RESUMEN
BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is a cystic tumor of the pancreas arising from abnormal papillary proliferation of ductal epithelial cells, and is a precancerous lesion of pancreatic malignancy. This study aimed to evaluate associations between acute pancreatitis (AP) and histologic subtypes of IPMN. METHODS: In the clinical study, patients with IPMN confirmed by surgical resection specimens at our institute between 2009 and 2021 were eligible for inclusion. Associations and predictive accuracy of AP on the presence of HGD were determined by logistic regressions. In addition, a systematic review and meta-analysis was conducted through literatures upon search in PubMed, Embase, CENTRAL, China National Knowledge Infrastructure (CKNI), and Wanfang database, up to June, 2023. Pooled effects of the associations between AP and HGD and intestinal epithelial subtype subtype, shown as odds ratios (ORs) with 95% confidence intervals (CIs), were calculated using random effects model. RESULTS: The retrospective cohort study included 47 patients (32 males, 15 females) diagnosed with IPMN at our center between 2009 and 2021, including 11 cases with AP (median 62 years) and 36 cases (median 64.5 years) without. Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of AP in predicting HGD were 78.7%, 57.1%, 82.5%, 36.4%, and 91.7%, respectively. Univariate logistic regression analysis showed that AP group had greater odds of presence of HGD (OR: 6.29,95% CI: 1.14-34.57) than non-AP group. Meta-analysis of five case-control studies in the literature included 930 patients and showed that AP-IPMN patients had higher odds for HGD (OR: 2.13, 95% CI 1.38-3.29) and intestinal epithelial subtype (OR: 5.38, 95% CI: 3.50-8.27) compared to non-AP IPMN. CONCLUSIONS: AP is predictive of malignancy in patients with IPMN.
Asunto(s)
Adenocarcinoma Mucinoso , Carcinoma Ductal Pancreático , Neoplasias Intraductales Pancreáticas , Neoplasias Pancreáticas , Pancreatitis , Masculino , Femenino , Humanos , Carcinoma Ductal Pancreático/patología , Pancreatitis/complicaciones , Pancreatitis/patología , Estudios Retrospectivos , Enfermedad Aguda , Adenocarcinoma Mucinoso/complicaciones , Adenocarcinoma Mucinoso/patología , Neoplasias Pancreáticas/patologíaRESUMEN
Butyrate is widely accepted as a proliferation inhibitor in colon cancer but less thoroughly characterized in the colonic epithelium of objects with type 2 diabetes mellitus. The present study investigated the regulatory effect of butyrate on proliferation, the related molecule high-mobility group box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE) in the colon of db/db type 2 diabetic model mice and non-cancerous NCM460 colon cells. Proliferation and the expression of HMGB1 and RAGE were increased and could be partially reversed by butyrate treatment in the colon of db/db mice, which were consistent in NCM460 cells under a high glucose state. In NCM460 cells, under the normal glucose state, proliferation increased by overexpression of HMGB1. Under a high glucose state, increased expression of HMGB1 was accompanied with a release from cell nuclei into the cytoplasm and extracellular matrix. Down-regulation of HMGB1 could lower the expression of RAGE and attenuate the abnormally increased proliferation. And overexpression of HMGB1 reversed the suppressing effect of butyrate on abnormally increased proliferation. Conclusively, butyrate suppressed the abnormally increased proliferation in colonic epithelial cells under diabetic state by targeting HMGB1.
Asunto(s)
Butiratos/farmacología , Proliferación Celular/efectos de los fármacos , Colon/citología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Células Epiteliales/fisiología , Expresión Génica/efectos de los fármacos , Proteína HMGB1/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Proteína HMGB1/genética , Masculino , Ratones Endogámicos , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismoRESUMEN
Diabetes mellitus (DM) is a group of metabolic diseases characterised by insulin deficiency/resistance and hyperglycaemia. We previously reported the presence of an impaired tight junction and decreased expression of occludin (Ocln) and zonula occludens-1 (ZO-1) in the intestinal epithelial cells (IECs) of type 1 DM mice, but the exact mechanism remains unclear. In this study, we investigated the role of microRNAs (miRNAs) in impairing the tight junction in IECs of DM mice. Using an integrated comparative miRNA microarray, miR-429 was found to be up-regulated in IECs of type 1 DM mice. Then, miR-429 was confirmed to directly target the 3'-UTR of Ocln, although it did not target ZO-1. Moreover, miR-429 down-regulated the Ocln expression in IEC-6 cells in vitro. Finally, exogenous agomiRNA-429 was shown to down-regulate Ocln and induce intestinal barrier dysfunction in normal mice, while exogenous antagomiRNA-429 up-regulated Ocln in vivo and improved intestinal barrier function in DM mice. In conclusion, increased miR-429 could down-regulate the expression of Ocln by targeting the Ocln 3'-UTR, which impaired intestinal barrier function in DM mice.
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Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Regulación hacia Abajo , Intestinos/patología , MicroARNs/metabolismo , Ocludina/genética , Regiones no Traducidas 3'/genética , Animales , Antagomirs/metabolismo , Secuencia de Bases , Sitios de Unión , Permeabilidad de la Membrana Celular , Regulación hacia Abajo/genética , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ocludina/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasRESUMEN
In previous studies, we have reported the abnormal proliferation and differentiation of intestinal epithelial cells (IECs) in diabetes mellitus (DM) mice. The insulin receptor (IR) and its downstream mitogen-activated protein kinase kinase (MAPKK also known as MEK)/extracellular-regulated protein kinase (ERK) pathway is a classic pathway associated with cell proliferation and differentiation. The purpose of the present study is to investigate the role of the MEK/ERK pathway in abnormal proliferation and differentiation of IECs in DM mice. DM mouse models were induced by intraperitoneal injection of streptozotocin. The expression levels of the IR and its isoforms in IECs of DM mice and in IEC-6 cells were investigated. To ensure that the downstream pathways were monitored, QPCR and Western blotting were performed to detect the expression levels of MEK1/2, ERK1/2, PI3K, and Akt. Moreover, siRNA for IR-A and U0126, a specific inhibitor of MEK, were used to further investigate the relationship between the IR/MEK/ERK pathway and abnormal proliferation and differentiation of IECs in DM mice. In DM mice, excessive proliferation, disturbed differentiation, and a high ratio of IR-A/IR-B were detected in IECs. The expression levels of MEK1, MEK2, and ERK1/2 and their phosphorylated proteins in DM mice were significantly higher than those in the control group (P < 0.05), which could be offset by using siRNA for IR-A. The abnormal proliferation and differentiation of IECs in DM mice were normalized after the in vivo administration of U0126. The abnormal proliferation and differentiation of IECs in DM mice are associated with high IR-A/IR-B ratio and increased IR/MEK/ERK pathway activity.
Asunto(s)
Diabetes Mellitus Experimental/patología , Células Epiteliales/citología , Mucosa Intestinal/citología , Sistema de Señalización de MAP Quinasas , Receptor de Insulina/metabolismo , Animales , Butadienos/administración & dosificación , Butadienos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Nitrilos/administración & dosificación , Nitrilos/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor de Insulina/genética , EstreptozocinaRESUMEN
BACKGROUND Type 1 diabetes mellitus (T1DM) is associated with increased risks of enteric infection. Paneth cells constitute the first line of the gut defense. Little is known about the impact of T1DM on the bactericidal function of intestinal Paneth cells. MATERIAL AND METHODS A T1DM mouse model was induced by intraperitoneal injection of streptozocin. The analysis of intestinal microbiota and the mucosal bactericidal assay were conducted to evaluate intestinal innate defense. Numbers of Paneth cells and their expression of related antimicrobial peptides were analyzed. Expression of total insulin receptor (IR) mRNA and relative levels of IR-A/IR-B were analyzed. The primary mouse small intestinal crypt culture was used to analyze the effect of insulin and glucose on the expression of related antimicrobial peptides of Paneth cells. RESULTS In T1DM mice, bacterial loads were increased and there was an alteration in the composition of the intestinal microflora. Exogenous bacteria had better survival in the small bowel of the T1DM mice. The expression of Paneth cell-derived antimicrobial peptides was significantly decreased in the T1DM mice, although the number of Paneth cells was increased. Relative levels of IR-A/IR-B in Paneth cells of diabetic mice were elevated, but the total IR mRNA did not change. Insulin treatment restored the expression of antimicrobial peptides and normalized the microbiota in the gut of T1DM mice. Subsequently, in vitro culture assay demonstrated that insulin rather than glucose was essential for the optimal expression of Paneth cell-derived antimicrobial peptides. CONCLUSIONS The bactericidal function of intestinal Paneth cells was impaired in STZ-induced diabetic mice, resulting in the altered intestinal flora, and insulin was essential for the optimal expression of Paneth cell-derived antimicrobial peptides.
Asunto(s)
Diabetes Mellitus Experimental/inmunología , Insulina/deficiencia , Células de Paneth/inmunología , Animales , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/inmunología , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/microbiología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/microbiología , Inmunidad Innata , Insulina/administración & dosificación , Insulina/sangre , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Ratones , Ratones Endogámicos C57BL , Microbiota , Células de Paneth/microbiología , Distribución Aleatoria , Receptor de Insulina/biosíntesis , Receptor de Insulina/deficiencia , Receptor de Insulina/metabolismoRESUMEN
Intestinal epithelial stem cells (IESCs) can differentiate into all types of intestinal epithelial cells (IECs) and Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a marker for IESC. Previous studies reported enhanced proliferation of IECs in diabetic mice. In this study, the in vitro differentiation of Lgr5 positive IESCs sorted from diabetic mice was further investigated. The diabetic mouse model was induced by streptozotocin (STZ), and crypt IECs were isolated from small intestines. Subsequently, Lgr5 positive IESCs were detected by flow cytometry (FCM) and sorted by magnetic activated cell sorting (MACS). Differentiation of the sorted IESCs was investigated by detecting the IEC markers in the diabetic mice using immunostaining, quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), and Western blot analysis, which was compared with normal mice. We found that the proportion of Lgr5 positive cells in the crypt IECs of diabetic mice was higher than that of control mice (P < 0.05). Lgr5 positive IESCs could be significantly enriched in Lgr5 positive cell fraction sorted by MACS. Furthermore, the absorptive cell marker sucrase-isomaltase (SI) and the Paneth cell marker lysozyme 1 (Lyz1) were more highly expressed in the differentiated cells derived from Lgr5 positive IESCs of diabetic mice in vitro (P < 0.05). We demonstrate that the number of Lgr5 positive IESCs is significantly increased in the small intestines of STZ-induced diabetic mice. Lgr5 positive IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro. We characterized the expression of Lgr5 in the small intestine of diabetic mice, and sorted Lgr5 positive intestinal epithelial stem cells (IESCs) for investigating their differentiation in vitro. We proved that the quantity of Lgr5 positive IESCs was significantly increased in the small intestines of diabetic mice. IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro.
Asunto(s)
Diferenciación Celular , Diabetes Mellitus Experimental/patología , Intestino Delgado/patología , Células de Paneth/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Células Madre , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Femenino , Separación Inmunomagnética , Absorción Intestinal , Mucosa Intestinal/patología , Mucosa Intestinal/fisiología , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Células de Paneth/patología , Células Madre/metabolismo , Células Madre/patología , Células Madre/fisiología , EstreptozocinaRESUMEN
Treatments for pancreatic injuries have been significantly improved recently, but full recovery of pancreatic function remains difficult. Embryonic stem cells have great potentialities for self-renewal and multiple differentiations. In this study, we explored an approach to induce the differentiation of pancreatic progenitor cells from embryonic stem cells in vitro. Male mouse embryonic stem cells were cultured by the hanging-drop method to form embryoid bodies. The definitive endoderm marked by CXCR4 in embryoid bodies was sorted by magnetic activated cell sorting and subsequently administrated with b-FGF, exendin-4, and cyclopamine to induce the differentiation of putative pancreatic progenitor cells, which was monitored by Pdx1, and Shh expressions. The putative pancreatic progenitor cells were transplanted into female BALB/c mice with pancreatitis induced by L-Arginine. Male donor cells were located by detecting sex-determining region of Y-chromosome DNA. Definitive endoderm cells (CXCR4(+) cells) were sorted from 5-day embryoid bodies. After 3-day administration with b-FGF, exendin-4, and cyclopamine, Pdx1-high/Shh-low cells were differentiated from CXCR4(+) cells. These cells developed into more amylase-secreted cells in vitro and could specifically reside in the damaged pancreas acinar area in mice with acute pancreatitis to enhance the regeneration. The putative pancreatic progenitor cells (Pdx1-high/Shh-low cells) derived from mouse embryonic stem cells through the administration of b-FGF, exendin-4, and cyclopamine on the CXCR4(+) cells in vitro could improve the regeneration of injured pancreatic acini in vivo.
Asunto(s)
Células Madre Embrionarias/citología , Proteínas Hedgehog/biosíntesis , Proteínas de Homeodominio/biosíntesis , Pancreatitis Aguda Necrotizante/terapia , Receptores CXCR4/biosíntesis , Trasplante de Células Madre/métodos , Transactivadores/biosíntesis , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Separación Celular/métodos , Modelos Animales de Enfermedad , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Autoimmune pancreatitis (AIP) is a distinct type of pancreatitis associated with a presumed autoimmune mechanism. The aim of this study was to analyze the clinical features and expressions of forkhead box P3 (Foxp3) and interleukin-17 (IL-17) in type 1 AIP in China and to identify factors for differentiation of AIP from non-AIP chronic pancreatitis (CP). MATERIAL AND METHODS: We retrospectively reviewed pancreatic specimens with diagnosis of type 1 AIP and non-AIP CP at Sun Yat-Sen Memorial Hospital in China from January 2000 to December 2013. The clinical symptoms, serological data, imaging findings, histopathology, and immunohistochemical findings of Foxp3 and IL-17 in the 2 groups were analyzed. RESULTS: Twenty-nine patients with type 1 AIP and 20 patients with non-AIP CP were enrolled. Obstructive jaundice was more common in type 1 AIP than in non-AIP CP (62.1% vs. 30.0%, P=0.042). The diffuse or segmental enlargement of the pancreas was more frequent in type 1 AIP than in non-AIP CP (72.4% vs. 40.0%, P=0.038). Histopathology of type 1 AIP presented dense lymphoplasmacytic infiltration, "snowstorm-like" fibrosis and abundant immunoglobulin (Ig) G4+ cells. Foxp3+ cells were more frequently observed in type 1 AIP than in non-AIP CP. IL-17+ cell infiltration was similar between the 2 groups. Furthermore, a positive correlation was found between Foxp3+ and IgG4+ cell counts in the pancreas of patients with type 1 AIP. CONCLUSIONS: Type 1 AIP has distinctive symptoms, image, and pathological characteristics, which could be used for differentiation from non-AIP CP. Foxp3+ cells might be helpful to distinguish type 1 AIP from non-AIP CP.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Factores de Transcripción Forkhead/metabolismo , Interleucina-17/metabolismo , Pancreatitis/metabolismo , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/diagnóstico por imagen , Enfermedades Autoinmunes/inmunología , China , Pancreatocolangiografía por Resonancia Magnética , Femenino , Humanos , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Pancreatitis/sangre , Pancreatitis/diagnóstico por imagen , Pancreatitis/inmunología , Pancreatitis Crónica/sangre , Pancreatitis Crónica/diagnóstico por imagen , Pancreatitis Crónica/inmunología , Pancreatitis Crónica/metabolismo , Tomografía Computarizada por Rayos XRESUMEN
OBJECTIVES: Embryonic stem cells (ESCs)-derived pancreatic precursor cells have great potential for pancreas repair. Expression of pancreatic duodenal homeobox 1 (Pdx1) in definitive endoderm (DE) cells is the premise that DE cells differentiate into pancreatic cells. To achieve the required number of Pdx1-expressing DE cells for cell transplantation therapy, a valid model must be established. Using this model, researchers investigated how Pdx1 regulates ESC differentiation into pancreatic cells. METHODS: Tet-On inducible lentiviral vector encoding Pdx1 or mock vector was transduced into mouse ESC (ES-E14TG2a). The mouse ESCs were divided into 3 groups: control (ESC), mock vector (Pdx1 - -ESC), and vector encoding Pdx1 (Pdx1 + -ESC). All groups were separately cocultured with the DE cells sorted by immune beads containing CXCR-4 + (C-X-C chemokine receptor type-4) antibody. Doxycycline induced the expression of Pdx1 on the Pdx1 + -ESC cells. The markers of cell differentiation and Notch pathway were examined. RESULTS: Significantly increased expression levels of Ptf1a, CK19, and amylase on day (d) 3 and d7, Neuro-D1 on d10 and d14, Pax6 and insulin on d14, as well as Notch1, Notch2, Hes1, and Hes5 on d3 and thereafter declined on d14 were observed in Pdx1 + -ESC group. CONCLUSIONS: Pdx1 + -ESC could differentiate into pancreatic-like cells with involvement of the Notch pathway.
Asunto(s)
Endodermo , Proteínas de Homeodominio , Células Madre Embrionarias de Ratones , Páncreas , Transactivadores , Animales , Diferenciación Celular , Endodermo/citología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Páncreas/citología , Receptores Notch/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Colorectal cancer (CRC) is the most common form of gastrointestinal malignancies. A growing number of reports focusing on oxaliplatin (OXA) resistance in CRC treatment have revealed that drug resistance is an urgent issue in clinical applications, especially for finding effective therapeutic targets. Recently, microRNAs (miRNAs) are reported to play a critical role in tumor progressions and multi-drug resistance. The main aim of this study is to establish whether miR-5000-3p is an oncogene that is resistant to OXA and further confirm its underlying regulatory role in CRC. The OXA-associated gene expression dataset in CRC cells was downloaded from Gene Expression Omnibus (GEO) database. Statistical software R was used for significance analysis of differentially expressed genes (DEGs) between OXA-resistant (OR)-CRC cells and CRC cells, and results indicated ubiquitin-specific peptidase 49 (USP49) was upregulated in OR-CRC cells. Luciferase reporter assay showed that USP49 was verified to act as a downstream target gene of miR-5000-3p. From the results of TCGA database, miR-5000-3p expression was upregulated and USP49 was downregulated in patients with CRC. The function of miR-5000-3p was detected using MTT assay, wound healing, Transwell, and flow cytometry assays. Moreover, through in vitro and in vivo experiments, miR-5000-3p expression was confirmed to be upregulated in CRC cells or OR-CRC cells comparing to normal cell lines. Molecular mechanism assays revealed that USP49 binds to the miR-5000-3p promoter to increase the expression of miR-5000-3p, resulting in cancer cells sensitized to OXA. To sum up, these results suggest that miR-5000-3p may be a novel biomarker involved in drug-resistance progression of CRC. Moreover, the drug-resistance mechanism of miR-5000-3p/USP49 axis provides new treatment strategies for CRC in clinical trials.
RESUMEN
Msi1 (Musashi 1) is regarded as a marker for neural and intestinal epithelial stem cells. However, it is still unclear whether Msi1-positive cells derived from mouse embryonic stem cells have the ability to differentiate into neural or intestinal epithelial cells. A pMsi1-GFP (green fluorescent protein) reporter plasmid was constructed in order to sort Msi1-positive cells out of the differentiated cell population. The GFP-positive cells (i.e. Msi1-positive cells) were sorted by FACS and were hypodermically engrafted into the backs of NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice. The presence of neural and intestinal epithelial cells in the grafts was detected. Msi1 was highly expressed in the GFP-positive cells, but not in the GFP-negative cells. The markers for neural cells (Nestin and Tubulin ß III) and intestinal epithelial cells [FABP2 (fatty acid binding protein 2), Lyz (lysozyme) and ChA (chromogranin A)] were more highly expressed in the grafts from Msi1-positive cells than those from Msi1-negative cells (P<0.05). The grafts from the Msi1-negative cells contained more mesodermal-like tissues than those from the Msi1-positive cells. The pMsi1-GFP vector can be used to sort Msi1-positive cells from a cell population derived from mouse embryonic stem cells. The Msi1-positive cells can differentiate into neural and intestinal epithelial-like cells in vivo.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Mucosa Intestinal/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Células Madre Embrionarias/trasplante , Regulación del Desarrollo de la Expresión Génica , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Neurogénesis/fisiología , Neuronas/metabolismo , Proteínas de Unión al ARN/genética , TransfecciónRESUMEN
BACKGROUND: Stem cells must be located and positively identified to elucidate their role in disease pathogenesis and therapy. In this study the expression patterns of the small intestinal stem cell (SISC) markers Musashi-1 (Msi1) and hairy and enhancer of split 1 (Hes1) were identified and the relationship between their expression and epithelial proliferation in the whole mouse small intestine was examined. MATERIAL/METHODS: Mouse small intestines were separated into four segments. Msi1 and Hes1 expression levels were quantified in each intestinal segment by immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and Western blot. Small intestinal epithelial proliferation was examined using the bromodeoxyuridine (BrdU) method and proliferating cell nuclear antigen (PCNA) immunostaining. RESULTS: Msi1- and Hes1-positive cells were predominantly detected at the crypt bases. Msi1 and Hes1 protein expression values were significantly higher in the jejunal segment than in the ileal (P<0.05) and mRNA was directly associated with protein expression values. Greater numbers of proliferative cells were detected in jejunal crypts with BrdU and PCNA labeling (P<0.05). CONCLUSIONS: Expression of the Msi1 and Hes1 SISC markers in the small intestine was not homogenous and the markers were more highly expressed in jejunal tissues. This expression pattern correlated with the small intestinal epithelial proliferation pattern measured by BrdU- and PCNA-labeling methods.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Yeyuno/citología , Yeyuno/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores/metabolismo , Proliferación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Microvellosidades/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Factor de Transcripción HES-1RESUMEN
OBJECTIVE: To determine the sensitivity and specificity of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) for papillary thyroid carcinoma (PTC) detection. DESIGN: The SELDI-TOF-MS protein profiles of patients with PTC, patients with benign nodular disease (BND), and healthy controls were analyzed to determine the sensitivity and specificity of SELDI-TOF-MS assay for PTC detection. Data analysis was performed to process the spectral data and classify the disease status of the patients. SETTING: Academic tertiary care hospital. PATIENTS: Serum samples were collected prospectively from 7 patients with PTC, 8 patients with BND, and 7 healthy control volunteers. INTERVENTION: All patients diagnosed as having PTC or BND underwent thyroidectomy from October 21, 2004, to January 31, 2006. MAIN OUTCOME MEASURES: Twenty-two serum samples were analyzed. RESULTS: Most protein peaks resolved by the SELDI-TOF-MS assay were in the range of 1 to 20 kDa. Classification tree analysis based on peak expression distinguished patients with PTC from those with BND with 85.7% sensitivity and 100% specificity. Serum samples from patients with PTC differed most significantly from those of patients with BND by the underexpression of a protein peak at 11 101 Da. CONCLUSIONS: This pilot study demonstrates that proteomic analysis of serum protein profiles distinguishes patients with PTC from patients with BND with a high degree of sensitivity and specificity. Further investigation into the clinical utility of this technology in PTC biomarker detection and surveillance is warranted.
Asunto(s)
Carcinoma Papilar/diagnóstico , Análisis por Matrices de Proteínas , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Proteómica , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVE: To determine the sensitivity and specificity of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) assay for head and neck squamous cell carcinoma (HNSCC) disease surveillance. DESIGN: The SELDI-TOF-MS serum protein profiles of patients with HNSCC were analyzed to determine the sensitivity and specificity of the SELDI assay for HNSCC detection following definitive treatment. SETTING: Academic research. PATIENTS: Thirty-two patients with previously untreated HNSCC. INTERVENTION: Serum samples were collected prospectively at 3-month intervals following treatment during a 24-month follow-up period. MAIN OUTCOME MEASURES: Ninety-three serum samples were analyzed. RESULTS: The SELDI-TOF-MS identified protein peaks in the range of 0 to 100 kDa. Classification tree analysis based on peak expression distinguished pretreatment from 6-month posttreatment samples with 75.0% sensitivity and 87.5% specificity. Samples collected at 3 months following treatment did not significantly differ from pretreatment samples. Serum samples from patients who were disease free at 6 months or longer following treatment differed from matched pretreatment samples by the overexpression of a protein peak at 6495 Da, while serum samples from patients with recurrence differed from matched pretreatment samples by the underexpression of a protein peak at 4493 Da. CONCLUSIONS: Proteomic analysis of serum protein profiles distinguishes pretreatment and posttreatment samples from patients with HNSCC with a high degree of sensitivity and specificity. After 6 months, serum protein profiles seem to have distinct differences in peak expression based on disease status. Further investigation of the clinical usefulness of this technology in HNSCC detection and surveillance is warranted.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Carcinoma de Células Escamosas/terapia , Estudios de Cohortes , Femenino , Neoplasias de Cabeza y Cuello/terapia , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
Colon cancer is one of the most common cancers in the world. Multidrug resistance is one of the main reasons for failure of therapy in patients with advanced colon cancer. In previous studies, multiple methods were investigated to reverse the multidrug resistance of colon cancer cells. However, to date, no clinical method has been identified to be satisfactory. Therefore, successful reversal of drug resistance in colon cancer cells still requires new therapeutic strategies or pharmaceuticals. Wild-type p53-induced phosphatase (Wip1), a member of the 2C type serine/threonine protein phosphatase family, is closely associated with the p53 gene, which is the most important tumor-suppressor gene. Wip1 was reported to be associated with the chemosensitivity of breast cancer cells. However, the correlation between the expression of Wip1 gene and the chemosensitivity of colon cancer cells has not been reported yet. In the present study, Wip1-811 small interfering RNA (siRNA) targeting Wip1 was investigated to reverse the multidrug resistance of colon cancer cells. The siRNA duplexes were transfected into RKO colon cancer cells. The messenger RNA (mRNA) expression of Wip1 was measured by reverse transcription-quantitative polymerase chain reaction. The protein level of Wip1 was detected by western blotting. The cell viability was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. Intracellular adriamycin cumulative concentration was determined using flow cytometry. Wip1-811 siRNA efficiently inhibited the expression of Wip1 at the mRNA and protein levels, and enhanced the sensitivity of RKO colon cancer cells towards chemotherapy, which was accompanied by increased cell apoptosis, following the inhibition of Wip1 gene expression. These results indicate that Wip1 gene silencing could enhance the chemosensitivity of colon cancer cells, which may provide a new potential approach for the reversal of multidrug resistance in colon cancer cells.
RESUMEN
BACKGROUND: Distinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. Mammalian IESCs and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. However, the association between Sox9 and DNA methylation in this process remains elusive. METHODS: The DNA methylation of small intestinal epithelial crypts in db/db mice was detected via combining methylated DNA immunoprecipitation with microarray hybridization. DNA methylation of Sox9 promoter in crypts and IESCs was validated using bisulfite sequence analysis. The target sequence of the transcription factor Sox9 in IESCs was investigated via chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq). RESULTS: Increased Sox9 expression is accompanied by the loss of methylation in its promoter in IESCs. Sox9 targets the enhancers of the Wnt signaling pathway-related genes. Sox9 predominantly acts as a transcriptional activator at proximal enhancers of Wnt4, Tab2, Sox4, and Fzd8, but also functions as a potential transcriptional inhibitor at a distant enhancer of Cdk1. Lack of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway leads to the loss of intrinsic inhibitory action and ultimately produces overactivation of this pathway in db/db mice. CONCLUSIONS: Our study sheds light on the connections among DNA methylation, transcription factor modulation, and Wnt signaling in IESCs in the diabetic state. Hypomethylation in the Sox9 promoter is correlated to increased Sox9 expression in db/db IESCs. Although there is increased expression of Sox9 in db/db IESCs, the loss of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway might result in abnormalities in this pathway.
Asunto(s)
Metilación de ADN/genética , Diabetes Mellitus/terapia , Factor de Transcripción SOX9/genética , Trasplante de Células Madre , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos NOD , Regiones Promotoras Genéticas , Células Madre/metabolismo , Activación Transcripcional/genética , Vía de Señalización Wnt/genéticaRESUMEN
Induced differentiation of definitive endoderm (DE) from embryonic stem cells (ESCs) has been the recent focus of studies investigating regeneration and transplantation of organs of the digestive system. Poor cell survival is the most important challenge to DE differentiation from ESCs. This study aimed to optimize culture conditions to promote the differentiation of mouse ESCs into DE, and to investigate the roles of the Wnt and Nodal signaling pathways in the DE differentiation. The mouse ESCs were treated with or without leukemia inhibitory factor, Wnt3a and Activin A alone or together, and examined the DE differentiation by the DE marker CXCR4 and the ESC marker Oct4. The result showed the optimal induction of differentiation was achieved in cells simultaneously treated with Wnt3a and Activin A. Induction of CXCR4 was also earlier when there was simultaneous activation of Wnt and Nodal signaling compared to the groups treated with only Wnt3a or Activin A alone. These findings provide the basis for the induced differentiation of ESCs for the generation of functional, mature cells of gastrointestinal lineage, which can be potentially used for cell replacement therapy, disease modeling, as well as drug discovery studies.
Asunto(s)
Endodermo/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteína Nodal/metabolismo , Proteína Wnt1/metabolismo , Animales , Diferenciación Celular , Humanos , Ratones , Transducción de SeñalRESUMEN
OBJECTIVES: To analyze surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein profiles of patients with head and neck squamous cell carcinoma (HNSCC) and healthy controls and to determine the sensitivity and specificity of SELDI assay for HNSCC detection before and after treatment. DESIGN: Proteomic analysis and comparison of serum samples. SETTING: Tertiary care academic medical center. SUBJECTS: Seventy-eight patients with HNSCC and 68 healthy controls. MAIN OUTCOME MEASURES: Serum samples were prospectively collected from 78 patients with HNSCC and 68 healthy control volunteers. SELDI-TOF-MS was performed on serum samples to identify protein peaks in the range of 0 to 100 kDa. Classification analysis of the spectral data was performed and used to classify the disease status of the patients. RESULTS: The SELDI-TOF-MS assay generated serum protein profiles ranging from 0 to 100 kDa. After background subtraction, mass calibration, and normalization, 545 protein peaks were identified. Classification tree analysis based on peak expression correctly classified patients with HNSCC with 82% sensitivity and 76% specificity. Subgroup analysis correctly classified 83% of oral cavity tumors, 81% of oropharyngeal tumors, and 88% of laryngeal tumors. Pretreatment and posttreatment samples were available from 12 patients, and the posttreatment samples were correctly classified in 86% of the patients at 3 months and 75% of the patients at 6 months. CONCLUSIONS: Proteomic SELDI-TOF-MS analysis of serum protein profiles distinguishes patients with HNSCC from controls with a high degree of sensitivity and specificity. Further investigation into the clinical utility of this technology in HNSCC detection and surveillance is warranted.
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Biomarcadores de Tumor/sangre , Proteínas Sanguíneas/análisis , Carcinoma de Células Escamosas/diagnóstico , Neoplasias de Cabeza y Cuello/diagnóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto , Anciano , Anciano de 80 o más Años , Diagnóstico Precoz , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Análisis de Regresión , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
OBJECTIVES: Depression of the Notch/Hes1 pathway has been reported to play a role in abnormal differentiation of intestinal epithelial cells (IECs) in diabetes mellitus (DM). However, the mechanism by which this pathway influences IEC differentiation has remained unclear. In this study, we have investigated the role of microRNAs (miRNAs) in regulating the Notch/Hes1 pathway in IECs of DM mice. MATERIALS AND METHODS: Integrated comparative miRNA microarray technology was used to determine the expression profile of miRNAs in IECs of DM mice. After bioinformatic analysis, an miRNA with altered expression levels, miRNA-30e, was identified as a candidate for regulating the Notch pathway in DM. A luciferase reporter assay confirmed that miRNA-30e targeted 3'-UTR of the Notch gene. The role of miRNA-30e in regulating Notch signalling was then explored by up- and downregulating its expression in vitro and in vivo. RESULTS: Abnormal differentiation of IECs in DM mice was associated with reduced activity of the Dll4/NICD/Hes1 signal pathway. Based on bioinformatic analyses, increased expression of miRNA-30e was identified as a potential candidate for regulating Notch signalling. miRNA-30e targeted the 3'-UTR of Dll4 and downregulated Dll4 expression in primary IECs and IEC-6 cells. Exogenous miRNA-30e reduced activity of the Dll4/NICD/Hes1 pathway, and induced abnormal differentiation of IECs in normal mice. Conversely, treatment with miRNA-30e antagonist upregu-lated activity of the Dll4/NICD/Hes1 pathway in vivo, and normalized IEC differentiation in DM mice. CONCLUSIONS: Increased levels of miRNA-30e downregulated activity of the Dll4/NICD/Hes1 signalling pathway by targeting the 3'-UTR of Dll4, which contributed to abnormal differentiation in small intestinal epithelia of DM mice.