RESUMEN
BACKGROUND: Alterations in DNA methylation occur during the pathogenesis of human tumors. In this study, we investigated the influence of DNA methyltransferase 3b (DNMT3b) on fragile histidine trial (FHIT) expression and on DNA methylation of the FHIT promoter region in the hepatoma cell line SMMC-7721. METHODS: DNMT3b siRNA was used to down-regulate DNMT3b expression. DNMT3b and FHIT proteins were determined by Western blotting. Methylation-specific PCR was used to analyze the methylation status of the FHIT gene. RESULTS: After DNMT3b siRNA transfection, the expression of DNMT3b was inhibited in SMMC-7721 cells, and the expression of FHIT was significantly higher than that in the control group. There was no significant difference in methylation status between the DNMT3b siRNA transfected cells and control cells. CONCLUSION: DNMT3b may play an important role in regulation of FHIT expression in hepatoma SMMC-7721 cells, but not through methylation of the FHIT promoter.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo , Carcinoma Hepatocelular/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/fisiopatología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasas/genética , Regulación hacia Abajo , Silenciador del Gen , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatología , Reacción en Cadena de la Polimerasa/métodos , ARN Interferente Pequeño/farmacología , Transfección , ADN Metiltransferasa 3BRESUMEN
OBJECTIVE: To investigate the influence of DNA methyltransferase (DNMT) 3b on the expression of cyclin D1 gene and methylation of its promoters and to investigate the function of DNMT3b. METHODS: Human hepatocellular carcinoma cells of the line SMMC7721 were cultured and randomly divided into 3 groups: experimental group transfected with siRNA to silence the DNMT3b, control group transfected with control siRNA, and normal group without transfection. The transfection rate of siRNA was detected by fluorescence microscopy. MTT method was used to measure the survival rate of the SMMC-7721 cells. Western blotting and cell proliferation assay were performed to evaluate the expression of cyclin D1 and cell growth. Methylation specific PCR (MSP) was performed to investigate whether the promoter of cyclin D1 was methylated. RESULTS: Fluorescence microscopy showed that the transfection rate of siRNA was over 90%. MTT method showed that 24 h and 36 h after transfection the A value and survival rate of the SMMC7721 cells of the experimental group were both significantly higher than those of the control d normal groups (all P < 0.05). Western blotting showed that the expression levels of DNMT3b and cyclin D1 of the experimental group decreased significantly compared with the control and the normal groups. MSP showed no obvious change of the state of methylation among the 3 groups. CONCLUSIONS: DNMT3b may regulate the expression and the function of cyclin D1 gene in the human hepatocellular carcinoma cells, but does not change its methylation state. DNMT3b may play their role as a signal transduction element rather than as a DNA methyltransferase.
Asunto(s)
Ciclina D1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Ciclina D1/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Transfección , ADN Metiltransferasa 3BRESUMEN
This study aimed to investigate the role and underlying mechanism of exosomes secreted by oxidized low-density lipoprotein (oxLDL)-stimulated macrophages in the progression of atherosclerosis (AS). Exosomes from peripheral blood of AS patients or oxLDL-treated macrophages were co-cultured with human neutrophils. Neutrophil extracellular traps (NETs) were detected by immunofluorescence staining. The levels of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). The expression levels of miR-146a and superoxide dismutase 2 (SOD2) were determined by quantitative real-time PCR (qRT-PCR) and western blot. The generation of intracellular reactive oxygen species (ROS) was observed by using dichlorofluorescin diacetate (DCFH-DA). ApoE-deficient mice were fed with high-fat diet (HFD) to induce AS. Atherosclerotic plaques were evaluated by Oil red O (ORO) and hematoxylin-eosin (HE) staining. Our results showed that miRNA-146a was enriched in serum-derived exosomes of AS patients and oxLDL-treated macrophage THP-1-derived exosomes. Importantly, exosomal miR-146a secreted by oxLDL-treated macrophages promoted ROS and NETs release via targeting SOD2. In addition, intravenous administration of oxLDL-treated THP-1 cells-derived exosomes into AS mice significantly deteriorated AS in vivo. Our findings indicate that exosomal miR-146a derived from oxLDL-treated macrophages promotes NETs formation via inducing oxidative stress, which might provide a novel scientific basis for the understanding of AS progression.
Asunto(s)
Aterosclerosis/sangre , Exosomas/metabolismo , Trampas Extracelulares/metabolismo , Lipoproteínas LDL/farmacología , Macrófagos/metabolismo , Neutrófilos/metabolismo , Anciano , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/metabolismo , Técnicas de Cocultivo , Citocinas/metabolismo , Progresión de la Enfermedad , Exosomas/ultraestructura , Trampas Extracelulares/efectos de los fármacos , Femenino , Humanos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Placa Aterosclerótica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Surgical site infection (SSI) is an important component of infections acquired from hospital. The most significant feature of vascular surgery different from other surgeries is frequent application of artificial grafts. Once SSI occurs after vascular operations with grafts, it might results in a serious disaster. Staphylococcus aureus and coagulase-negative Staphylococcus are the most common pathogenic bacteria for SSI after vascular surgery. Although SSI in vascular surgery often lacks of typical clinical characters, some clinical symptoms, laboratory data and certain imaging procedures may help to diagnose. In most cases of SSI after vascular procedures, the artificial grafts must be removed and sensitive antibiotics should be administered. However, for different cases, personalized management plan should be made depending on the severity and location of SSI.