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1.
Biol Pharm Bull ; 39(11): 1802-1808, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27803451

RESUMO

Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor ß ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.


Assuntos
Carcinoma Embrionário/genética , Fator 3 de Diferenciação de Crescimento/genética , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Testiculares/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Fator 3 de Transcrição de Octâmero/metabolismo , Transcrição Gênica
2.
Biochem Biophys Res Commun ; 450(2): 984-90, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-24971534

RESUMO

The epithelium-specific ETS transcription factor-1 (ESE-1) is physiologically important in the pathogenesis of various diseases. Recently, OCT4, a transcription factor involved in stem cell pluripotency, has been implicated in tumorigenesis. In this study, we invested the molecular mechanism by which ESE-1 regulates transcription of OCT4 in NCCIT human embryonic carcinoma cells. Real-time PCR analysis revealed that OCT4 levels were high in undifferentiated NCCIT cells but significantly decreased upon retinoic acid-mediated differentiation, concomitant with up-regulation of ESE-1 expression. OCT4 mRNA level rose following shRNA-mediated knockdown of ESE-1, but declined when ESE-1 was overexpressed, suggesting that the expression levels of OCT4 and ESE-1 may be coordinated in an opposite manner. Promoter-reporter assays revealed that induced OCT4 promoter activity in NCCIT cells was significantly down-regulated by ESE-1 overexpression in a dose-dependent manner. The inhibitory effect of ESE-1 on OCT4 promoter activity was relieved by co-expression of an ESE-1 mutant lacking the transactivation domain, but not by mutants lacking other domains. Serial deletion and site-directed mutagenesis of the OCT4 promoter revealed that a potential ETS binding site (EBS) is present in the conserved region 2 (CR2). ESE-1 interacted with the EBS element in CR2 and enrichment of CR2 significantly increased upon RA-mediated differentiation of NCCIT cells, suggesting that this binding is likely to be involved in ESE-1-mediated repression of OCT4 promoter activity upon differentiation. Taken together, the results of this study reveal the molecular details of the mechanism by which the oncogenic factor ESE-1 regulates expression of the stem cell transcription factor OCT4 in pluripotent NCCIT cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco de Carcinoma Embrionário/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Células-Tronco de Carcinoma Embrionário/citologia , Técnicas de Silenciamento de Genes , Humanos , Mutação , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-ets/genética , Fatores de Transcrição/genética , Transcrição Gênica , Ativação Transcricional
3.
Biol Pharm Bull ; 37(4): 659-65, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24694612

RESUMO

E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.


Assuntos
Carcinoma Embrionário/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ets/biossíntese , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Carcinoma Embrionário/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Tamanho Celular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Proto-Oncogênicas c-ets/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transfecção
4.
Reprod Sci ; 27(6): 1318-1329, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32046453

RESUMO

We aimed to identify novel biomarkers in amniotic fluid (AF) that predict the outcome of emergency cerclage in women with cervical insufficiency. This retrospective cohort study included 40 singleton pregnant women who received emergency cerclage for cervical insufficiency (17-25 weeks) and underwent amniocentesis. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify AF proteins in pooled samples (n = 16) using a nested case-control approach. The six candidate biomarkers of interest were validated by enzyme-linked immunosorbent assays (ELISA) in the final cohort (n = 40). The differentially expressed proteins (DEPs) were analyzed by pathway analysis software. The primary outcome measure was failure of emergency cerclage [defined as spontaneous preterm delivery (SPTD) at < 34 weeks of gestation after cerclage placement]. Sixty-eight proteins were differentially expressed (P < 0.001) in AF from SPTD cases and near-term controls, of which 44 (64.7%) were upregulated and 24 (35.3%) were downregulated. Validation by ELISA confirmed that AF from women with cerclage failure contained significantly higher levels of myeloperoxidase, lactoferrin, glucose-6-phosphate isomerase, lipocalin-2, and lymphocyte cytosolic protein 1, the first four of which were independent of cervical dilatation at presentation. The five pathways with the most differentially regulated proteins were actin cytoskeleton signaling, acute phase response signaling, ILK signaling, glycolysis, and gluconeogenesis. Proteomic analyses of AF in this study identified DEPs and specific protein pathways related to poor prognosis after emergency cerclage for cervical insufficiency. Four novel independent biomarkers in AF for cerclage failure have been identified using proteomics.


Assuntos
Líquido Amniótico/metabolismo , Cerclagem Cervical/métodos , Nascimento Prematuro/prevenção & controle , Incompetência do Colo do Útero/cirurgia , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida , Feminino , Humanos , Gravidez , Nascimento Prematuro/metabolismo , Prognóstico , Proteômica , Estudos Retrospectivos , Espectrometria de Massas em Tandem , Incompetência do Colo do Útero/metabolismo , Adulto Jovem
5.
FEBS Lett ; 592(1): 24-35, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29223130

RESUMO

Cripto-1 and OCT4, expressed in stem cells and cancers, play important roles in tumorigenesis. Here, we demonstrate that Cripto-1 expression is regulated by OCT4 in human embryonic carcinoma NCCIT cells. The endogenous expression of Cripto-1 and OCT4 is significantly reduced during differentiation. Cripto-1 expression is increased by OCT4 overexpression, but decreased by shRNA-mediated OCT4 knockdown. OCT4 overexpression significantly activates Cripto-1 transcriptional activity. A 5'-upstream minimal promoter sequence in the gene-encoding Cripto-1 is significantly activated by OCT4 overexpression. Mutation of the putative OCT4-binding site abolishes OCT4-mediated activation of the Cripto-1 promoter. The OCT4 transactivation domains mediate transcriptional activity of the Cripto-1 minimal promoter through direct interaction. Taken together, OCT4 plays an important role as a transcriptional activator of Cripto-1 expression in NCCIT cells.


Assuntos
Carcinoma Embrionário/genética , Proteínas Ligadas por GPI/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Neoplasias/genética , Fator 3 de Transcrição de Octâmero/genética , Sítios de Ligação/genética , Carcinoma Embrionário/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Ativação Transcricional
6.
Resuscitation ; 128: 6-10, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29698750

RESUMO

AIM OF THE STUDY: To identify proteins of which depletion are associated with the poor 6-month neurological outcome of out-of-hospital cardiac arrest survivors. METHODS: Seven healthy volunteers and 34 out-of-hospital cardiac arrest survivors admitted to the intensive care unit (ICU) and underwent targeted-temperature management were enrolled. According to the 6-month cerebral performance category (CPC) scale, patients were divided into the good (CPC 1-2) and poor (CPC 3-5) outcome groups. Blood samples were obtained at 0, 24, and 72 h after admission to the ICU. RESULTS: With proteomic approaches, we found 23 proteins that showed group-differences between the sera pooled from 7 study groups: healthy volunteers, the good outcome groups (0, 24, and 72 h), and the poor outcome groups (0, 24, and 72 h). We selected 7 candidate proteins of which intensities were different between the good and poor outcome groups (>2-fold change) and excluded 5 proteins related to haemolysis or remaining high abundant proteins. To confirm the 2 identified proteins: retinal dehydrogenase 1 and Kallistatin, we performed enzyme-linked immunosorbent assay with individual serum. Finally, old age (odds ratio = 1.055; 95% confidence interval, 1.002-1.112; p = 0.043) and low serum kallistatin level at 0 h (odds ratio = 0.784; 95% confidence interval, 0.618-0.995; p = 0.046) were independently associated with the poor 6-month neurological outcome. CONCLUSION: The depletion of serum kallistatin at admission to the ICU was associated with the poor neurological outcome of out-of-hospital cardiac arrest survivors.


Assuntos
Parada Cardíaca Extra-Hospitalar/sangue , Serpinas/sangue , Adulto , Fatores Etários , Análise de Variância , Biomarcadores/sangue , Reanimação Cardiopulmonar , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Hipotermia Induzida/métodos , Proteômica , Recuperação de Função Fisiológica , Retinal Desidrogenase/sangue
7.
FEBS Lett ; 588(17): 3129-36, 2014 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-24983502

RESUMO

We examined the molecular mechanism of OCT4 gene regulation by polyomavirus enhancer activator 3 (PEA3) in NCCIT cells. Endogenous PEA3 and OCT4 were significantly elevated in undifferentiated cells and reduced upon differentiation. PEA3 knockdown led to a reduction in OCT4 levels. OCT4 promoter activity was significantly up-regulated by dose-dependent PEA3 overexpression. Deletion and site-directed mutagenesis of the OCT4 promoter revealed a putative binding site within the conserved region 2 (CR2). PEA3 interacted with the binding element within CR2 in NCCIT cells. This study reveals the molecular details of the mechanism by which the oncogenic factor PEA3 regulates OCT4 gene expression as a transcriptional activator.


Assuntos
Desenvolvimento Embrionário , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Bases , Carcinogênese , Linhagem Celular Tumoral , Humanos , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas/genética , Seminoma/patologia , Deleção de Sequência
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