the expression of BMAL1, which upregulated MUC4. Thus, we generated a novel dry eye mouse model induced by circadian disruption, elucidated the underlying mechanism, and identified a potential clinical treatment.

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds.

The aim of this study was to identify effective genetic markers for the Antigen Processing Associated Transporter 1 (TAP1), α (1,2) Fucosyltransferase 1 (FUT1), Natural Resistance Associated Macrophage Protein 1 (NRAMP1), Mucin 4 (MUC4) and Mucin 13 (MUC13) diarrhea-resistance genes in the local pig breeds, namely Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, to provide a reference for the characterization of local pig breed resources in Shanghai. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLR) and sequence sequencing were applied to analyze the polymorphisms of the above genes and to explore the effects on the immunity of Shanghai local pig breeds in conjunction with some immunity factors. The results showed that both TAP1 and MUC4 genes had antidiarrheal genotype GG in the five pig breeds, AG and GG genotypes of the FUT1 gene were detected in Pudong white pigs, AA antidiarrheal genes of the NRAMP1 gene were detected in Meishan pigs, the AB type of the NRAMP1 gene was detected in Pudong white pigs, and antidiarrheal genotype GG of the MUC13 gene was only detected in Shanghai white pigs. The MUC13 antidiarrhea genotype GG was only detected in Shanghai white pigs. The TAP1 gene was moderately polymorphic in Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, among which TAP1 in Shanghai white pigs and Shawutou pigs did not satisfy the Hardy-Weinberg equilibrium. The FUT1 gene of Pudong white pigs was in a state of low polymorphism. NRAMP1 of Meishan pigs and Pudong white pigs was in a state of moderate polymorphism, which did not satisfy the Hardy-Weinberg equilibrium. The MUC4 genes of Shanghai white pigs and Pudong white pigs were in a state of low polymorphism, and the MUC4 genes of Fengjing pigs and Shawutou pigs were in a state of moderate polymorphism, and the MUC4 genes of Fengjing pigs and Pudong white pigs did not satisfy the Hardy-Weinberg equilibrium. The MUC13 gene of Shanghai white pigs and Pudong white pigs was in a state of moderate polymorphism. Meishan pigs had higher levels of IL-2, IL-10, IgG and TNF-α, and Pudong white pigs had higher levels of IL-12 than the other pigs. The level of interleukin 12 (IL-12) was significantly higher in the AA genotype of the MUC13 gene of Shanghai white pigs than in the AG genotype. The indicator of tumor necrosis factor alpha (TNF-α) in the AA genotype of the TAP1 gene of Fengjing pigs was significantly higher than that of the GG and AG genotypes. The indicator of IL-12 in the AG genotype of the Shawutou pig TAP1 gene was significantly higher than that of the GG genotype. The level of TNF-α in the AA genotype of the NRAMP1 gene of Meishan pigs was markedly higher than that of the AB genotype. The IL-2 level of the AG type of the FUT1 gene was obviously higher than that of the GG type of Pudong white pigs, the IL-2 level of the AA type of the MUC4 gene was dramatically higher than that of the AG type, and the IgG level of the GG type of the MUC13 gene was apparently higher than that of the AG type. The results of this study are of great significance in guiding the antidiarrhea breeding and molecular selection of Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs and laying the foundation for future antidiarrhea breeding of various local pig breeds in Shanghai.

A novel fatty acid metabolism-related signature identifies MUC4 as a novel therapy target for esophageal squamous cell carcinoma.

Fatty acid metabolism has been identified as an emerging hallmark of cancer, which was closely associated with cancer prognosis. Whether fatty acid metabolism-related genes (FMGs) signature play a more crucial role in biological behavior of esophageal squamous cell carcinoma (ESCC) prognosis remains unknown. Thus, we aimed to identify a reliable FMGs signature for assisting treatment decisions and prognosis evaluation of ESCC. In the present study, we conducted consensus clustering analysis on 259 publicly available ESCC samples. The clinical information was downloaded from The Cancer Genome Atlas (TCGA, 80 ESCC samples) and Gene Expression Omnibus (GEO) database (GSE53625, 179 ESCC samples). A consensus clustering arithmetic was used to determine the FMGs molecular subtypes, and survival outcomes and immune features were evaluated among the different subtypes. Kaplan-Meier analysis and the receiver operating characteristic (ROC) was applied to evaluate the reliability of the risk model in training cohort, validation cohort and all cohorts. A nomogram to predict patients' 1-year, 3-year and 5-year survival rate was also studied. Finally, CCK-8 assay, wound healing assay, and transwell assay were implemented to evaluate the inherent mechanisms of FMGs for tumorigenesis in ESCC. Two subtypes were identified by consensus clustering, of which cluster 2 is preferentially associated with poor prognosis, lower immune cell infiltration. A fatty acid (FA) metabolism-related risk model containing eight genes (FZD10, TACSTD2, MUC4, PDLIM1, PRSS12, BAALC, DNAJA2 and ALOX12B) was established. High-risk group patients displayed worse survival, higher stromal, immune and ESTIMATE scores than in the low-risk group. Moreover, a nomogram revealed good predictive ability of clinical outcomes in ESCC patients. The results of qRT-PCR analysis revealed that the MUC4 and BAALC had high expression level, and FZD10, PDLIM1, TACSTD2, ALOX12B had low expression level in ESCC cells. In vitro, silencing MUC4 remarkably inhibited ESCC cell proliferation, invasion and migration. Our study fills the gap of FMGs signature in predicting the prognosis of ESCC patients. These findings revealed that cluster subtypes and risk model of FMGs had effects on survival prediction, and were expected to be the potential promising targets for ESCC.

Surface plasmon resonance microscopy identifies glycan heterogeneity in pancreatic cancer cells that influences mucin-4 binding interactions.

Membrane proteins are the main targets of therapeutic drugs and most of them are glycosylated. Glycans play pivotal roles in several biological processes, and glycosylation changes are a well-established hallmark of several types of cancer, including pancreatic cancer, that contribute to tumor growth. Mucin-4 (MUC-4) is a membrane glycoprotein which is associated with pancreatic cancer and metastasis, and it has been targeted as a promising vaccine candidate. In this study, Surface Plasmon Resonance Microscopy (SPRM) was implemented to study complex influences of the native N-glycan cellular environment on binding interactions to the MUC-4 receptor as this is currently the only commercially available label-free technique with high enough sensitivity and resolution to measure binding kinetics and heterogeneity on single cells. Such unique capability enables for a more accurate understanding of the "true" binding interactions on human cancer cells without disrupting the native environment of the target MUC-4 receptor. Removal of N-linked glycans in pancreatic cancer cells using PNGase F exposed heterogeneity in Concanavalin (Con A) binding by revealing three new binding populations with higher affinities than the glycosylated control cells. Anti-MUC-4 binding interactions of enzymatically N-linked deglycosylated pancreatic cancer cells produced a 25x faster association and 37x higher affinity relative to the glycosylated control cells. Lastly, four interaction modes were observed for Helix Pomatia Agglutinin (HPA) binding to the glycosylated control cells, but shifted and increased in activity upon removal of N-linked glycans. These results identified predominant interaction modes of glycan and MUC-4 in pancreatic cancer cells, the kinetics of their binding interactions were quantified, and the influence of N-linked glycans in MUC-4 binding interactions was revealed.

Clinical Significance of MUC4 and Associated Proteins in Pancreatic and Periampullary Cancers.

OBJECTIVE: This study primarily aimed to assess the expression of MUC4 in patients with pancreatic ductal adenocarcinoma (PDAC) as compared with controls and assess its clinical relevance. MATERIALS AND METHODS: Serum MUC4 levels and MUC4 gene expression in snap-frozen tissue were analyzed through surface plasmon resonance and quantitative polymerase chain reaction, respectively. Tumor tissues and control tissues were analyzed for MUC4 and other mucins through immunohistochemistry. RESULT: MUC4 expression in tumor tissue was found to be significantly elevated in PDAC patients as compared with chronic pancreatitis tissues and normal pancreatic tissues. Periampullary carcinoma and cholangiocarcinoma tissue also showed increased expression of MUC4 and other mucins. CONCLUSIONS: Differential expression of MUC4 in pancreatic tumor tissues can help to differentiate PDAC from benign conditions.

Mutational profile evaluates metastatic capacity of Chinese colorectal cancer patients, revealed by whole-exome sequencing.

Colorectal cancer (CRC) is the third most common cancer and the prevalence rate of CRC is increasing in the China. In this study, whole-exome sequencing (WES) was performed on primary tissues of 47 CRC Chinese patients including 22 metastatic and 25 non-metastatic patients. By comparison with data from western colorectal cancer patients in the Cancer Genome Atlas (TCGA), we identified a number of genes that are more likely to be mutated in Chinese colorectal cancer patients, such as MUC12, MUC12, MUC2, MUC4, HYDIN and KMT2C. Interestingly, MUC family genes including MUC12, MUC2 and MUC4, have mutation rates of >20%, while the mutation frequency was extremely low in western colorectal cancer patients, which were <3% in TCGA and 0% in Memorial Sloan Kettering Cancer Center (MSKCC). We detected metastasis-specific mutated genes including TCF7L2, MST1L, HRNR and SMAD4, while MUC4, NEB, FLG and RFPL4A alteration is more prevalent in the non-metastasis group. Further analysis reveals mutation genes in metastasis group are more focus in the Wnt and Hippo signaling pathway. APC, SMAD4 and TCF7L2 accounted for the major genetic abnormalities in this pathway. In conclusion, this study identified the unique characteristics of gene mutations in Chinese patients with colorectal cancer, and is a valuable reference for personalized treatment in Chinese CRC patients.

Novel KMT2B gene mutation in MUC4 positive low-grade fibromyxoid sarcoma.

BACKGROUND: Low-grade Fibromyxoid Sarcoma(LGFM)is a rare fibrosarcoma, which mainly occurs in young people and is mostly seen in the trunk and limbs. The tumor is usually FUS-CREB3L2 fusion caused by t(7;16)(q32-34;p11)chromosome translocation, and rarely FUS-CREB3L1 and EWSR1-CREB3L1 fusion. MUC4 diffuse strong positive can be used as a specific index of LGFM. LGFM is similar to Sclerosing Epithelioid Fibrosarcoma(SEF) and may have the same origin. CASE PRESENTATION: We report a case of LGFM in the chest wall. A female who is 59 years old. In 2016, CT showed dense nodule shadow and focal thickening of the left pleura, the patient underwent surgery, Pathological report that low to moderate malignant fibrosarcoma(fibromyxoid type). The CT re-examination in 2021 showed that the tumors on the left chest wall were significantly larger than before. Pathological examination showed the disease is composed of alternating collagen like and mucinous areas. Under high-power microscope, the tumor cells are consistent in shape, spindle or short spindle, and the tumor cells are arranged in bundles. In local areas, the density of tumor cells is significantly increased, mixed with collagen fibers, and small focal SEF appear. The result of immunohistochemistry showed that SMA, Desmin, CD34, STAT6, S100, SOX10, HMB45 and Melan A were negative, EMA was weakly positive, MUC4 was diffuse and strongly positive, and Ki67 index was low (3%). CONCLUSION: Sequencing results showed that MET, EGFR, KMT2B and RET gene were mutated in LGFM, and KMT2B gene had cancer promoting effect, but there was no literature report in LGFM, which may be of certain significance for the diagnosis and treatment of LGFM.

Sialylated IVIg promotes clinical improvements in a rabbit dry eye model by regulating inflammatory cytokines.

Dry eye disease (DED) is caused by a loss of homeostasis of the tear film, which results in visual disturbance, ocular surface inflammation and damage, and neurosensory abnormalities. Although it is prevalent in 5-50% of the global population, there are limited clinical options for its treatment. This study explored the potential use of human intravenous immunoglobulin (IVIg) and its enriched fractions of sialylation, sialylated IVIg (sIVIg), as a treatment for DED. Fifteen female New Zealand white rabbits were topically instilled with 0.2% benzalkonium chloride (BAC) twice daily for five consecutive days to induce experimental dry eye. Saline, 0.4% IVIg, or 0.04% sIVIg eye drops were instilled twice daily for 20 consecutive days. Clinical evaluations, such as non-invasive tear break-up time (NIBUT) and corneal fluorescein staining (CFS), were conducted. mRNA levels of mucin 4, mucin 16, TNF-α, IL-1ß, MMP9, IL-10, TGF-ß, and CD209 in rabbit conjunctival tissues were examined using reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). The relationships between CD209 family members in rabbits and various mammalian species were analyzed using a phylogenetic tree. IVIg or sIVIg treatment resulted in clinical improvements in the rabbit DED model. The inflammatory cytokines, TNF-α and IL-1ß, were increased and mucin 4 and mucin 16, cell surface-associated mucins, were decreased in BAC-induced dry eye. Following IVIg or sIVIg treatment, inflammatory cytokines decreased, whereas the anti-inflammatory cytokine, IL-10, increased substantially. Moreover, a 10-fold lower sIVIg treatment dose resulted in prolonged IL-10 production, representing a significantly improved DED compared to IVIg. Furthermore, the expression of rabbit CD209 mRNA in the rabbit conjunctiva and its close relationship with primate homologs suggest that it may interact with IVIg or sIVIg to promote IL-10 expression, as previously described in humans. At a lower dosage, sIVIg showed a more efficient improvement in DED, making it a promising new candidate medication for DED.

Interfaz inmunológica de la superficie ocular: expresión de MUC1, MUC4 y MUC16 y producción de citoquinas proinflamatorias en pacientes con síndrome de ojo seco secundario a enfermedad de Sjögren / Ocular surface immunological interface: expression of MUC1, MUC4 and MUC16 and production of proinflammatory cytokines in patients with dry eye syndrome secondary to Sjögren's disease

El síndrome de ojo seco (SOS) constituye una de las patologías oculares más frecuentes y con mayor repercusión en la calidad de vida de pacientes en el mundo actual. A su vez, dado cambios en los hábitos como el mayor uso de pantallas, la mayor polución y el envejecimiento poblacional hacen que la proyección sea en aumento. Se estima que el 10% de los SOS son pacientes con síndrome de Sjögren (SS). Este puede ser primario o secundario cuando se encuentra asociado a otras enfermedades del tejido conectivo como el lupus eritematoso sistémico (LES), la artritis reumatoide (AR) o la esclerosis sistémica (ES). El SOS secundario a SS (SS-SOS) nos resulta de particular interés por ser generalmente severo y de difícil tratamiento. El suero autólogo (SA) constituye una herramienta poco utilizada en nuestro medio hasta la realización de este proyecto. Sus componentes, que incluyen factores de crecimiento epitelial y vitamina A constituyen bloques fundamentales del mantenimiento de las barreras epiteliales haciéndolo una terapia prometedora y accesible. El SS es la tercera enfermedad reumática más prevalente junto con AR y el LES. Se estima que la prevalencia del SSP va del 0.1 al 4.8% de la población dependiendo de la edad, y 90% de estos pacientes son mujeres. A pesar de su alta prevalencia, aún hay gran desconocimiento de los mecanismos moleculares involucrados en el SS-SOS. Tanto la inmunidad innata como adaptativa participan en la patogenia del SOS asociado a SS. Estudios previos encontraron que tanto un perfil Th1 como Th17 serían los principales actores. Por este motivo, elegimos el estudio de las citoquinas IL-17 e IL-22 las cuales son las principales citoquinas efectoras de este perfil linfocitario. Estas citoquinas mantienen la integridad de la barrera epitelial estimulando la formación de proteínas que conforman las uniones estrechas.En especial IL-22 tiene un rol importante en la supervivencia y proliferación celular. Por su parte IL-17 tendría un rol estimulando la producción de péptidos antimicrobianos y quimioquinas que atraen leucocitos a la zona afectada cuando la barrera epitelial es penetrada. En la escasa literatura publicada se ha observado que los niveles de IL-17 e IL­22 se encuentran aumentados significativamente en pacientes con SS-SOS con relación a los no SS y a los controles sanos. A su vez, el nivel de estas citoquinas se correlacionaba positivamente con el cuestionario de queratopatía del índice de enfermedad de la superficie ocular (OSDI por su sigla en inglés) y negativamente con el tiempo de rotura del film lagrimal (TBUT por su sigla en inglés) y el resultado de la prueba de Schirmer. Debemos destacar que estos resultados fueron vistos en un único estudio que incluyó un pequeño número de pacientes. Las mucinas jugarían un rol fundamental en la homeostasis de la superficie ocular (SO). En particular las mucinas transmembrana MUC1, MUC4 y MUC16 estarían involucradas en la activación de factores de transcripción de citoquinas proinflamatorias. Nuestra hipótesis inicial era que se podría observar una disminución en la expresión de estas mucinas en relación con el daño de la SO. Esta hipótesis se basó en el conocido rol protector que las mucinas 12 juegan normalmente en las superficies epiteliales, atrapando agentes patógenos y su estudiada naturaleza inmunorreguladora. A lo largo de este trabajo de doctorado PROINBIO, se utilizó una combinación de diversas estrategias experimentales y clínicas para estudiar la superficie ocular en pacientes con síndrome de Sjögren (SS) y síndrome de ojo seco (SOS) y la correlación de estos hallazgos con la clínica del paciente.Para el desarrollo de este proyecto de investigación, conseguimos articular un servicio dirigido a los pacientes con SOS con la participación de un equipo multidisciplinario integrado por médicos oftalmólogos pertenecientes a la cátedra de oftalmología (valoración- seguimiento - indicación terapéutica), hemoterapia (desarrollo del colirio de suero autólogo) y unidad de enfermedades autoinmunes (captación de los pacientes con síndrome de Sjögren primario o secundario) del Hospital de Clínicas en Montevideo, Uruguay. Al mismo tiempo, desarrollamos y pusimos a punto la toma de biopsias de células conjuntivales mediante impresión conjuntival. Este procedimiento se había realizado anteriormente en nuestro medio, pero nunca para el estudio posterior con técnica de RT-PCR (reacción en cadena de la polimerasa en tiempo real). También conseguimos sistematizar el correcto almacenamiento y procesamiento de las muestras para la realización de esta técnica. Esto facilitará en el futuro continuar con el estudio de nuevos marcadores en esta y otras patologías de la SO. Nuestros resultados nos permitieron arribar a las siguientes conclusiones: Determinamos la primera estimación de prevalencia de SOS en una población de pacientes con diagnóstico de otras patologías autoinmunes en nuestra población la cual fue discretamente menor a la reportada en la literatura. Esta fue de 22% de los pacientes, constituyendo la primera estimación en Uruguay de SOS en pacientes con patología autoinmune. Logramos medir la expresión de las tres principales mucinas transmembrana de la SO en nuestro grupo de pacientes estudiados e individuos sanos. Contrario a nuestra hipótesis, observamos un aumento significativo de la expresión de MUC1 y MUC4 en nuestro grupo de pacientes con SOS.A pesar de que es conocido el rol fundamental de las mucinas en mantener la homeostasis de la SO, se ha descrito el aumento en la expresión de MUC1 y MUC4 frente al daño inflamatorio o en casos de neoplasias tanto en el epitelio digestivo como respiratorio. Detectamos una diferencia en la expresión de estas mucinas entre pacientes con SSP donde observamos un aumento de las tres mucinas de estudio (MUC1, MUC4 y MUC16) y SSS donde únicamente MUC1 se vio significativamente elevada. Asimismo, correlacionamos estos hallazgos con la clínica (síntomas y signos de ojo seco) observando que MUC4 correlaciona significativamente tanto con menor producción del film lagrimal como con un mayor grado de queratopatía superficial. La correlación de MUC4 con los síntomas de SOS no fue significativa lo cual pensamos sí se observaría probablemente incluyendo un mayor número de pacientes en el estudio. Por otra parte, MUC1 y MUC16 se correlacionaron positivamente con una presencia de síntomas más severos de SOS pero su correlación con los signos clínicos de SOS no llegó al nivel de significancia excepto en el caso de MUC1 con relación al grado de queratopatía. Evaluamos la expresión de citoquinas proinflamatorias del perfil Th17 como son IL-17 e IL-22. Observamos como la IL 22 se correlaciona con la expresión de MUC16, la cual a su vez aumenta en pacientes con mayor sintomatología de SOS. Encontramos una correlación significativa entre la expresión de ambas citoquinas como era esperable ya que ambas son citoquinas que corresponden a un mismo perfil de respuesta linfocitaria. No pudimos reproducir los resultados vistos en la literatura, aunque escasa, la cual reporta que los pacientes con SOS presentan un aumento de estas citoquinas en relación a una mayor severidad clínica del SS-SOS.En nuestro estudio no observamos diferencia entre nuestro grupo de pacientes y el grupo control ni una correlación con los síntomas o signos del SOS. Analizamos la expresión de MUC1, MUC4 y MUC16 comparando el grupo de pacientes con SS tratados con inmunomoduladores versus los no tratados observando una diferencia interesante entre estos dos grupos. Mientras ambos grupos sobre expresan MUC1. Los pacientes en tratamiento presentan un aumento de MUC4 y no de MUC16 y lo inverso se observa en los pacientes no tratados. Evidenciamos la mejoría sintomática del tratamiento de estos pacientes con suero autólogo al 20% el cual es una opción terapéutica disponible en nuestro medio que está subutilizada por la falta de evidencia clínica hasta el momento. Estos resultados son un sustento objetivo de la efectividad del tratamiento para este grupo de pacientes. En suma, este trabajo constituye una aproximación a comprender como varia la expresión a nivel de ARN mensajero de mucinas transmembranarias específicas y su asociación con citoquinas de perfil Th17, así como síntomas y signos clínicos del SS-SOS. Constituye un gran paso inicial a la caracterización molecular de la SO de estos pacientes y con esta la potencialidad de desarrollar terapias dirigidas. Según nuestro saber y entender, éste constituye el primer trabajo de estas características en nuestro medio y nos brinda más información de la prevalencia y características de estos pacientes en Uruguay. La puesta a punto de la técnica de impresión conjuntival, conocida en el mundo, pero no utilizada en nuestro país habitualmente para la extracción de ARN, abre las puertas para ampliar nuestras investigaciones focalizándonos en otras moléculas posiblemente cruciales en el desarrollo de SOS como por ejemplo el estudio de glicosil transferasas.

Effects of 17β-Estradiol on Colonic Permeability and Inflammation in an Azoxymethane/Dextran Sulfate Sodium-Induced Colitis Mouse Model

BACKGROUND/AIMS: Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases (IBDs) such as ulcerative colitis. This dysfunction is caused by increased permeability and the loss of tight junctions in intestinal epithelial cells. The aim of this study was to investigate whether estradiol treatment reduces colonic permeability, tight junction disruption, and inflammation in an azoxymethane (AOM)/dextran sodium sulfate (DSS) colon cancer mouse model. METHODS: The effects of 17β-estradiol (E2) were evaluated in ICR male mice 4 weeks after AOM/DSS treatment. Histological damage was scored by hematoxylin and eosin staining and the levels of the colonic mucosal cytokine myeloperoxidase (MPO) were assessed by enzyme-linked immunosorbent assay (ELISA). To evaluate the effects of E2 on intestinal permeability, tight junctions, and inflammation, we performed quantitative real-time polymerase chain reaction and Western blot analysis. Furthermore, the expression levels of mucin 2 (MUC2) and mucin 4 (MUC4) were measured as target genes for intestinal permeability, whereas zonula occludens 1 (ZO-1), occludin (OCLN), and claudin 4 (CLDN4) served as target genes for the tight junctions. RESULTS: The colitis-mediated induced damage score and MPO activity were reduced by E2 treatment (p < 0.05). In addition, the mRNA expression levels of intestinal barrier-related molecules (i.e., MUC2, ZO-1, OCLN, and CLDN4) were decreased by AOM/DSS-treatment; furthermore, this inhibition was rescued by E2 supplementation. The mRNA and protein expression of inflammation-related genes (i.e., KLF4, NF-κB, iNOS, and COX-2) was increased by AOM/DSS-treatment and ameliorated by E2. CONCLUSIONS: E2 acts through the estrogen receptor β signaling pathway to elicit anti-inflammatory effects on intestinal barrier by inducing the expression of MUC2 and tight junction molecules and inhibiting pro-inflammatory cytokines.

MUC4 research progress in tumor molecular markers / 生物医学工程学杂志

Mucin antigen 4 (MUC4) is a molecular marker for some malignant tumors for early tumor diagnosis, prognosis and targeted therapy. It provides a new research direction in tumor diagnosis and treatment that will have a wide application prospect. In recent years, there has been a large number of research reports on the basic and clini-a wide application prospect. In recent years, there has been a large number of research reports on the basic and clinical studies about MUC4, but the molecular imaging study about MUC4 is seldom reported. In this paper the recentcal studies about MUC4, but the molecular imaging study about MUC4 is seldom reported. In this paper the recent research about MUC4 on basic and clinical studies is briefly reviewed, and it is expected to promote the development of tumor molecular imaging.

Study on anti-tumor effect of cyanidin-3-glucoside on ovarian cancer / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect and the mechanism of cyanidin-3-glucoside (C3G) in the growth inhibition of ovarian cancer in vitro and in vivo.</p><p><b>METHOD</b>After human ovarian cancer cell line HO-8910PM was treated with C3G, cell growth was determined by the Cell Counting Kit-8 (CCK-8) assay and apoptosis was evaluated by flow cytometry analysis stained with Annexin V-FITC/PI. The protein expression in HO-8910PM cells was analyzed by Western blot assay. HO-8910PM cells were injected subcutaneously into nude mice to establish xenograft model. After 3 weeks of implantation, mice were randomized into 2 groups (n = 8): control group, feed with 0.2 mL double distilled water; C3G group, feed with C3G at a dose of 5 mg x kg(-1). All treatment lasted for two weeks, thrice per week. Eight weeks after implantation, tumor weight and inhibition rate were evaluated respectively after the mice were sacrificed. Immunohistochemistry was used to detect the positive expression of Ki-67 and Mucin-4 in the tumors.</p><p><b>RESULT</b>The proliferation of ovarian cancer cells was inhibited significantly by C3G with IC50 being 13.82 mg x L(-1). Apoptosis rate induced by C3G was markedly highter than that of control. The expression of Mucin4 was down-regulated in HO-8910PM cells after treatment of C3G. C3G inhibited the growth of ovarian xenograft tumors in nude mice. Furthermore, the positive expression of Ki-67 and Mucin-4 were both decreased in tumors after administration of C3G.</p><p><b>CONCLUSION</b>C3G exerts anti-tumor activity in ovarian cancer both in vitro and in vivo, which may be related to down-regulation of Mucin-4 protein.</p>

Pentamer guided HLA-restricted epitope identification for mucoprotein 4 antigen of pancreatic ductal adenocarcinoma / 中华外科杂志

<p><b>OBJECTIVES</b>To identify HLA-restricted epitope of mucoprotein 4 (MUC4) antigen as a tumor associated antigen of pancreatic ductal adenocarcinoma (PDAC), and to validate its natural presentation in PDAC patient peripheral blood.</p><p><b>METHODS</b>Two epitope prediction databases (SYFPEITHI and ProPred-I) were used to predict HLA-A*0201 restricted MUC4 epitope, T2 cell assay was used to determine the peptide binding affinity with HLA-A*0201 molecule. Dendritic cells (DCs) were induced from the HLA-A* 0201-positive healthy individuals' peripheral blood mononuclear cells (PBMC). Mature DCs were pulsed with synthesized peptides. Autologous CD8(+) T cells from the HLA-A* 0201 healthy donor were stimulated with the peptide-pulsed DCs as CTL. CTL activity was assessed by lactate dehydrogenase release assay and IFN-γ released by enzyme-linked immunospot assay. Pentamer was synthesized for HLA-A* 0201 restricted epitope P1126, then was used to detect specific CTL in PBMC of PDAC patients.</p><p><b>RESULTS</b>Five candidate HLA-A*0201 epitopes were predicted, LLLGVGTFV (P1125) and LLGVGTFVV (P1126) were determined as the two with more HLA-A*0201 affinity. Mature DCs could be induced from PBMCs. CTL induced by peptide P1126 could lyses T2 cells pulsed with peptide P1126 and HCT-116 cells [MUC4(+), HLA-A2(+)]. The number of CTL induced by peptide P1126 which could secret IFN-γ (130.3 ± 6.6) was obviously higher than that in the negative group. By Pentamer assay, P1126-pentamer and CD8 double positive CTL could be detected in PBMC of PDAC patients with MUC4(+) than patients with MUC4(-), but no significant difference of CTL frequency between patients with HLA-A2(+) and with HLA-A2(-) in MUC4(+) PDAC patients.</p><p><b>CONCLUSIONS</b>Tumor associated antigen MUC4-derived HLA-A* 0201-restrictive cytotoxic T lymphocyte (CTL) epitope P1126 can induce CTL reaction. The CTL can secret immunologic active material to induce the specific target cells lysis. P1126 epitope can be naturally presented in PBMC of PDAC patients, but its HLA-restriction may not be perfect.</p>

Expression of MUC1 and MUC4 and Its Prognostic Significance in Non-Small Cell Lung Carcinoma

BACKGROUND: Mucin (MUC)1 and MUC4 (MUC1, 4) are high molecular weight glycoproteins expressed in normal and malignant epithelial cells, and these expressions are related to the prognosis of some carcinomas. In non-small cell lung carcinoma (NSCLC), the relationship between MUC1, 4 expressions and their prognostic significance is not well known. We evaluated these relationships in a series of NSCLC: 1) between MUC1, 4 expression levels and histologic subtypes, and 2) between high expression of MUC1, 4 and their prognostic significance. METHODS: We performed immunohistochemical staining for MUC1, 4 in paraffin-embedded tissues from 165 NSCLC cases arranged in a tissue microarray. RESULTS: We found a significant correlation between MUC1, 4 expressions and NSCLC histologic subtypes (p < 0.05). High MUC1 expression was characteristic of adenocarcinoma. Low MUC1, 4 expressions were characteristic of squamous cell carcinoma. In adenocarcinoma, we found significant association between diffuse MUC1 expression and short patient survival (p = 0.005). In squamous cell carcinoma, diffuse MUC4 expression showed long patient survival trend (p = 0.128). CONCLUSIONS: MUC1, 4 expression levels were significantly correlated with NSCLC histologic subtypes. Diffuse MUC1 expression was significantly associated with shortened survival in NSCLC patients, especially in adenocarcinoma.