RESUMEN
Methylation of HBV cccDNA has been detected in vivo and in vitro; however, the mechanism and its effects on HBV replication remain unclear. HBx derived from a 1.2-mer HBV replicon upregulated protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), 3a, and 3b, resulting in methylation of the negative regulatory region (NRE) in cccDNA, while none of these effects were observed with an HBx-null mutant. The HBx-positive HBV cccDNA expressed higher levels of HBc and produced about 4-fold higher levels of HBV particles than those from the HBx-null counterpart. For these effects, HBx interrupted the action of NRE binding protein via methylation of the C-1619 within NRE, resulting in activation of the core promoter. Treatment with 5-Aza-2'dC or DNMT1 knock-down drastically impaired the ability of HBx to activate the core promoter and stimulate HBV replication in 1.2-mer HBV replicon and in vitro infection systems, indicating the positive role of HBx-mediated cccDNA methylation in HBV replication.
Asunto(s)
Metilación de ADN/genética , ADN Circular/metabolismo , Transactivadores/metabolismo , Replicación Viral/fisiología , Sitios de Unión , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas , Proteínas de Unión al ADN/metabolismo , Genoma Viral , Células Hep G2 , Virus de la Hepatitis B/genética , Humanos , Regiones Promotoras Genéticas , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Allele frequencies for 23 autosomal short tandem repeat loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, TH01, FGA, D5S818, D13S317, D7S820, D2S441, D19S433, D22S1045, D10S1248, D1S1656, D12S391, D2S1338, SE33, Penta D, Penta E), 1 Y-chromosome short tandem repeat locus (DYS391) and Y indel were obtained from 1000 unrelated individuals of the Korean population.
Asunto(s)
Dermatoglifia del ADN/métodos , Genes Ligados a Y/genética , Sitios Genéticos/genética , Genética de Población , Mutación INDEL/genética , Repeticiones de Microsatélite/genética , Femenino , Humanos , Masculino , República de Corea/etnologíaRESUMEN
Hippocampal synaptic function and plasticity deteriorate with age, often resulting in learning and memory deficits. As MicroRNAs (miRNAs) are important regulators of neuronal protein expression, we examined whether miRNAs may contribute to this age-associated decline in hippocampal function. We first compared the small RNA transcriptome of hippocampal tissues from young and old mice. Among 269 hippocampal miRNAs, 80 were differentially expressed (≥ twofold) among the age groups. We focused on 36 miRNAs upregulated in the old mice compared with those in the young mice. The potential targets of these 36 miRNAs included 11 critical Eph/Ephrin synaptic signaling components. The expression levels of several genes in the Eph/Ephrin pathway, including EphB2, were significantly downregulated in the aged hippocampus. EphB2 is a known regulator of synaptic plasticity in hippocampal neurons, in part by regulating the surface expression of the NMDA receptor NR1 subunit. We found that EphB2 is a direct target of miR-204 among miRNAs that were upregulated with age. The transfection of primary hippocampal neurons with a miR-204 mimic suppressed both EphB2 mRNA and protein expression and reduced the surface expression of NR1. Transfection of miR-204 also decreased the total expression of NR1. miR-204 induces senescence-like phenotype in fully matured neurons as evidenced by an increase in p16-positive cells. We suggest that aging is accompanied by the upregulation of miR-204 in the hippocampus, which downregulates EphB2 and results in reduced surface and total NR1 expression. This mechanism may contribute to age-associated decline in hippocampal synaptic plasticity and the related cognitive functions.
Asunto(s)
Envejecimiento/metabolismo , Hipocampo/metabolismo , MicroARNs/metabolismo , Neuronas/metabolismo , Receptor EphB2/metabolismo , Envejecimiento/genética , Animales , Regulación hacia Abajo , Hipocampo/citología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/biosíntesis , MicroARNs/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal , Neuronas/citología , Receptor EphB2/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de SeñalRESUMEN
We here report a simple assay system for DNA methyltransferase (DNMT) inhibitors based on the HBx-induced DNA methylation of E-cadherin. A stable cell line named G1 was generated by co-transfecting E-cadherin luciferase reporter and HBx-expression plasmid into HepG2 cells. Treatment of G1 cells with DNMT inhibitors, 5-azacytidine, 5-aza-2'-deoxycytidine, and procainamaid, dose-dependently inhibited DNA methylation of E-cadherin promoter in the reporter, resulting in up-regulation of luciferase levels and its enzyme activity. Treatment with all-trans retinoic acid that is known to inhibit DNMT expression, also induced similar effects. Our system can be useful for development of epi-drugs targeting DNA methylation in malignancies.
Asunto(s)
Cadherinas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Metilación de ADN/efectos de los fármacos , Inhibidores Enzimáticos/análisis , Transactivadores/metabolismo , Cadherinas/biosíntesis , Regulación hacia Abajo , Células Hep G2 , Humanos , Luciferasas/genética , Regiones Promotoras Genéticas , Transfección , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Aberrant promoter methylation of tumor suppressor genes including retinoic acid receptor-ß2 (RAR-ß2) is frequently detected in hepatitis C virus (HCV)-associated hepatocellular carcinoma; however, the mechanism and its significance are relatively unknown. Here, we showed that HCV Core induced promoter hypermethylation of RAR-ß2 to inhibit its expression via up-regulation of DNA methyltransferases 1 and 3b. Under the condition, all-trans retinoic acid (ATRA) failed to activate p16 expression and thus could not inactivate the Rb-E2F pathway. Accordingly, Core-expressing cells exhibited resistance to ATRA-induced growth inhibition. Taken together, HCV Core antagonizes ATRA, a natural anti-cancer compound, to stimulate cell growth via epigenetic down-regulation of RAR-ß2.
Asunto(s)
Metilación de ADN , Antígenos de la Hepatitis C/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/farmacología , Proteínas del Núcleo Viral/metabolismo , Carcinoma Hepatocelular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Hepacivirus , Humanos , Neoplasias Hepáticas , Metiltransferasas/biosíntesis , Proteínas de Neoplasias/biosíntesis , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Ácido Retinoico/genéticaRESUMEN
Nasopharyngeal carcinoma is closely associated with infection with Epstein-Barr Virus (EBV); however, the mechanism is still unclear. Here, we report that the EBV oncoprotein, latent membrane protein 1 (LMP1), suppresses apoptotic cell death provoked by all-trans retinoic acid (ATRA) in NPC cells. For this purpose, LMP1 downregulated levels of Bak whilst it upregulated levels of Bcl2, lowering the ratio of Bak to Bcl2. In addition, LMP1 suppressed ATRA-mediated activation of Caspase 9, Caspase 3, and PARP but not Caspase 8 in Ad-AH cells, suggesting that LMP1 acts by blocking the activation of intrinsic apoptosis pathway by ATRA. These effects were almost completely abolished when levels of retinoic acid receptor-ß(2) (RAR-ß(2)) in the LMP1-expressing cells were recovered by either exogenous gene expression or treatment with a universal DNMT inhibitor, 5-Aza-2'dC, indicating that LMP1 executes its antiapoptotic effects by downregulating levels of RAR-ß(2) via DNA methylation.
