RESUMO
Large studies in humans and animals have demonstrated a clear association of an adverse intrauterine environment with an increased risk of cardiovascular disease later in life. Yet mechanisms remain largely elusive. The present study tested the hypothesis that gestational hypoxia leads to promoter hypermethylation and epigenetic repression of the glucocorticoid receptor (GR) gene in the developing heart, resulting in increased heart susceptibility to ischemia and reperfusion injury in offspring. Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in a significant decrease of GR exon 14, 15, 16, and 17 transcripts, leading to down-regulation of GR mRNA and protein in the fetal heart. Functional cAMP-response elements (CREs) at -4408 and -3896 and Sp1 binding sites at -3425 and -3034 were identified at GR untranslated exon 1 promoters. Hypoxia significantly increased CpG methylation at the CREs and Sp1 binding sites and decreased transcription factor binding to GR exon 1 promoter, accounting for the repression of the GR gene in the developing heart. Of importance, treatment of newborn pups with 5-aza-2'-deoxycytidine reversed hypoxia-induced promoter methylation, restored GR expression and prevented hypoxia-mediated increase in ischemia and reperfusion injury of the heart in offspring. The findings demonstrate a novel mechanism of epigenetic repression of the GR gene in fetal stress-mediated programming of ischemic-sensitive phenotype in the heart.
Assuntos
Epigênese Genética , Hipóxia/genética , Traumatismo por Reperfusão Miocárdica/genética , Oxigênio/farmacologia , Receptores de Glucocorticoides/genética , Fator de Transcrição Sp1/genética , Animais , Animais Recém-Nascidos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sítios de Ligação , Metilação de DNA/efeitos dos fármacos , Decitabina , Éxons , Feminino , Hipóxia/tratamento farmacológico , Hipóxia/metabolismo , Hipóxia/patologia , Masculino , Exposição Materna , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fenótipo , Gravidez , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Fator de Transcrição Sp1/metabolismoRESUMO
Perinatal hypoxic-ischemic encephalopathy (HIE) is associated with high neonatal mortality and severe long-term neurologic morbidity. Yet the mechanisms of brain injury in infants with HIE remain largely elusive. The present study determined a novel mechanism of microRNA-210 (miR-210) in silencing endogenous neuroprotection and increasing hypoxic-ischemic brain injury in neonatal rats. The study further revealed a potential therapeutic effect of miR-210 inhibition using complementary locked nucleic acid oligonucleotides (miR-210-LNA) in 10-day-old neonatal rats in the Rice-Vannucci model. The underlying mechanisms were investigated with intracerebroventricular injection (i.c.v) of miR-210 mimic, miR-210-LNA, glucocorticoid receptor (GR) agonist and antagonist. Luciferase reporter gene assay was conducted for identification of miR-210 targeting GR 3'untranslated region. The results showed that the HI treatment significantly increased miR-210 levels in the brain, and miR-210 mimic significantly decreased GR protein abundance and exacerbated HI brain injury in the pups. MiR-210-LNA administration via i.c.v. 4h after the HI insult significantly decreased brain miR-210 levels, increased GR protein abundance, reduced HI-induced neuronal death and brain infarct size, and improved long-term neurological function recovery. Of importance, the intranasal delivery of miR-210-LNA 4h after the HI insult produced similar effects in decreasing HI-induced neonatal brain injury and improving neurological function later in life. Altogether, the present study provides evidence of a novel mechanism of miR-210 in a neonatal HI brain injury model, and suggests a potential therapeutic approach of miR-210 inhibition in the treatment of neonatal HIE.
Assuntos
Encéfalo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/prevenção & controle , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Oligonucleotídeos/administração & dosagem , Regiões 3' não Traduzidas , Animais , Animais Recém-Nascidos , Ratos , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMO
Gestational hypoxia is a common stress to the fetal development and increases the risk of neonatal morbidity. The present study tested the hypothesis that fetal hypoxia results in heightened brain vulnerability to hypoxic-ischemic (HI) injury in neonatal rats via down-regulation of glucocorticoid receptor (GR) in the developing brain. Time-dated pregnant rats were exposed to hypoxia (10.5% O2) from days 15 to 21 of gestation. Brain HI injury was determined in day 10 pups. Maternal hypoxia resulted in asymmetric intrauterine growth restriction in the fetus. The brain HI injury was significantly increased in maternal hypoxia-treated pups as compared with the normoxia control in both males and females. Activation of brain GR by dexamethasone injection into the right lateral ventricle produced a concentration-dependent reduction of HI-induced brain injury in control pups. Maternal hypoxia significantly decreased GR mRNA and protein abundance in the fetal brain and neonatal hippocampus and abolished the dexamethasone-mediated neuroprotective effect in pup brains. This decreased GR expression was resulted from increased DNA methylation, decreased binding of transcription factors Egr-1 and Sp1 to GR gene exon 17 and 111 promoters, and reduced expression of GR exon 17 and 111 mRNA variants. The results demonstrate that gestational hypoxia causes epigenetic repression of GR gene expression in the developing brain resulting in the heightened brain vulnerability to HI injury in neonatal rats.
Assuntos
Hipóxia Fetal/complicações , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipóxia-Isquemia Encefálica/etiologia , Hipóxia-Isquemia Encefálica/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Peso Corporal , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Infarto Encefálico/etiologia , Infarto Encefálico/patologia , Metilação de DNA , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Hipóxia Fetal/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucocorticoides/uso terapêutico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/genética , Fatores SexuaisRESUMO
Adverse environments during the fetal and neonatal development period may permanently program physiology and metabolism, and lead to increased risk of diseases in later life. Programming of the hypothalamic-pituitary-adrenal (HPA) axis is one of the key mechanisms that contribute to altered metabolism and response to stress. Programming of the HPA axis often involves epigenetic modification of the glucocorticoid receptor (GR) gene promoter, which influences tissue-specific GR expression patterns and response to stimuli. This review summarizes the current state of research on the HPA axis and programming of health and disease in the adult, focusing on the epigenetic regulation of GR gene expression patterns in response to fetal and neonatal stress. Aberrant GR gene expression patterns in the developing brain may have a significant negative impact on protection of the immature brain against hypoxic-ischemic encephalopathy in the critical period of development during and immediately after birth.
Assuntos
Epigênese Genética/fisiologia , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Estresse Fisiológico/fisiologia , Animais , Epigênese Genética/genética , Humanos , Sistema Hipotálamo-Hipofisário/embriologia , Sistema Hipotálamo-Hipofisário/crescimento & desenvolvimento , Sistema Hipófise-Suprarrenal/embriologia , Sistema Hipófise-Suprarrenal/crescimento & desenvolvimento , Receptores de Glucocorticoides/genéticaRESUMO
Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Transportador de Glucose Tipo 3/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Apoptose/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Técnicas de Silenciamento de Genes , Transportador de Glucose Tipo 3/metabolismo , Humanos , Hibridização In Situ , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Proteínas de Peixe-Zebra/metabolismoRESUMO
Fetal hypoxia causes protein kinase Cε (PKCε) gene repression in the heart resulting in heightened ischemic injury in male offspring in a sex-dependent manner. The present study tested the hypothesis that heightened methylation of the early growth response factor-1 (Egr-1) binding site at PKCε gene promoter contributes to sex dimorphism of hypoxia-induced programming of PKCε gene repression in the developing heart. Pregnant rats were divided into normoxic and hypoxic (10.5% O2 from day 15 to 21 of gestation) groups. Hypoxia selectively decreased PKCε mRNA and protein abundance in the heart of male, but not female, near-term (21 days) fetuses. Methylation of the CpG site at the Egr-1 binding site of PKCε promoter was significantly increased in the male hearts by hypoxia, resulting in decreased Egr-1 binding affinity and reduced Egr-1 binding to the PKCε promoter. Nuclear Egr-1 levels were not affected by hypoxia. There was significantly higher abundance of estrogen receptor α (ERα) and ß (ERß) isoforms in female than in male fetal hearts, which were not significantly altered by hypoxia. Both ERα and ERß bind to the Egr-1 binding site with significant greater levels in the female fetal hearts. The increased methylation with reduced Egr-1 binding and PKCε gene repression persisted in 3-mo-old adult male hearts in a sex-dependent manner. The results indicate a key role for heightened methylation of the Egr-1 binding site in hypoxia-mediated programming of PKCε gene repression in the developing heart and suggest a novel protective mechanism of ER by binding to the Egr-1 binding site in epigenetic regulation of PKCε gene expression patterns in the early developmental stage.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Epigênese Genética/fisiologia , Hipóxia Fetal/genética , Coração/crescimento & desenvolvimento , Miocárdio/enzimologia , Proteína Quinase C-épsilon/genética , Animais , Animais Recém-Nascidos , Antimetabólitos/farmacologia , Azacitidina/farmacologia , Sítios de Ligação , Western Blotting , Imunoprecipitação da Cromatina , Metilação de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Epigênese Genética/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Caracteres SexuaisRESUMO
Maternal hypoxia inhibits cardiomyocyte proliferation in the heart of fetal and neonatal rats. The present study tested the hypothesis that hypoxia has a direct effect inhibiting cardiomyocyte proliferation via upregulating tissue inhibitors of metalloproteinases (TIMP) in fetal rat hearts. Isolated fetal rat hearts and rat embryonic ventricular myocyte H9c2 cells were treated ex vivo with 20% or 1% O(2) for 48 or 24 h, respectively. Hypoxia caused a significant reduction in cardiomyocyte Ki-67 expression and bromodeoxyuridine incorporation in fetal hearts and H9c2 cells. In both fetal hearts and H9c2 cells, hypoxia resulted in a significant decrease in a cell division marker cyclin D2 but an increase in a cell division inhibitor p27. Additionally, hypoxia caused an upregulation of TIMP-3 and TIMP-4 in fetal hearts and H9c2 cells. Knockdown of TIMP-3 in H9c2 cells significantly increased cyclin D2 and Ki-67 and partially blocked the hypoxia-induced inhibition of cyclin D2 and Ki-67 in H9c2 cells. Unlike TIMP-3, TIMP-4 knockdown had no significant effects on the basal levels of cell proliferation but completely abrogated the hypoxia-mediated effects. These findings provide evidence of a novel causal role of TIMP-4 and TIMP-3 in the direct inhibitory effect of hypoxia on cardiomyocyte proliferation in the developing heart.
Assuntos
Proliferação de Células , Coração Fetal/fisiologia , Hipóxia Fetal/patologia , Miócitos Cardíacos/fisiologia , Inibidores Teciduais de Metaloproteinases/fisiologia , Animais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Linhagem Celular , Tamanho Celular , Ciclina D2/biossíntese , Ciclina D2/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Feminino , Imunofluorescência , Antígeno Ki-67/biossíntese , Gravidez , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Inibidores Teciduais de Metaloproteinases/genética , Transfecção , Regulação para Cima/fisiologia , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
Heart disease is the leading cause of death in the United States. Recent studies demonstrate that fetal programming of PKCε gene repression results in ischemia-sensitive phenotype in the heart. The present study tests the hypothesis that increased norepinephrine causes epigenetic repression of PKCε gene in the heart via Nox1-dependent reactive oxygen species (ROS) production. Prolonged norepinephrine treatment increased ROS production in fetal rat hearts and embryonic ventricular myocyte H9c2 cells via a selective increase in Nox1 expression. Norepinephrine-induced ROS resulted in an increase in PKCε promoter methylation at Egr-1 and Sp-1 binding sites, leading to PKCε gene repression. N-acetylcysteine, diphenyleneiodonium, and apocynin blocked norepinephrine-induced ROS production and the promoter methylation, and also restored PKCε mRNA and protein to control levels in vivo in fetal hearts and in vitro in embryonic myocyte cells. Accordingly, norepinephrine-induced ROS production, promoter methylation, and PKCε gene repression were completely abrogated by knockdown of Nox1 in cardiomyocytes. These findings provide evidence of a novel interaction between elevated norepinephrine and epigenetic repression of PKCε gene in the heart mediated by Nox1-dependent oxidative stress and suggest new insights of molecular mechanisms linking the heightened sympathetic activity to aberrant cardioprotection and increased ischemic vulnerability in the heart.
Assuntos
Coração Fetal/efeitos dos fármacos , Coração Fetal/metabolismo , NADH NADPH Oxirredutases/metabolismo , Norepinefrina/farmacologia , Proteína Quinase C-épsilon/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Epigênese Genética/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Modelos Cardiovasculares , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , Regiões Promotoras Genéticas , Proteína Quinase C-épsilon/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição Sp1/metabolismoRESUMO
BACKGROUND AND PURPOSE: Maternal cigarette smoking increases the risk of neonatal morbidity. We tested the hypothesis that perinatal nicotine exposure causes heightened brain vulnerability to hypoxic-ischemic (HI) injury in neonatal rats through aberrant expression patterns of angiotensin II type 1 (AT(1)R) and type 2 (AT(2)R) receptors in the developing brain. METHODS: Nicotine was administered to pregnant rats through subcutaneous osmotic minipumps. HI brain injury was determined in 10-day-old pups. AT(1)R and AT(2)R expression patterns were assessed through Western blotting, quantitative polymerase chain reaction, immunofluorescence, and confocal imaging. RESULTS: Perinatal nicotine exposure significantly increased HI brain infarct size in male, but not female, pups. In fetal brains, nicotine caused a decrease in mRNA and protein abundance of AT(2)R but not AT(1)R. The downregulation of AT(2)R persisted in brains of male pups, and nicotine treatment resulted in a significant increase in methylation of CpG locus 3 bases upstream of TATA-box at the AT(2)R gene promoter. In female brains, there was an increase in AT(2)R but a decrease in AT(1)R expression. Both AT(1)R and AT(2)R expressed in neurons but not in astrocytes in the cortex and hippocampus. Central application of AT(1)R antagonist losartan or AT(2)R antagonist PD123319 increased HI brain infarct size in both male and female pups. In male pups, AT(2)R agonist CGP42112 abrogated nicotine-induced increase in HI brain infarction. In females, PD123319 uncovered the nicotine's effect on HI brain infarction. CONCLUSIONS: Perinatal nicotine exposure causes epigenetic repression of the AT(2)R gene in the developing brain resulting in heightened brain vulnerability to HI injury in neonatal male rats in a sex-dependent manner.
Assuntos
Lesão Encefálica Crônica/patologia , Isquemia Encefálica/patologia , Hipóxia Encefálica/patologia , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Receptores de Angiotensina/fisiologia , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Encéfalo/patologia , Metilação de DNA/efeitos dos fármacos , Feminino , Retardo do Crescimento Fetal/induzido quimicamente , Retardo do Crescimento Fetal/patologia , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Imuno-Histoquímica , Masculino , Microscopia Confocal , Gravidez , Piridinas/farmacologia , Piridinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/biossíntese , Receptor Tipo 2 de Angiotensina/genéticaRESUMO
PURPOSE: The newly-identified lysosomal protein transmembrane 4 beta-35 (LAPTM4B-35) plays important roles in tumor progression and metastasis, while argininosuccinate synthetase (ASS) provides arginine as an indispensable nutrient for hepatocellular carcinoma (HCC). The present study investigated the clinical significance of the coexpression of LAPTM4B-35 and ASS in HCC patients on determining the prognosis. METHODS: Immunohistochemistry was used to evaluate the expression of LAPTM4B-35 and ASS in HCC tissues and paired noncancerous liver samples from 71 patients. The correlation of combined LAPTM4B-35 and ASS expression with selected clinicopathologic parameters was assessed with the chi-squared test. Patient survival and differences in survival were determined by the Kaplan-Meier method and the log-rank test. A Cox regression analysis was adopted for a multivariate analysis of the prognostic factors. RESULTS: Combined LAPTM4B-35 and ASS expression was significantly associated with TNM stage and portal vein invasion. In addition, patients with HCCs expressing both LAPTM4B-35 and ASS exhibited both markedly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001). According to the multivariate analyses, combined LAPTM4B-35 and ASS expression was found to be an independent prognostic factor for OS and DFS (P = 0.039 and P = 0.035, respectively). CONCLUSION: The overexpression of LAPTM4B-35 in combination with positive ASS expression is a negative prognostic marker for HCC.
Assuntos
Argininossuccinato Sintase/biossíntese , Biomarcadores Tumorais/biossíntese , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/biossíntese , Proteínas Oncogênicas/biossíntese , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Análise de SobrevidaRESUMO
BACKGROUND: LAPTM4B-35 is a 35-kDa tetra-transmembrane protein overexpressed in hepatocellular carcinoma (HCC) and promotes cell survival, proliferation, and tumorigenesis. However, the potential clinical implications of LAPTM4B-35 in HCC are still unclear. This study is aimed to investigate the correlations between LAPTM4B-35 expression and prognosis in patients with HCC. METHODS: Western blot and immunohistochemistry assays were used to determine the expression of LAPTM4B-35 in HCCs and their paired noncancerous liver tissues from 65 patients. The correlations of LAPTM4B-35 expression with clinicopathological parameters were assessed by Chi-square test. Patient survival was determined by Kaplan-Meier method and log-rank test. Cox regression was adopted for multivariate analysis of prognostic factors. RESULTS: LAPTM4B-35 overexpression occurred in 76.9% of HCC tissues, while only in 4.6% of noncancerous liver tissues. Overexpression of LAPTM4B-35 was significantly associated with TNM staging and invasive tumors. Patients with higher LAPTM4B-35 expression had significantly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001). On multivariate analysis, elevated expression of LAPTM4B-35 was found to be an independent prognostic factor for OS and DFS (P = 0.009, 0.043, respectively). CONCLUSIONS: LAPTM4B-35 overexpression is an independent prognostic factor for OS and DFS of HCC.
Assuntos
Carcinoma Hepatocelular/mortalidade , Neoplasias Hepáticas/mortalidade , Proteínas de Membrana/análise , Proteínas Oncogênicas/análise , Adulto , Animais , Western Blotting , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
BACKGROUND/AIMS: It was previously established that LAPTM4B-35 highly expressed in gallbladder carcinoma and being of clinicopathological and prognostic significances. However, expression of LAPTM4B gene in gallbladder carcinoma (GBC-SD), a gallbladder carcinoma cell line, and its role in invasive potential remain unclear. METHODOLOGY: Expression of LAPTM4B in GBC-SD cells was first detected. Plasmids, pcDNA3-AE (containing complete open reading frame of LAPTM4B) and Mock (pcDNA3), were transiently transfected into GBC-SD cells. Invasive phenotypes (migration and invasion) and relative molecules were then shown by transwell assay, crossing river test and Western blot analysis. RESULTS: Immunocytochemical staining revealed that LAPTM4B-35 positively expressed in cytoplasm of GBC-SD cells. But LAPTM4B-35 expression was obviously weaker in GBC-SD cells than that in BEL-7402 cells (positive control). Besides, cells transfected with pcDNA3-AE presented shorter crossing river time, less migrated and invaded cell numbers, compared with cells transfected with the Mock plasmid and parent cells. Finally, increased expressions of active uPA, MMP-9, pro MMP-2 and active MMP-2 were also observed in cells transfected with pcDNA3-AE. CONCLUSIONS: Our data suggested that LAPTM4B expressed in GBC-SD cells at a relatively low level. Forced overexpression of LAPTM4B increased invasive potential of GBC-SD cells, through modulating molecules associated with degradation of extracellular matrix.
Assuntos
Neoplasias da Vesícula Biliar/genética , Proteínas de Membrana/genética , Proteínas Oncogênicas/genética , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Fosfatos de Dinucleosídeos/metabolismo , Matriz Extracelular/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Humanos , Técnicas Imunoenzimáticas , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/metabolismo , Fases de Leitura Aberta , Fenótipo , Plasmídeos , Estatísticas não Paramétricas , Transfecção , Células Tumorais CultivadasRESUMO
Lysosomal protein transmembrane 4 beta (LAPTM4B) was originally identified as a hepatocellular carcinoma (HCC)-associated gene. This gene and its protein product LAPTM4B-35, are both overexpressed in a variety of human cancers. However, its specific role in cell transformation and malignancy has remained elusive. In the present study we investigated the effects of LAPTM4B-35 overexpression on the malignant phenotypic features in the HLE cell line. Our data show that overexpression of LAPTM4B-35 promotes cell proliferation, exogenous growth-stimulating factor-independent and anchorage-independent growth, and enhances metastatic potential, including promotion of both cell migration and invasion. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes, which included upregulation of proliferation-promoting transcription factors such as c-Myc, c-Jun, and c-Fos, and cell cycle-promoting proteins such as cyclin D1 and cyclin E. In addition, mutagenesis study showed that the PPRP motif in the N-terminal region of LAPTM4B-35 plays a critical role in promoting proliferation, migration, and invasion, as well as in the upregulation of the oncoproteins noted above. These data offer insight into the mechanism by which this novel tetratransmembrane protein contributes to the pathogenesis of liver cancer, and suggest that it may be a potential target for cancer therapy.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas de Membrana/fisiologia , Proteínas Oncogênicas/fisiologia , Idoso , Motivos de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas de Membrana/química , Metástase Neoplásica , Proteínas Oncogênicas/química , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologiaRESUMO
Lysosome-associated protein transmembrane 4beta (LAPTM4B) is a member of the mammalian 4-tetratransmembrane spanning protein superfamily, which is strongly expressed in some solid malignancies. The present study investigated the correlation of LAPTM4B expression with clinicopathological features and prognosis in ovarian carcinoma. Expression of LAPTM4B was evaluated semiquantitatively by immunohistochemistry in 85 cases of ovarian carcinoma. High LAPTM4B expression was found in 54 (63.5%) of these 85 carcinomas, and was positively correlated with FIGO stage, histological subtype and residual tumor after primary surgery, but not with age and tumor grade. Patients with high LAPTM4B expression had significantly poorer overall survival (OS) and progression-free survival (PFS) (P<0.001 and P<0.001, respectively) compared to patients with low expression of LAPTM4B. On multivariate analysis, LAPTM4B expression was found to be an independent prognostic factor for OS and PFS (P=0.01 and P=0.03, respectively). These results showed that high LAPTM4B expression was associated with progression of ovarian carcinoma and correlated with unfavorable clinical outcome, suggesting that LAPTM4B may be a clinically useful prognostic indicator for ovarian carcinoma.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Membrana/biossíntese , Proteínas Oncogênicas/biossíntese , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Adulto , Idoso , Western Blotting , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , PrognósticoRESUMO
The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.
Assuntos
Dexametasona/administração & dosagem , Epigênese Genética , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/patologia , Receptores de Glucocorticoides/biossíntese , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Ciclina D2/biossíntese , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Metilação de DNA/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Humanos , Mifepristona/administração & dosagem , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RatosRESUMO
Adverse environmental conditions faced by an individual early during its life, such as gestational hypoxia, can have a profound influence on the risk of diseases, such as neurological disorders, in later life. Clinical and preclinical studies suggest that epigenetic programming of gene expression patterns in response to maternal stress have a crucial role in the fetal origins of neurological diseases. Herein, we summarize recent studies regarding the role of epigenetic mechanisms in the developmental programming of neurological diseases in offspring, primarily focusing on DNA methylation/demethylation and miRNAs. Such information could increase our understanding of the fetal origins of adult diseases and help develop effective prevention and intervention against neurological diseases.
Assuntos
Encefalopatias/genética , Hipóxia Fetal/genética , Animais , Encefalopatias/etiologia , Epigênese Genética , Hipóxia Fetal/complicações , HumanosRESUMO
Our previous study demonstrated that pregnancy increased large-conductance Ca(2+)-activated potassium channel ß1 subunit (BKß1) expression and large-conductance Ca(2+)-activated potassium channel activity in uterine arteries, which were abrogated by chronic hypoxia. The present study tested the hypothesis that promoter methylation/demethylation is a key mechanism in epigenetic reprogramming of BKß1 expression patterns in uterine arteries. Ovine BKß1 promoter of 2315 bp spanning from -2211 to +104 of the transcription start site was cloned, and an Sp1-380 binding site that contains CpG dinucleotide in its core binding sequences was identified. Site-directed deletion of the Sp1 site significantly decreased the BKß1 promoter activity. Estrogen receptor-α bound to the Sp1 site through tethering to Sp1 and upregulated the expression of BKß1. The Sp1 binding site at BKß1 promoter was highly methylated in uterine arteries of nonpregnant sheep, and methylation inhibited transcription factor binding and BKß1 promoter activity. Pregnancy caused a significant decrease in CpG methylation at the Sp1 binding site and increased Sp1 binding to the BKß1 promoter and BKß1 mRNA abundance. Chronic hypoxia during gestation abrogated this pregnancy-induced demethylation and upregulation of BKß1 expression. The results provide evidence of a novel mechanism of promoter demethylation in pregnancy-induced reprogramming of large-conductance Ca(2+)-activated potassium channel expression and function in uterine arteries and suggest new insights of epigenetic mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.
Assuntos
Adaptação Fisiológica/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Prenhez/fisiologia , Artéria Uterina/fisiologia , Animais , Ilhas de CpG/genética , Ilhas de CpG/fisiologia , Metilação de DNA , Epigênese Genética/fisiologia , Feminino , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Modelos Animais , Gravidez , Glicoproteínas beta 1 Específicas da Gravidez/fisiologia , Ligação Proteica/fisiologia , Ovinos , Regulação para CimaRESUMO
AIMS: Hypoxia causes protein kinase C epsilon (PKCε) gene repression in foetal hearts, resulting in heightened cardiac susceptibility to ischaemic injury in offspring. We tested the hypothesis that hypoxia inducible factor 1 (HIF-1) and/or reactive oxygen species (ROS) mediate hypoxia-induced PKCε gene repression. METHODS AND RESULTS: Hypoxia induced in vivo to pregnant rats, ex vivo to isolated foetal rat hearts, and in vitro in the rat embryonic ventricular myocyte cell line H9c2 resulted in a comparable decrease in PKCε protein and mRNA abundance in foetal hearts and H9c2 cells, which was associated with a significant increase in CpG methylation of the SP1-binding sites at the PKCε promoter. In H9c2 cells and foetal hearts, hypoxia caused nuclear accumulation of HIF-1α, which was inhibited by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole and 2-methoxy estradiol. The HIF-1α inhibitors had no significant effect on hypoxia-induced PKCε mRNA repression. Hypoxia produced a time-dependent increase in ROS production in H9c2 cells and foetal hearts that was blocked by ROS scavengers N-acetyl-cysteine or tempol. In accordance, N-acetyl-cysteine and tempol, but not apocynin, inhibited the hypoxic effect and restored PKCε protein and mRNA expression to the control values in foetal hearts and H9c2 cells. The ROS scavengers blocked hypoxia-induced CpG methylation of the SP1-binding sites, restored SP1 binding to the PKCε promoter, and abrogated the hypoxia-induced increase in the susceptibility of the heart to ischaemic injury in offspring. CONCLUSIONS: The results demonstrate that hypoxia induces epigenetic repression of the PKCε gene through a NADPH oxidase-independent ROS-mediated pathway in the foetal heart, leading to heightened heart vulnerability to ischaemic injury in offspring.
Assuntos
Epigênese Genética , Coração Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipóxia/metabolismo , Estresse Oxidativo , Proteína Quinase C-épsilon/genética , Acetilcisteína/farmacologia , Animais , Óxidos N-Cíclicos/farmacologia , Metilação de DNA , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/etiologia , NADPH Oxidases/fisiologia , Gravidez , Regiões Promotoras Genéticas , Proteína Quinase C-épsilon/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Marcadores de SpinRESUMO
Gallbladder carcinoma (GBC) is a malignancy with an extremely poor prognosis. In order to improve the survival rate, identification of new susceptibility risk factors is of importance. Here, we report findings on the novel cancer-related gene lysosomal protein transmembrane 4 beta (LAPTM4B) that has two alleles designated LAPTM4B*1 and LAPTM4B*2. Allele *1 differs from allele *2 in that it contains one copy of a 19-bp sequence, whereas this sequence is duplicated in exon 1 of allele *2. This study aimed to investigate the relationship of LAPTM4B allelic variation and GBC susceptibility. LAPTM4B genotype was analyzed in 155 healthy individuals and 91 GBC patients by PCR, and the genotypic distribution of LAPTM4B was analyzed with the chi-squared test. The frequency of allele *2 was 37.9 and 24.8% in the GBC and the control groups, respectively, representing a significant difference between these two groups (P<0.001). LAPTM4B allele *2 may be a risk factor associated with genetic susceptibility to GBC.
Assuntos
Povo Asiático/genética , Neoplasias da Vesícula Biliar/genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Proteínas Oncogênicas/genética , Polimorfismo Genético , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Sequência de Bases , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
BACKGROUND: Lysosomal protein transmembrane 4 beta (LAPTM4B) is a gene related to hepatocellular carcinoma that has two alleles designated LAPTM4B*1 and LAPTM4B*2. This study aimed to investigate the correlation of LAPTM4B genotype with prognosis and clinicopathologic features in patients who have undergone resection for hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: The LAPTM4B genotype was analyzed by PCR in 68 patients who had undergone curative hepatic resection for hepatocellular carcinoma. The correlation of LAPTM4B genotype with clinicopathologic parameters was assessed with the Chi-squared test. Differences in patient survival were determined by the Kaplan-Meier method. Multivariate analysis of prognostic factors was carried out with Cox regression analysis. Patients with LAPTM4B *2 had both significantly shorter overall survival (OS) and shorter disease-free survival (DFS) (both P<0.001). Multivariate analysis showed that LAPTM4B genotype is an independent prognostic factor for OS and DFS (both P<0.001). CONCLUSIONS/SIGNIFICANCE: Allele *2 of LAPTM4B is a risk factor associated with poor prognosis in patients with resected HCC. LAPTM4B status may be useful preoperatively as an adjunct in evaluation of the operability of HCC.