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1.
Cell Tissue Res ; 364(3): 599-610, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26841878

RESUMO

To investigate the role of macrophages in oxygen-induced retinal neovascularization (NV) in mice, particularly the involvement of bone marrow-derived cells (BMCs) and the underlying mechanisms, BMCs from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P) 1 mice after irradiation. The mice were exposed to 75 % oxygen from P7 to P12 to initiate oxygen-induced retinopathy (OIR). The macrophages were depleted by injection of clodronate-liposomes (lip) intraperitoneally. The eyes were collected at P12 and P17. Retinal flatmounts and histopathological cross-sections were performed to analyze the severity of retinal NV and BMC recruitment. BMCs immunopositive for CD31 (PECAM-1; endothelial cell marker) and α-SMA (smooth muscle cell marker) antigens were detected using a confocal microscope. Expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) mRNA was detected by RT-PCR. The VEGF, SDF-1, CXCR4 and CD45 protein expression was detected by western blot examination. The retinal avascular area in OIR mice at P12 was unaffected after macrophage depletion carried out twice (38.27 ± 1.92 % reduction) using clodronate-lip. The retinal avascular area and the NV area at P17 were reduced after macrophage depletion four times (79.53 ± 1.02 % reduction); these findings were supported by retinal flatmounts and histopathological cross-sections. Macrophage depletion led to significant inhibition of BMC recruitment into the NV tufts at P17, with decreased expression of retinal VEGF, SDF-1, CXCR4 and CD45. The recruited BMCs differentiated primarily into CD31-positive endothelial cells (ECs) and α-SMA-positive smooth muscle cells (SMCs). This study suggested that macrophages promoted the vasculogenesis of retinal NV, particularly the contribution of BMCs in the mouse OIR model, which might be triggered by VEGF and SDF-1 production.


Assuntos
Macrófagos/metabolismo , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Administração Intravenosa , Animais , Animais Recém-Nascidos , Células da Medula Óssea/patologia , Diferenciação Celular , Movimento Celular , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Oxigênio , Retina/patologia , Neovascularização Retiniana/complicações , Retinopatia da Prematuridade/complicações
2.
Hepatology ; 59(5): 1850-63, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24002871

RESUMO

UNLABELLED: The MYC oncogene is overexpressed in hepatocellular carcinoma (HCC) and has been associated with widespread microRNA (miRNA) repression; however, the underlying mechanisms are largely unknown. Here, we report that the c-Myc oncogenic transcription factor physically interacts with enhancer of zeste homolog 2 (EZH2), a core enzymatic unit of polycomb repressive complex 2 (PRC2). Furthermore, miR-101, an important tumor-suppressive miRNA in human hepatocarcinomas, is epigenetically repressed by PRC2 complex in a c-Myc-mediated manner. miR-101, in turn, inhibits the expression of two subunits of PRC2 (EZH2 and EED), thus creating a double-negative feedback loop that regulates the process of hepatocarcinogenesis. Restoration of miR-101 expression suppresses multiple malignant phenotypes of HCC cells by coordinate repression of a cohort of oncogenes, including STMN1, JUNB, and CXCR7, and further increases expression of endogenous miR-101 by inhibition of PRC2 activation. In addition, co-overexpression of c-Myc and EZH2 in HCC samples was closely associated with lower expression of miR-101 (P < 0.0001) and poorer prognosis of HCC patients (P < 0.01). CONCLUSIONS: c-Myc collaborates with EZH2-containing PRC2 complex in silencing tumor-suppressive miRNAs during hepatocarcinogenesis and provides promising therapeutic candidates for human HCC.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Hepáticas/genética , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Receptores CXCR/fisiologia
3.
Carcinogenesis ; 34(8): 1773-81, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23615404

RESUMO

The POU transcription factor OCT4 is a pleiotropic regulator of gene expression in embryonic stem cells. Recent studies demonstrated that OCT4 is aberrantly expressed in multiple types of human cancer; however, the underlying molecular mechanism remains largely unknown. In this study, we report that OCT4-pg4, a pseudogene of OCT4, is abnormally activated in hepatocellular carcinoma (HCC). The expression level of OCT4-pg4 is positively correlated with that of OCT4, and both gene transcripts can be directly targeted by a tumor-suppressive micro RNA miR-145. We find that the non-coding RNA OCT4-pg4 is biologically active, as it can upregulate OCT4 protein level in HCC. Mechanistic analysis revealed that OCT4-pg4 functions as a natural micro RNA sponge to protect OCT4 transcript from being inhibited by miR-145. In addition, our study also showed that OCT4-pg4 can promote growth and tumorigenicity of HCC cells, thus exerting an oncogenic role in hepatocarcinogenesis. Furthermore, survival analysis suggests that high OCT4-pg4 level is significantly correlated with poor prognosis of HCC patients. Taken together, our finding adds a new layer of post-transcriptional regulation of OCT4 and sheds new light on the treatment of human HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Fator 3 de Transcrição de Octâmero/genética , Pseudogenes , Animais , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Fator 3 de Transcrição de Octâmero/biossíntese , Prognóstico , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Regulação para Cima
4.
Biochim Biophys Acta ; 1822(2): 301-13, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22138128

RESUMO

Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-κB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague-Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-κB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility.


Assuntos
Apoptose/genética , Infertilidade Masculina/metabolismo , Células Intersticiais do Testículo/metabolismo , NF-kappa B/metabolismo , Proteínas/genética , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Infertilidade Masculina/genética , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Mesilatos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Síndrome de Células de Sertoli/genética , Síndrome de Células de Sertoli/metabolismo , Espermatogênese/efeitos dos fármacos , Espermatogênese/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
Gastroenterology ; 141(6): 2076-2087.e6, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21925125

RESUMO

BACKGROUND & AIMS: Human epidermal growth factor receptor 2 (HER2) (neu/ERBB2) is overexpressed on many types of cancer cells, including gastric cancer cells; HER2 overexpression has been associated with metastasis and poor prognosis. We investigated the mechanisms by which HER2 regulates cell migration and invasion. METHODS: HER2 expression or activity was reduced in gastric cancer cell lines using small interfering RNAs or the monoclonal antibody, trastuzumab. We identified proteins that interact with HER2 or microRNAs (miRNAs) involved in HER2 signaling. We used various software programs to identify miRNAs that regulate factors in the HER2 signaling pathway. We analyzed expression patterns of these miRNAs in gastric cancer cell lines and tumor samples from patients. RESULTS: We found that CD44 binds directly to HER2, which up-regulates the expression of metastasis-associated protein-1, induces deacetylation of histone H3 lysine 9, and suppresses transcription of microRNA139 (miR-139) to inhibit expression of its target gene, C-X-C chemokine receptor type 4 (CXCR4). Knockdown of HER2 and CD44 reduced invasive activity of cultured gastric cancer cells and suppressed tumor growth in nude mice. Lymph node metastasis was associated with high levels of HER2, CD44, and CXCR4, and reduced levels of miR-139 in human metastatic gastric tumors. Cultures of different types of metastatic cancer cells with histone deacetylase inhibitors and/or DNA methyltransferase resulted in up-regulation of miR-139. CONCLUSIONS: HER2 interaction with CD44 up-regulates CXCR4 by inhibiting expression of miR-139, at the epigenetic level, in gastric cancer cells. These findings indicate how HER2 signaling might promote gastric tumor progression and metastasis.


Assuntos
Epigênese Genética/genética , Receptores de Hialuronatos/metabolismo , MicroRNAs/genética , Receptor ErbB-2/metabolismo , Receptores CXCR4/metabolismo , Neoplasias Gástricas/genética , Animais , Northern Blotting , Movimento Celular , Primers do DNA/química , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Técnicas de Amplificação de Ácido Nucleico , Células Tumorais Cultivadas , Regulação para Cima
6.
Mol Cell Biochem ; 364(1-2): 209-16, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22278385

RESUMO

Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress and neural degeneration are suggested to be involved in the pathogenesis of PD. Previous studies have revealed that Astragaloside IV (AS-IV) can reduce inflammation and oxidation, making it a potential therapeutic agent for neurodegenerative disease. In this study, we investigated whether AS-IV protect against 1-methyl-4-phenylpyridnium ion (MPP(+))-induced dopaminergic neurotoxicity in SH-SY5Y cells and determined the mechanism of AS-IV neuroprotection. We found that pretreatment with AS-IV significantly reversed the loss of cell viability, nuclear condensation, the generation of intracellular reactive oxygen species (ROS), and the increase in Bax/Bcl-2 ratio and the activity of caspase-3 induced by MPP(+). Our study suggests that the neuroprotective effect of AS-IV is related to mechanisms including ROS production and the inhibition of Bax-mediated pathway. The present study supports the notion that AS-IV may be a promising neuroprotective agent for the treatment of neurodegenerative disorders such as PD.


Assuntos
Doença de Parkinson/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Proteína X Associada a bcl-2/metabolismo , 1-Metil-4-fenilpiridínio/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética
7.
Zhongguo Dang Dai Er Ke Za Zhi ; 14(4): 267-70, 2012 Apr.
Artigo em Zh | MEDLINE | ID: mdl-22537954

RESUMO

OBJECTIVE: To study the characteristics of genotype spectrum and hematologic parameters in children with HbH disease in the North Guangxi region. METHODS: HbH disease was identified by clinical manifestations, routine blood tests and hemoglobin electrophoresis in 166 children who came form the North Guangxi region. Genotypes were determined by Multi-PCR combined with PCR reverse dot blot. DNA sequencing was used when the genotype could not be identified by regular methods. RESULTS: Of the 166 children with HbH disease, 8 genotypes were identified: --SEA/-α3.7 (82 cases), --SEA/-α4.2 (40 cases), --SEA/αCSα (38 cases), --SEA/αQSα (1 case), --SEA/αWSα (1 case), --SEA/αCD43/44 (-C) α (1 case), --SEA/-α3.7 plus CD17 (A→T) (1 case) and --SEA/-α4.2 plus CD41-42(-TTCT) (1 case). One case was confirmed as the heterozygote of --SEA and an unknown mutation. In the 134 cases with complete medical data, 2 had normal hemoglobin levels, 36 manifested mild anemia, 90 manifested moderate anemia, and 6 (genotype: --SEA/αCSα) showed severe anemia because of the coexistence of infection. Children with the genotype of --SEA/-α3.7 (69 cases), --SEA/-α4.2 (31 cases) and --SEA/αCSα (34 cases) had hemoglobin levels of 62-120, 69-127 and 34-110 g/L respectively. The hemoglobin level in the --SEA/αCSα group was significantly lower than in the deletional HbH disease group (genotypes: --SEA/-α3.7 and --SEA/-α4.2 ) (P<0.05). In contrast, MCV levels in the --SEA/αCSα group were significantly higher than in the deletional HbH disease group (P<0.05). CONCLUSIONS: The genotype spectrum of HbH disease is diverse in the North Guangxi region. Deletional genotype is prevalent. The disease is heterogeneous. The children with --SEA/αCSα HbH disease have severer anemia and higher MCV levels than those with deletional HbH disease.


Assuntos
Hemoglobina H/genética , Talassemia alfa/genética , Adolescente , Criança , Pré-Escolar , China , Feminino , Genética Populacional , Genótipo , Humanos , Lactente , Masculino , Mutação , Talassemia alfa/sangue
8.
Arch Biochem Biophys ; 499(1-2): 49-55, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20460098

RESUMO

HER2 overexpression is associated with metastasis-the main cause of death in individuals with gastric cancer. In this study, we demonstrated that vector-based shRNA significantly knocked down the expression of HER2 and considerably inhibited both the migration and invasion of gastric cancer cells. HER2 knockdown resulted in the downregulation of the expression of MMP-1, while HER2 overexpression improved the transcription of MMP-1 through the activation of an MMP-1 promoter. The promoter region of MMP-1 between -2500 and -2000 bp was found to be crucial for the upregulation of HER2-mediated transcription. Furthermore, a truncated promoter (-70 to+63) did not display any transcriptional activity. Cell invasion activity was almost completely inhibited when MMP-1 was knocked down. Conversely, the overexpression of MMP-1 partly rescued the invasion ability of cell strains with knocked-down HER2. These findings help further understanding of the molecular mechanisms through which HER2 promotes malignancy, and suggest that targeting both HER2 and MMP-1 may be required to effectively block HER2 signaling in gastric cancer therapy.


Assuntos
Metaloproteinase 1 da Matriz/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Genes erbB-2 , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Receptor ErbB-2/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Regulação para Cima
9.
Cell Tissue Res ; 337(2): 257-67, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19471968

RESUMO

N-Myc downstream regulated gene 2 (NDRG2) is expressed in the testis of adult animals and is involved in cell differentiation and development. However, little is known about the expression pattern of NDRG2 in the testis during postnatal development. Here, we show that NDRG2 is consistently expressed in Leydig cells in the rat testis during postnatal development. However, its expression has also been detected at a high frequency in spermatogenic cells of the seminiferous tubules in young rats but at a much lower frequency in adult rats. Furthermore, high levels of NDRG2 expression have been found in methoxyacetic-acid-induced apoptotic germ cells, particularly at stages X-XIII of the seminiferous epithelium cycle of adult rats. Interestingly, high levels of NDRG2 expression have also been observed in spontaneously apoptotic germ cells in the seminiferous tubules of young and adult rats. Thus, the expression of NDRG2 in germ cells seems to alter during spermatogenesis. These findings suggest that NDRG2 regulates testicular development and spermatogenesis in rats and is involved in the physiological and pathological apoptosis of germ cells.


Assuntos
Células Intersticiais do Testículo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espermatogênese/fisiologia , Testículo/crescimento & desenvolvimento , Acetatos/farmacologia , Fatores Etários , Animais , Apoptose , Imunossupressores/farmacologia , Masculino , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo
10.
Exp Eye Res ; 88(5): 910-8, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19111720

RESUMO

Choroidal neovascularization (CNV) is one of the most frequent causes of severe and progressive vision loss, while its pathogenesis is still poorly understood. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by both the extracellular matrix (ECM) and soluble signaling factors and controls essential cellular processes. Extensive evidence has shown that FAK is activated in angiogenic response. This study aims to investigate the effect of FAK on CNV formation. The Brown-Norway (BN) rats underwent laser rupture of Bruch's membrane to induce CNV and were then killed at 1, 3, 7, and 14 days following laser injury. Immunofluorescence and Western blot were processed to detect FAK protein. Retinal pigment epithelial (RPE) cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1 (HIF-1alpha) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot. Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal microvascular endothelial cells (CECs), respectively. FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. In vitro experiment showed that FAK was involved in hypoxia signaling in cultured RPE cells. The absence of FAK effectively reduced the expression of hypoxia-induced HIF-1alpha and VEGF in RPE cells, resulting in the inhibition of proliferation and migration of CECs. Our results suggest that FAK pathway activation plays a role in the development of CNV, and regulates the proliferation and migration of CECs by acting through HIF-1 and then up-regulating the expression of the angiogenic factor VEGF in RPE cells. It is reasonable to propose that FAK siRNA will potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorders, which could present a therapeutically relevant strategy for the inhibition of CNV.


Assuntos
Neovascularização de Coroide/enzimologia , Quinase 1 de Adesão Focal/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Hipóxia Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Neovascularização de Coroide/metabolismo , Técnicas de Cocultura , Modelos Animais de Doenças , Endotélio Vascular/citologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos BN , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/fisiologia , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
FASEB J ; 22(5): 1606-17, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18096813

RESUMO

In adults, angiogenic abnormalities are involved in not only tumor growth but several human inherited diseases as well. It is unclear, however, concerning how the normal vascular structure is maintained and how angiogenesis is initiated in normal adults. Using the Cre-LoxP-mediated conditional gene deletion, we show in the present study that in adult mice disruption of the transcription factor recombination signal-binding protein Jkappa (RBP-J) in endothelial cells strikingly induced spontaneous angiogenesis in multiple tissues, including retina and cornea, as well as in internal organs, such as liver and lung. In a choroidal neovascularization model, which mimics the angiogenic process in tumor growth and age-related macular degeneration, RBP-J deficiency induced a more intensive angiogenic response to injury. This could be transmitted by bone marrow, indicating that RBP-J could modulate bone marrow-derived endothelial progenitor cells in adult angiogenesis. In addition, in the absence of RBP-J, proliferation of endothelial cells increased significantly, leading to accumulative vessel outgrowth. These findings suggest that in adults RBP-J-mediated Notch signaling may play an essential role in the maintenance of vascular homeostasis by repressing endothelial cell proliferation.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Neovascularização Fisiológica/fisiologia , Receptor Notch1/fisiologia , Animais , Proliferação de Células , Corioide/irrigação sanguínea , Homeostase/efeitos dos fármacos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Mesoderma/irrigação sanguínea , Camundongos , Camundongos Knockout , Transdução de Sinais , Regulação para Cima , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
12.
Mol Cancer Ther ; 7(7): 1890-9, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18644999

RESUMO

BH3-interacting domain death agonist (BID) is a crucial element in death signaling pathways and is recognized as an intracellular link connecting the intrinsic mitochondrial apoptotic and extrinsic death receptor-mediated apoptotic pathways. Herein, we describe experiments conducted with a fusion protein, which was generated by fusing a human epidermal growth factor receptor-2 (HER2)-specific single-chain antibody with domain II of Pseudomonas exotoxin A and the truncated active BID (tBID). These experiments extend our previous work on several other immuno-proapoptotic proteins. Specifically, by excluding cells with undetectable HER2, we showed that the secreted immuno-tBID molecule selectively recognized and killed HER2-overexpressing tumor cells in vitro by attacking their mitochondria and inducing their apoptotic death. This apoptosis could only be inhibited partially by caspase pan-inhibitor zVAD and mitochondrial protector TAT-BH4. Subsequently, we transferred the immuno-tbid gene into BALB/c athymic mice bearing HER2-positive tumors together with other immuno-proapoptotic proteins using i.m. injections of liposome-encapsulated vectors. The expression of the immuno-tbid gene suppressed tumor growth and prolonged animal survival significantly. We also shortened the translocation domain of Pseudomonas exotoxin A II to only 10-amino acid sequence, which were crucial for furin cleavage. The new recombinant molecule retained the translocation efficiency and the ability of specific killing HER2-positive tumor cells. Our data showed that, compared with the toxins employed before, the chimeric immuno-tBID molecule can not only specifically recognize HER2-positive tumor cells but also certainly induce apoptosis even in the presence of zVAD and TAT-BH4, thereby suggesting an alternative approach to treating HER2/neu-positive tumors.


Assuntos
Anticorpos Antineoplásicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/farmacologia , Neoplasias/patologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Fator de Indução de Apoptose/metabolismo , Células COS , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Citocromos c/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Peptídeos/química , Estrutura Terciária de Proteína , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Neuroreport ; 19(9): 927-31, 2008 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-18520995

RESUMO

NDRG2, a member of the N-myc downstream-regulated gene (NDRG) family, is involved in cell differentiation and development. However, the distribution and function of Ndrg2 in the central nervous system remains unclear. Here, we analyzed the expression and distribution of Ndrg2 in the mouse brain and explored the potential physiological functions of Ndrg2. Ndrg2 was expressed in different regions of the brain, including the cerebral cortex, olfactory bulb, midbrain, hippocampus, and thalamus, with high levels in the midbrain and thalamus. Immunohistochemistry assay revealed that Ndrg2-positive cells distributed widely in the adult mouse brain and some of them showed nuclear staining. Indirect immunofluorescence and confocal microscopy studies showed that Ndrg2 protein colocalized with glial fibrillary acidic protein, indicating that Ndrg2 is expressed in astrocytes. Furthermore, Ndrg2 expression increased in glioma cells that were differentiating into astrocytes. Taken together, these findings suggest that Ndrg2 is possibly associated with glial cell proliferation and differentiation based on its immunolocalization in this study.


Assuntos
Sistema Nervoso/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/metabolismo , Imuno-Histoquímica/métodos , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso/anatomia & histologia , Neuroblastoma/metabolismo , Proteínas/genética , Fatores de Tempo
14.
Theranostics ; 8(10): 2846-2861, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29774079

RESUMO

Transforming growth factor-ß (TGF-ß) has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) during cancer progression, mainly by activating a set of pleiotropic transcription factors including SNAI2/Slug. However, the involvement of long non-coding RNAs (lncRNAs) in TGF-ß-induced Slug activation and EMT remains largely unknown. Methods: In this study, we used microarray analysis to compare lncRNA expression profiles between TGF-ß treated and untreated breast cancer cells. Then, the clinical significance of lncRNAs in breast cancer was investigated by qPCR and Kaplan-Meier survival analysis. The molecular mechanisms and EMT-promoting effects in vitro were analyzed by confocal laser microscopy, Western blotting, chromosome conformation capture (3C), chromatin isolation by RNA purification (ChIRP), ChIP, luciferase reporter assay and transwell migration assay. Lastly, the pro-metastatic effects in vivo were evaluated by bioluminescent imaging and hematoxylin and eosin (H&E) staining. Results: We observed that TGF-ß induced genome-wide changes in lncRNA levels in breast cancer cells, among which AC026904.1 and UCA1 were highly expressed in metastatic breast cancer and closely associated with poor prognosis. Mechanistic study revealed that AC026904.1 and UCA1 were upregulated by non-canonical and canonical TGF-ß pathways, respectively. Further analysis showed that AC026904.1 functions as an enhancer RNA in the nucleus, whereas UCA1 exerts a competitive endogenous RNA (ceRNA) activity in the cytoplasm. In addition, the biological functions of these two lncRNAs converged on the activation and maintenance of Slug, constituting a one-two punch in promoting EMT and tumor metastasis. Conclusion: These findings uncover for the first time that AC026904.1 and UCA1 could cooperatively upregulate Slug expression at both transcriptional and post-transcriptional levels, exerting critical roles in TGF-ß-induced EMT. The present work provides new evidence that lncRNAs function as key regulators of EMT and hold great promise to be used as novel biomarkers and therapeutic targets for metastatic breast cancer.


Assuntos
Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/genética , Fatores de Transcrição da Família Snail/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , RNA Longo não Codificante/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima
15.
Cancer Biol Ther ; 5(7): 842-7, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16760653

RESUMO

Breast cancer, the most common malignancy in women, has a known association with the steroid hormone estrogen. Estrogen receptor alpha (ERalpha) plays an important role in the clinical care of breast cancer patients, both as a prognostic factor and as a therapeutic target. Here, we show that a small interfering RNA (siRNA) against ERalpha downregulates ERalpha expression in human MCF-7 and Bcap-37 breast cancer cells, causing a significant decrease in breast cancer cell proliferation. Tumor cells lacking ERalpha expression grew at a much slower rate than did control cells in vitro. Moreover, ERalpha knockdown in breast cancer cells resulted in decreased, even completely abrogated tumor growth in BALB/c nude mice, providing direct evidence for an essential role of ERalpha in breast cancer growth. Our results suggest siRNA-mediated gene silencing of ERalpha may impair tumorigenicity, and even suppress the tumor growth.


Assuntos
Neoplasias da Mama/terapia , Receptor alfa de Estrogênio/antagonistas & inibidores , Terapia Genética/métodos , Interferência de RNA , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Receptor alfa de Estrogênio/genética , Feminino , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cancer Biol Ther ; 5(7): 860-6, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16861916

RESUMO

Increased resistance to apoptosis is a hallmark of many tumor cells. Survivin, a member of IAP family protein, is expressed in many human cancers and plays an important role in protecting cells from apoptosis. Here we show that vector-based small interfering RNAs (siRNA) stably knockdown survivin expression in several cancer cell lines, leading to increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin or TNF-alpha. The apoptotic susceptibility was dependent on divergent levels of survivin expression. The stable transfectants exhibited abnormal morphology, suppressed cell growth, enhanced spontaneous apoptosis and cell cycle hindrance. Furthermore, in nude mice xenografts of survivin-positive tumors, cells expressing survivin-targeted siRNAs exhibited decreased tumor formation and reduced angiogenesis. Results from these studies: (1) provide direct evidence that intracellular silencing of survivin by siRNA sensitizes human tumor cells to apoptosis; (2) define survivin as a promising molecular target for cancer therapy; and (3) suggest the potential applicability of survivin-targeted siRNA for treating human tumors, probably in combination with chemotherapy.


Assuntos
Neoplasias da Mama/terapia , Terapia Genética , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Neovascularização Patológica/terapia , RNA Interferente Pequeno/genética , Neoplasias do Colo do Útero/terapia , Animais , Apoptose , Neoplasias da Mama/irrigação sanguínea , Proliferação de Células , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Vetores Genéticos/genética , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Survivina , Neoplasias do Colo do Útero/irrigação sanguínea , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Sheng Li Xue Bao ; 58(4): 331-6, 2006 Aug 25.
Artigo em Zh | MEDLINE | ID: mdl-16906333

RESUMO

NDRG2, one of the new N-Myc downstream-regulated gene (NDRG) gene families, is believed to be involved in cell growth event. However, the exact function is still unknown. Identification of the tissue or cell types expressing this gene in vivo will provide clues in clarifying its physiological roles. Using RT-PCR and Western blot, we analyzed the expression level of NDRG2 mRNA and protein in human fetal tissues from different gestational ages. The anti-NDRG2 monoclonal antibody, which has been proved to react specifically with NDRG2 protein, was further used to analyze the cellular location of NDRG2 protein in various human fetal tissues by immunohistochemistry. We found that NDRG2 expression was developmentally dynamic, being generally lower in the early stages of development and markedly increasing during the later stages. NDRG2 mRNA and protein distribution were generally consistent in heart and lung. One of the differences was that NDRG2 protein appeared later than mRNA in kidney. Another unmatched expression was found in liver. NDRG2 mRNA appeared later than protein in liver. In human fetal tissues at sixteen and twenty-eight weeks of gestation, NDRG2 protein immunoreactions could be seen in epithelium of small intestine, epithelium of large intestine, superficial layer of epidermis, whisker follicles, epithelium of small bronchus, hepatocytes, cardiac myocytes, thymus corpuscles and epithelium of renal tubule, and the immunoreactions in those tissues from twenty-eight weeks of gestation was stronger than that from sixteen weeks of gestation. In the present study, we demonstrate the expression pattern and cellular location of NDRG2 protein in a large set of human fetal tissues. This is the first demonstration of NDRG2 protein expression in human fetal tissues. Taken together, the results suggest that NDRG2 protein found in a variety of tissues is not a tissue-specific protein, and may play important roles in histogenesis and organogenesis.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Supressoras de Tumor/metabolismo , Feto , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Proteínas Supressoras de Tumor/genética
18.
Int J Ophthalmol ; 9(4): 519-27, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27162722

RESUMO

AIM: To investigate the influence of hyperglycemia on the severity of choroidal neovascularization (CNV), especially the involvement of bone marrow-derived cells (BMCs) and underlying mechanisms. METHODS: BMCs from firefly luciferase (Fluc)/green fluorescent protein (GFP) double transgenic mice were transplanted into C57BL/6J wide-type mice. The recipient mice were injected intraperitoneally with streptozotocin (STZ) daily for 5 consecutive days to induce diabetes mellitus (DM), followed by CNV laser photocoagulation. The BMCs recruitment in CNV exposed to hyperglycemia was firstly examined in Fluc/GFP chimeric mice by in vivo optical bioluminescence imaging (BLI) and in vitro Fluc assays. The CNV severity was evaluated by H&E staining and choroidal flatmount. The expression of vascular endothelial growth factor (VEGF) and stromal cell derived factor-1 (SDF-1) was detected by Western Blot. RESULTS: BLI showed that the BMCs exerted dynamic effects in CNV model in Fluc/GFP chimeric mice exposed to hyperglycemia. The signal intensity of transplanted Fluc(+)GFP(+) BMCs in the DM chimeric mice was significantly higher than that in the control chimeric mice with CNV induction at days 5, 7, 14 and 21 (121861.67±9948.81 vs 144998.33±13787.13 photons/second/cm(2)/sr for control and DM mice, P 5d<0.05; 178791.67±30350.8 vs 240166.67±22605.3, P 7d<0.05; 124176.67±16253.52 vs 196376.67±18556.79, P 14d<0.05; 97951.60±10343.09 vs 119510.00±14383.76, P 21d<0.05), which was consistent with in vitro Fluc assay at day 7 [relative light units of Fluc (RLU1)], 215.00±52.05 vs 707.33±88.65, P<0.05; RLU1/ relative light units of renilla luciferase (RLU2), 0.90±0.17 vs 1.83±0.17, P<0.05]. The CNVs in the DM mice were wider than those in the control group at days 5, 7, 14 and 21 (147.83±17.36 vs 220.33±20.17 µm, P 5d<0.05; 212.17±24.63 vs 326.83±19.49, P 7d<0.05; 163.17±18.24 vs 265.17±20.55, P 14d<0.05; 132.00±10.88 vs 205.33±12.98, P 21d<0.05). The average area of CNV in the DM group was larger at 7d (20688.67±3644.96 vs 32218.00±4132.69 µm(2), P<0.05). The expression of VEGF and SDF-1 was enhanced in the DM mice. CONCLUSION: Hyperglycemia promots the vasculogenesis of CNV, especially the contribution of BMCs, which might be triggered by VEGF and SDF-1 production.

20.
World J Gastroenterol ; 11(14): 2130-5, 2005 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-15810079

RESUMO

AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line. METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line. p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot. MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index. RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined. The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58, P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell). CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/genética , Terapia Genética/métodos , Leucemia Eritroblástica Aguda/tratamento farmacológico , Piperazinas/farmacologia , Pirimidinas/farmacologia , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Sequência de Bases , Benzamidas , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Mesilato de Imatinib , Técnicas In Vitro , Células K562 , Dados de Sequência Molecular , Transfecção
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