RESUMEN
Ascl2 has been shown to be involved in tumorigenesis in colorectal cancer (CRC), although its epigenetic regulatory mechanism is largely unknown. Here, we found that methylation of the Ascl2 promoter (bp -1670 â¼ -1139) was significantly increased compared to the other regions of the Ascl2 locus in CRC cells and was associated with elevated Ascl2 mRNA expression. Furthermore, we found that promoter methylation was predictive of CRC patient survival after analyzing DNA methylation data, RNA-Seq data, and clinical data of 410 CRC patient samples from the MethHC database, the MEXPRESS database, and the Cbioportal website. Using the established TET methylcytosine dioxygenase 2 (TET2) knockdown and ectopic TET2 catalytic domain-expression cell models, we performed glucosylated hydroxymethyl-sensitive quatitative PCR (qPCR), real-time PCR, and Western blot assays to further confirm that hypermethylation of the Ascl2 promoter, and elevated Ascl2 expression in CRC cells was partly due to the decreased expression of TET2. Furthermore, BCLAF1 was identified as a TET2 interactor in CRC cells by LC-MS/MS, coimmunoprecipitation, immunofluorescence colocalization, and proximity ligation assays. Subsequently, we found the TET2-BCLAF1 complex bound to multiple elements around CCGG sites at the Ascl2 promoter and further restrained its hypermethylation by inducing its hydroxymethylation using chromatin immunoprecipitation-qPCR and glucosylated hydroxymethyl-qPCR assays. Finally, we demonstrate that TET2-modulated Ascl2-targeted stem gene expression in CRC cells was independent of Wnt signaling. Taken together, our data suggest an additional option for inhibiting Ascl2 expression in CRC cells through TET2-BCLAF1-mediated promoter methylation, Ascl2-dependent self-renewal of CRC progenitor cells, and TET2-BCLAF1-related CRC progression.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias Colorrectales , Metilación de ADN , Dioxigenasas , Proteínas Represoras , Proteínas Supresoras de Tumor , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/genética , Dioxigenasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Espectrometría de Masas en Tándem , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
We have reported that Achaete scute-like 2 (Ascl2) transcriptionally repressed miR-200 family members and affected the epithelial-mesenchymal transition (EMT)-mesenchymal-epithelial transition (MET) plasticity in colorectal cancer (CRC) cells. However, little is known about the regulation of the Ascl2/miR-200 axis. Here, we found that hypoxia inducible factor-1α (HIF-1α) mRNA levels were positively correlated with Ascl2 mRNA levels and inversely correlated with miR-200b in CRC samples. Mechanistically, we showed that Ascl2 was a downstream target of HIF-1α and had a critical role in the EMT phenotype induced by hypoxia or HIF-1α over-expression. Hypoxia or HIF-1α over-expression activated Ascl2 expression in CRC cells in a direct transcriptional mechanism via binding with the hypoxia-response element (HRE) at the proximal Ascl2 promoter. HIF-1α-induced Ascl2 expression repressed miR-200b expression to induce EMT occurrence. Furthermore, we found HIF-1α was a direct target of miR-200b. MiR-200b bound with the 3'-UTR of HIF-1α in CRC cells. HIF-1α/Ascl2/miR-200b regulatory feedback circuit modulated the EMT-MET plasticity of CRC cells. Our results confirmed a novel HIF-1α/Ascl2/miR-200b regulatory feedback circuit in modulating EMT-MET plasticity of CRC cells, which could serve as a possible therapeutic target.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal/genética , Retroalimentación Fisiológica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , MicroARNs/fisiología , Línea Celular Tumoral , Plasticidad de la Célula/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Transducción de Señal/genética , Hipoxia Tumoral/genéticaRESUMEN
The myogenic regulatory factors (MRFs) and myocyte enhancer factor 2 (MEF2) transcription factors have been extensively studied as key transcription factors that regulate myogenic gene expression. However, few reports on the molecular mechanism that modulates chromatin remodeling during skeletal muscle differentiation are available. We reported here that the expression of the H3-K9 methyltransferase Suv39h1 was decreased during myoblast differentiation. Ectopic expression of Suv39h1 could inhibit myoblast differentiation, increasing H3-K9 methylation levels, whereas knockdown of Suv39h1 stimulated myoblast differentiation. Furthermore, Suv39h1 interacted with MEF2C directly and inhibited MEF2 transcription activity in a dose-dependent manner. Together, our studies revealed a molecular mechanism wherein Suv39h1 modulated myogenic gene expression and activation during skeletal muscle differentiation.
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Diferenciación Celular/fisiología , Metiltransferasas/metabolismo , Desarrollo de Músculos/fisiología , Mioblastos/citología , Mioblastos/metabolismo , Proteínas Represoras/metabolismo , Animales , Western Blotting , Diferenciación Celular/genética , Línea Celular , Inmunoprecipitación de Cromatina , Epigénesis Genética/genética , Inmunoprecipitación , Metiltransferasas/genética , Ratones , Desarrollo de Músculos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genéticaRESUMEN
Ascl2, a basic helix-loop-helix transcription factor, is a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. However, its involvement in colon cancer and downstream molecular events is largely undefined; in particular, the mechanism by which Ascl2 regulates the plasticity of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) programs in colon cancer cells remains unknown. In this study, we systematically demonstrate that Ascl2 loss of function in colon cancer cells promotes MET by derepressing the expression of microRNA (miR)-200s (i.e. miR-200b, miR-200a, miR-429, miR-200c, and miR-141) and further activating their expression through a transcriptional mechanism that involves direct binding to the most proximal E-box (E-box2) in the miR-200b-a-429 promoter. Activation of miR-200s due to Ascl2 deficiency led to the inhibition of ZEB1/2 expression and the alteration of epithelial and mesenchymal features. Transfection of miR-200b, miR-200a, and miR-429 inhibitors into Ascl2-deficient colon cancer cells promoted the epithelial-mesenchymal transition in a reversible manner. Transfection of miR-200a or miR-429 inhibitors into Ascl2-deficient colon cancer cells increased cellular proliferation and migration. Ascl2 mRNA levels and the miR-200a, miR-200b, miR-200c, miR-141, or miR-429 levels in the colon cancerous samples were inversely correlated. These results provide the first evidence of a link between Ascl2 and miR-200s in the regulation of EMT-MET plasticity in colon cancer.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Neoplasias del Colon/metabolismo , Transición Epitelial-Mesenquimal , MicroARNs/genética , Anciano , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HT29 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Metástasis Linfática , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Familia de Multigenes , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Myozenin-1 (Myoz1) gene-encoded calsarcin-2 protein was expressed exclusively in fast-twitch muscles. Peroxisome proliferator-activated receptor γ2 (PPAR-γ2) is a key regulator of adipocyte differentiation, fatty acid uptake and storage in mammals. In this study, transgenic (TG) mice were generated by injecting linearized DNA that contained mouse creatine kinase M-type enhancer, Myoz1 core promoter, swine PPAR-γ2 (sPPAR-γ2) and SV40 polyadenylation sequences into pronuclei of fertilized FVB/NJ mouse embryos using microinjection technology. Then, the TG mice were used to identify whether swine Myoz1 (sMyoz1) promoter could upregulate sPPAR-γ2 expression in skeletal muscle in a TG mouse model. The results showed that the sMyoz1 promoter indeed upregulated sPPAR-γ2 expression on both the RNA and protein levels. The target genes of PPAR-γ in fat formation pathways, such as fatty acid-binding protein 4 (FABP4) and lipoprotein lipase (LPL), were also overexpressed on the RNA level. Meanwhile, the level of skeletal muscle triacylglycerol in TG mice was increased (P < 0.05), and the result of Oil Red-O staining in the skeletal muscle sections also showed that the number of lipid droplets was significantly increased in TG mice compared to wild-type mice, which might improve the intramuscular fat (IMF) content. For pork, the quality was mostly influenced by the IMF; the identification of swine muscle-specific promoter, sMyoz1, could further serve to develop transgenic pigs with higher intramuscular fat contents and improve pork quality.
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Ratones Transgénicos , Proteínas Musculares/genética , Músculo Esquelético/fisiología , PPAR gamma/genética , Adipocitos/fisiología , Tejido Adiposo/fisiología , Animales , Femenino , Regulación de la Expresión Génica , Masculino , Regiones Promotoras Genéticas , Sus scrofa/genética , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND AND AIM: The roles of host glycosylation in interactions with EPEC and EHEC O157:H7 are largely unclear; this study examined whether O-glycans are involved in EPEC and EHEC O157:H7 adherence to HT-29 cells. METHODS: Bacterial adherence to the cultured cells was determined using the direct co-staining of adherent bacteria and host cells, the adherent bacteria plating, and/or the direct fluorescent observation of the adherent GFP-labeled bacteria. RESULTS: A comparison of the adherence of EPEC and EHEC O157:H7 to HT-29-Gal and HT-29 cells indicated that the differentiation of HT-29 cells led to a reduction in the adherence of EPEC and EHEC O157:H7. EPEC and EHEC O157:H7 adhesion decreased after the abrogation of O-glycan biosynthesis mediated by benzyl-α-GalNAc treatment. Core 2 O-glycan-deficient HT-29 cells induced by C2GnT2 knockdown had a significant reduction in EPEC and EHEC O157:H7 adhesion in C2GnT2-sh2/HT-29 cells compared with HT-29 and shRNA-Ctr/HT-29 cells. MUC2 expression in benzyl-α-GalNAc-treated HT-29 cells was significantly reduced but unchanged in C2GnT2-deficient HT-29 cells. EPEC or EHEC O157:H7 infection in C2GnT2-deficient HT-29 cells deteriorated the epithelial barrier function. The occludin expression in the shRNA-Ctr/HT-29 and C2GnT2-sh2/HT-29 cells after infection with EPEC or EHEC O157:H7 was pyknic and discontinuous at the cell surface compared with its continuous distribution of control cells. These data indicate that EPEC and EHEC O157:H7 adherence to HT-29 cells is related to mucin-type core 2 O-glycan. CONCLUSIONS: This study provides the concepts toward the design of carbohydrate-dependent inhibition of EPEC and EHEC O157:H7 adhesion to human intestinal epithelial cells.
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Adhesión Bacteriana/fisiología , Escherichia coli Enterohemorrágica/metabolismo , Escherichia coli Enteropatógena/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/farmacología , Anticuerpos , Compuestos de Bencilo/farmacología , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enteropatógena/genética , Células HT29 , Humanos , Mucina 2/genética , Mucina 2/metabolismo , N-Acetilglucosaminiltransferasas/genética , Interferencia de ARNRESUMEN
Yes-associated protein1 (YAP1), a downstream effector of the Hippo pathway, is over-expressed in several types of malignancies. We analyzed retrospectively the TCGA database using 447 colorectal cancer (CRC) samples to determine the correlation between YAP1 expression level and CRC patient prognosis. YAP1-enforced expressed CRC cell lines were constructed using the lentivirus particles containing a YAP1 insert. YAP1 was highly expressed in CRC cancerous tissues and is associated with distant metastasis of CRC patients. Kaplan - Meier analysis indicated that CRC patients with a higher YAP1 expression group (n = 104) had worse disease-free survival (DFS) and overall survival (OS) than lower YAP1 expression group (n = 343) (p = 0.008 and p = 0.022). Univariate and multivariate analysis indicated that the elevated YAP1 expression predicted the aggressive phenotype and was an independent indicator for OS and DFS of CRC patients. YAP1 over-expression in CRC cells enhanced their migration and invasion significantly which can be reversed by AXL, CTGF, or CYR61 interference. The study suggested that YAP1 affected the prognosis of CRC patients and controlled the abilities of invasion and migration of CRC cells via its target genes AXL, CTGF, and CYR61.
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Neoplasias Colorrectales , Proteínas Señalizadoras YAP , Humanos , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Fenotipo , Estudios RetrospectivosRESUMEN
The PD-1/PD-L1 pathway in mucosal immunity is currently actively explored and considered as a target for inflammatory bowel disease (IBD) treatment. However, systemic PD-L1 administration may cause unpredictable adverse effects due to immunosuppression. Here we show that reactive oxygen species (ROS)-responsive nanoparticles enhance the efficacy and safety of PD-L1 in a mouse colitis model. The nanoparticles control the accumulation and release of PD-L1 fused to Fc (PD-L1-Fc) at inflammatory sites in the colon. The nanotherapeutics shows superiority in alleviating inflammatory symptoms over systemic PD-L1-Fc administration and mitigates the adverse effects of PD-L1-Fc administration. The nanoparticles-formulated PD-L1-Fc affects production of proinflammatory and anti-inflammatory cytokines, attenuates the infiltration of macrophages, neutrophils, and dendritic cells, increases the frequencies of Treg, Th1 and Tfh cells, reshapes the gut microbiota composition; and increases short-chain fatty acid production. In summary, PD-L1-Fc-decorated nanoparticles may provide an effective and safe strategy for the targeted treatment of IBD.
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Colitis , Enfermedades Inflamatorias del Intestino , Ratones , Animales , Antígeno B7-H1/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Citocinas/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Macrófagos/metabolismo , Modelos Animales de EnfermedadRESUMEN
The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.
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Carcinogénesis , Centro Organizador de los Microtúbulos , Animales , Ratones , Cuerpos Basales , Diferenciación Celular , Transformación Celular Neoplásica , HiperplasiaRESUMEN
OBJECTIVE: To evaluate the effect of ultrasound interventional injection of cisplatin in the treatment of hepatocellular carcinoma (HCC). METHODS: 68 patients with HCC admitted to our hospital from February 2016 to February 2018 were enrolled. According to the different treatment methods, they were divided into a study group and a control group. The control group was treated with hepatic artery embolization chemotherapy (n=34), the study group adopted interventional ultrasound injection of cisplatin (n=34). The clinical treatment effects of the two groups of patients were compared; the liver function and the occurrence of adverse reactions were compared; all patients were followed up for 24 months, and their progressive-free survival (PFS) was also compared. RESULTS: The study group had higher total effective rate compared with the control group (91.18% vs 67.65%) (P<0.05); the AST, ALT, T-BIL and D-BIL levels of the two groups decreased remarkably after treatment, and the reduction in the study group was more obvious (P<0.05); the levels of VEGF, AFP and CEA of the two groups reduced noticeably after treatment, and the decrease in the study group was more significant (P<0.05); the total incidence of adverse reactions in the study group and the control group was 2.94% and 23.53% respectively, (P<0.05); the two groups were followed up for 24 months, and the disease control rate (DCR) of the control group was 58.82%, significantly lower than 85.29% in the study group; the PFS time of the control group was (15.68±4.23) lower than that of the study group (18.12±5.42) (P<0.05). CONCLUSION: Interventional ultrasound injection of cisplatin in the treatment of HCC has a definite effect. It can effectively relieve liver damage, reduce adverse reactions, improve serum tumor marker levels, and boost the DCR and PFS time of tumor patients.
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Brassinosteroids (BRs) are a group of plant steroid hormones that regulate many important agronomic traits. Studies on the functional mechanisms of BR-related genes in crop plants are necessary for the application of BRs in agriculture. In this study, ZmD11, an ortholog of rice DWARF11 (D11), and 42 other BR biosynthesis-related genes were identified in maize (Zea mays). Complementary experiments confirmed that ZmD11 completely rescued the abnormal panicle architecture and plant height of the rice cpb1 mutant. A phylogenetic analysis indicated that ZmD11-like proteins were found in other monocots and dicots, but not in lower plants and that alternative splicing variants of these homologues mainly exist in Triticeae crops. A subcellular localization analysis showed that ZmD11 localized to the endoplasmic reticulum. The ZmD11 gene was predominantly expressed in young ears and seeds from 10 to 16 days after pollination, especially in the scutellar aleurone layer and pericarp. Furthermore, the constitutive expression of the ZmD11 gene significantly increased seed length, seed area, seed weight and both seed starch and protein contents in rice and maize. Our results suggest that ZmD11 is a key gene in the regulation of seed size and quality and that it has a potential application value in the molecular breeding of crops.
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Brasinoesteroides/biosíntesis , Oryza/genética , Proteínas de Plantas/genética , Semillas/crecimiento & desarrollo , Zea mays/genética , Empalme Alternativo , Regulación de la Expresión Génica de las Plantas , Oryza/fisiología , Filogenia , Proteínas de Plantas/fisiología , Semillas/genética , Zea mays/fisiologíaRESUMEN
HYPOTHESIS: Recently, molybdenum disulfide (MoS2) as a new type of catalyst, has been used for hydrogen evolution. In order to overcome the shortcomings of MoS2, such as poor conductivity and lack of catalytic activity sites, various materials were added to prepare composites, such as graphene with good conductivity. EXPERIMENTS: In this work, we combined MoS2 with graphene through a very simple liquid-phase salt-assisted co-exfoliation method. The obtained MoS2/graphene nano-composites have been further applied to electrocatalyst of hydrogen evolution reaction. The influences of different ratio of MoS2 to graphene and different solvents of exfoliation on the electrocatalytic activity for the hydrogen evolution reaction have been investigated in detail. FINDINGS: In general, the obtained MoS2/graphene nano-catalysts exhibits the smallest Tafel slope of 61â¯mV/dec with the exfoliation solvent of isopropanol, and 65â¯mV/dec in N-methyl pyrrolidone. All the MoS2/graphene composites exhibit much better catalytic activity than either MoS2 or graphene single substance due to the synergetic effect between MoS2 and graphene nanosheets.
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The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and cancer stem cells (CSCs). The transcription factor Ascl2 (Wnt target gene) is fate decider of intestinal cryptic stem cells and colon cancer stem cells. It is unclear how Wnt signaling is translated into Ascl2 expression and keeping the self-renewal of CRC progenitor cells. We showed that the exogenous Ascl2 in colorectal cancer (CRC) cells activated the endogenous Ascl2 expression via a direct autoactivatory loop, including Ascl2 binding to its own promoter and further transcriptional activation. Higher Ascl2 expression in human CRC cancerous tissues led to greater enrichment in Ascl2 immunoprecipitated DNA within the Ascl2 promoter in the CRC cancerous sample than the peri-cancerous mucosa. Ascl2 binding to its own promoter and inducing further transcriptional activation of the Ascl2 gene was predominant in the CD133+CD44+ CRC population. R-spondin1/Wnt activated Ascl2 expression dose-dependently in the CD133+CD44+ CRC population, but not in the CD133-CD44- CRC population, which was caused by differences in Ascl2 autoregulation under R-spondin1/Wnt activation. R-spondin1/Wnt treatment in the CD133+CD44+ or CRC CD133-CD44- populations exerted a different pattern of stemness maintenance, which was defined by alterations of the mRNA levels of stemness-associated genes, the protein expression levels (Bmi1, C-myc, Oct-4 and Nanog) and tumorsphere formation. The results indicated that Ascl2 autoregulation formed a transcriptional switch that was enhanced by Wnt signaling in the CD133+CD44+ CRC population, thus conferring their self-renewal.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Autorrenovación de las Células , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Homeostasis , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Antígenos CD/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Trombospondinas/metabolismo , Transcripción Genética , Activación Transcripcional/genéticaRESUMEN
In this paper, a graphene/nickel-cobalt hydroxide ternary hydrogel (G-Ni-Co) with superior electrochemical performances was prepared by a simple hydrothermal method using Ni(NO3)2·6H2O, Co(NO3)2·6H2O, and graphene oxide as the starting materials. The mass fraction and the pH value of the reaction system were optimized. The prepared G-Ni-Co was assembled into a symmetric supercapacitor and its electrochemical performance was estimated. In a symmetric supercapacitor, the specific capacitance of G-Ni-Co is 551.3â¯Fâ¯g-1 at the scan rate of 10â¯mVâ¯s-1 and 646.1â¯Fâ¯g-1 at the current density of 0.5â¯Aâ¯g-1, respectively. The specific capacitance still retains 70.8% after 5000 cycles at the scan rate of 100â¯mVâ¯s-1. The energy density reaches 108.6â¯Wâ¯hâ¯kg-1 at a power density of 550.0â¯Wâ¯kg-1 and remains 72.4â¯Wâ¯hâ¯kg-1 at 7600.0â¯Wâ¯kg-1, respectively.
RESUMEN
Achaete scute-like 2 (Ascl2) is the Wnt signaling target, its regulation by other signaling is undefined. Now we demonstrated that CD133+/CD44+ cell population from HT-29 or Caco-2 cells exhibited cancer stem cell (CSC) properties with highly expressed Ascl2, which is related to the Hippo signaling pathway. YAP1 interference in CD133+/CD44+ HT-29 or Caco-2 cells reduced their proliferation, colony-forming ability and tumorsphere formation in vitro and inhibited the 'stemness'-associated genes and Ascl2 expression. Enforcing YAP1 expression in HT-29 or Caco-2 cells triggered the opposite changes. Ascl2 interference reversed the phenotype of YAP1-enforced expressed HT-29 or Caco-2 cells. Krüppel-like factor 5 (KLF5) protein, not KLF5 mRNA levels, were increased due to YAP1 overexpression which is reported to prevent KLF5 degradation. Co-immunoprecipitation (Co-IP) assays demonstrated that YAP1 bound with KLF5 in HT-29 and Caco-2 cells. Luciferase and chromatin immunoprecipitation (ChIP) assays indicated that both YAP1 and KLF5 bound to the first two loci with GC-boxes in Ascl2 promoter and induced Ascl2 transcription. The decreased Ascl2 transcription by YAP1 interference required an intact KLF5 binding site (GC-box) within Ascl2 promoter, KLF5 knockdown reduced YAP1 binding and Ascl2 luciferase reporter activity upon YAP1 overexpression. Positive correlation among YAP1 and Ascl2 mRNA levels was observed in colorectal cancer (CRC) samples. Thus, our study demonstrated that Ascl2, a fate decider of CRC progenitor cells can be activated by the Hippo signaling pathway in CRC progenitor cells, and ensured their self-renewability.
RESUMEN
Nanog is an important embryonic stem cell (ESC) gene that does not function as a classical oncogene, but needs to cooperate with other molecules to potentiate tumorigenic activity. The question addressed by the present study was whether a miRNA link exists between Nanog and epithelial-mesenchymal transition (EMT)-mesenchymal-epithelial transition (MET) plasticity. Here, we found that Nanog mRNA expression level was inversely correlated with miR-200c and miR-200b expression levels in colon cancer cell lines and human colorectal cancer tissues. Forced Nanog expression in low-Nanog colon cancer cells inhibited miR-200c and miR-200b expression, and interfered Nanog expression in high-Nanog colon cancer cells promoted miR-200c and miR-200b expression. Furthermore, we confirmed that Nanog directly repressed transcription of the miR-200c and miR-200b genes, and miR-200c and miR-200b mediated Nanog-induced EMT occurrence. Luciferase and ChIP assays determined that Nanog bound directly to the potential Nanog binding sites in the miR-200c and miR-200b promoters and repressed their transcription. In conclusion, our findings suggest that Nanog modulates EMT-MET plasticity by regulating miR-200 clusters via a direct transcriptional mechanism, and the Nanog-miR-200 axis may be a good therapeutic target for CRC control.
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Neoplasias del Colon/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteína Homeótica Nanog/genética , Transcripción Genética , Animales , Sitios de Unión , Células CACO-2 , Proliferación Celular , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Regulación hacia Abajo , Células HCT116 , Células HT29 , Humanos , Ratones Desnudos , MicroARNs/metabolismo , Proteína Homeótica Nanog/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
BACKGROUND: How host cell glycosylation affects EPEC or EHEC O157:H7 invasion is unclear. This study investigated whether and how O-glycans were involved in EPEC or EHEC O157:H7 invasion into HT-29 cells. RESULTS: Lectin histochemical staining confirmed stronger staining with PNA, which labeled Galß1, 3 GalNAc (core 1 structure) in HT-29-Gal-OBN and C2GnT2-sh2/HT-29 cells, compared with control cells. EPEC or EHEC O157:H7 invasion into HT-29 and its derived cells was based on the intracellular presence of GFP-labeled bacteria. The differentiation of HT-29 cells led to a reduction in EPEC internalization compared with HT-29 cells (p < 0.01). EPEC or EHEC O157:H7 invasion into HT-29-OBN and HT-29-Gal-OBN cells increased compared with HT-29 and HT-29-Gal cells (p < 0.05 and p < 0.01). Core 2 O-glycan-deficient HT-29 cells underwent a significant increase in EPEC (p < 0.01) or EHEC O157:H7 (p < 0.05) invasion compared with control cells. METHODS: Bacterial invasion into cultured cells was determined by a gentamicin protection assay and a GFP-labeled bacteria invasion assay. O-glycans biosynthesis was inhibited by benzyl-α-GalNAc, and core 2 O-glycan-deficient HT-29 cells were induced by C2GnT2 interference. CONCLUSION: These data indicated that EPEC or EHEC O157:H7 invasion into HT-29 cells was related to their O-glycosylation status. This study provided the first evidence of carbohydrate-dependent EPEC or EHEC O157:H7 invasion into host cells.
RESUMEN
The role of Achaete scute-like 2 (Ascl2) in colorectal cancer (CRC) cell differentiation is unknown. LS174T, HT-29 and Caco-2 cells have high Ascl2 expression, while Lovo and SW480 cells have low Ascl2 expression. LS174T and HT-29 cells with Ascl2 knockdown were transfected with caudal type homeobox 2 (CDX2) promoter constructs and used for luciferase assays and chromatin immunoprecipitation (ChIP) assays. Ascl2 knockdown promoted differentiation of CRC cells into a goblet cell phenotype, as determined by increased expression of MUC2, TFF3, and CDX2. Ascl2 knockdown activated CDX2 expression through a transcriptional mechanism via direct binding of Ascl2 to the proximal E-box of the CDX2 promoter. Ascl2 over-expression in Lovo and SW480 cells inhibited a goblet cell phenotype, as determined by reduced CDX2 and MUC2 expression. Inverse correlations between Ascl2 and CDX2, and Ascl2 and MUC2 mRNA levels, as well as Ascl2 and CDX2 protein levels were observed in CRC cancerous samples. This study demonstrates CDX2 repression by Ascl2 and highlights a role for Ascl2 in CRC cell differentiation. These findings suggest that the Ascl2/CDX2 axis may serve as a potential therapeutic target in colorectal cancer.