RESUMO
Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding protein family of transcription factors. ATF5 regulates stress responses and cell survival, proliferation, and differentiation and also plays a role in viral infections, cancer, diabetes, schizophrenia, and the olfactory system. Moreover, it was found to also have a critical cell cycle-dependent structural function at the centrosome. However, the mechanism that controls the localization of ATF5 at the centrosome is unclear. Here we report that ATF5 is small ubiquitin-like modifier (SUMO) 2/3-modified at a conserved SUMO-targeting consensus site in various types of mammalian cells. We found that SUMOylation of ATF5 is elevated in the G1 phase of the cell cycle and diminished in the G2/M phase. ATF5 SUMOylation disrupted the interaction of ATF5 with several centrosomal proteins and dislodged ATF5 from the centrosome at the end of the M phase. Of note, blockade of ATF5 SUMOylation deregulated the centrosome cycle, impeded ATF5 translocation from the centrosome, and caused genomic instability and G2/M arrest in HeLa cells. Our results indicate that ATF5 SUMOylation is an essential mechanism that regulates ATF5 localization and function at the centrosome.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Centrossomo/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitinas/metabolismo , Fatores Ativadores da Transcrição/química , Fatores Ativadores da Transcrição/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Centrossomo/enzimologia , Sequência Consenso , Sequência Conservada , Deleção de Genes , Instabilidade Genômica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/química , Ubiquitinas/genéticaRESUMO
Glioblastoma (GBM), the most common type of primary tumor in the central nervous system, is a very aggressive brain tumor with poor prognosis and a high recurrence rate. Increasing evidence suggests that human cytomegalovirus (HCMV) infection is related to GBM and leads to GBM cell growth and metastasis. MicroRNAs are important regulators in the growth and metastasis of glioblastoma. This study aimed to demonstrate the role of miR-144-3p in HCMV-positive glioblastoma. We found that, after HCMV infection, the expression of miR-144-3p decreased, whereas the expression of TOP2A increased. Bioinformatics analyses indicated that miR-144-3p directly targets the TOP2A 3'-UTR (Untranslated Region). We discovered that the overexpression of miR-144-3p downregulated the overexpression of TOP2A and inhibited the proliferation, clone formation, and invasion of HCMV-positive glioma in vitro. Taken together, these results show that miR-144-3p inhibited growth and promoted apoptosis in glioma cells by targeting TOP2A.
Assuntos
Neoplasias Encefálicas/patologia , Infecções por Citomegalovirus/genética , DNA Topoisomerases Tipo II/genética , Glioblastoma/patologia , MicroRNAs/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões 3' não Traduzidas , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/patologia , DNA Topoisomerases Tipo II/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/virologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Proteínas de Ligação a Poli-ADP-Ribose/metabolismoRESUMO
Glioma is the most common malignant primary brain tumor which arises from the central nervous system. Our studies reported that an anti-apoptotic factor, activating transcription factor 5 (ATF5), is highly expressed in malignant glioma specimens and cell lines. Downregulation by dominant-negetive ATF5 could repress glioma cell proliferation and accelerate apoptosis. Here, we further investigate the upstream factor which regulates ATF5 expression. Bioinformatic analysis showed that ATF5 was a potential target of miR-141-3p. Luciferase reporter assay verified that miR-141-3p specifically targeted the ATF5 3'-UTR in glioma cells. Functional studied suggested that miR-141-3p overexpression inhibited proliferation and promoted apoptosis of glioma cells (U87MG and U251). Xenograft experiments proved the inhibition of miR-141-3p on glioma growth in vivo. Moreover, exogenous ATF5 without 3'-UTR restored the cell proliferation inhibition triggered by miR-141-3p. Taken together, we put forward that miR-141-3p is a new upstream target towards ATF5. It can serve as a crucial tumor suppressor in regulating the ATF5-regulated growth of malignant glioma.
Assuntos
Antineoplásicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Glioma/tratamento farmacológico , Glioma/patologia , MicroRNAs/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Glioma/metabolismo , Humanos , Relação Estrutura-AtividadeRESUMO
Glioblastoma (GBM) are the most common and aggressive tumors of human brain. Recent studies showed that human cytomegalovirus (HCMV) can induce malignant transformation of tumor cells to maintain stemness. Transcription factor 5 (ATF5) is an anti-apoptotic protein that is highly expressed in malignant glioma. The aim of this study is to investigate the effect of HCMV infection on the stem cell makers of U251 cells. U251 cells were infected by AD169 HCMV strain (MOI = 1). The expression of stem cell makers (CD133, NES, Notch1) in infected U251 cells were compared with the expression in uninfected U251 cell to see the difference between them. Then, the changes of cell proliferation activity and the expression level of Notch intracellular domain (NICD), Notch1, ATF5, and IE protein were detected in the infected cells, and the expressions of Notch1 and NICD were increased. Cell proliferation assay showed that HCMV infection significantly increased the proliferation. These cells could form tumor spheres in non-adherent conditions. Consistent with these findings, the effect of silencing ATF5 on the proliferation of HCMV-infected U251 cells was also examined. The result shows that short interfering RNA-mediated ATF5 downregulation inhibited this process. These findings imply that HCMV infection may regulate ATF5 signaling pathway to increase cell malignant traits and maintain stemness. J. Med. Virol. 89:878-886, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Antígeno AC133/análise , Transformação Celular Viral , Citomegalovirus/crescimento & desenvolvimento , Nestina/análise , Neuroglia/virologia , Receptor Notch1/análise , Fatores Ativadores da Transcrição/análise , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , HumanosRESUMO
The prominent feature of human cytomegalovirus (HCMV) is cell tropism specificity for human fetal nervous system, which leads to severe fetal nervous system damage especially in first-trimester gestation. In this study, human astrocytes isolated from fetal brain were infected with HCMV AD169 and whole genome transcriptome profile was performed. The results showed that the gene expression of interferon stimulated genes (ISGs), chemokine and chemokine receptors were significantly up-regulated (P < 0.01). The antiviral replication effects of IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1, Fc = 148.17) was investigated. Lentivirus with IFIT1 overexpression or knockdown was transduced into astrocytes, respectively. The viral mRNA, protein expression and HCMV titers were determined. The results showed that IE1, IE2, pp65, and viral titers were significantly decreased in IFIT1 overexpression group and enhanced in the knockdown group compared with control one (P < 0.01). Taken together, this study revealed IFIT1 played an important antiviral role in HCMV infected fetal astrocytes. The prominent feature of human cytomegalovirus (HCMV) is cellular tropism specificity for human fetal brain nervous system leading to severe fetal nervous damage especially in first-trimester gestation. In this study, human astrocytes isolated from first-trimester fetal brain were infected with HCMV AD169 and IFIT1 was studied for its antiviral replication effects. The results provided insights into the function of IFIT1 as a key factor in antiviral defense contributing to development of targeted therapeutics to fetal brain with HCMV infection. J. Med. Virol. 89:672-684, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Astrócitos/imunologia , Astrócitos/virologia , Proteínas de Transporte/metabolismo , Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Células Cultivadas , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Gravidez , RNA Viral/análise , Proteínas de Ligação a RNA , Carga Viral , Proteínas Virais/análiseRESUMO
Glioblastoma is the most common and malignant tumour that occurs primarily in nervous system and has a high morbidity. Research on glioblastoma has recently focused on human cytomegalovirus, belonging to the beta subfamily of Herpesviridae that plays crucial roles in cancer development and progression. This study aimed to investigate the role of human cytomegalovirus-associated microRNA-613 in glioblastoma. In this study, we demonstrate that microRNA-613 expression was frequently reduced in human cytomegalovirus-positive glioblastoma specimens/cells compared with human cytomegalovirus-negative glioblastoma tissue/cells, and a significant correlation was observed between the reduction in microRNA-613 expression and the presence of unfavourable variables, including tumour size (p = 0.0118), World Health Organization stage (p = 0.0169), the overall survival (p = 0.0107) and disease-free (p = 0.0159) survival of patients. Overexpression of microRNA-613 in the glioblastoma cell lines U87 and U251 retarded cell growth and induced cell apoptosis. Upregulation of microRNA-613 inhibited glioblastoma cell clone formation, invasion and migration. Furthermore, we demonstrated that arginase-2 was directly regulated by microRNA-613 and played an essential role in mediating the biological effects of microRNA-613 in glioblastoma. Re-expression of arginase-2 markedly reversed the inhibitory properties of microRNA-613 in glioblastoma cells. Taken together, our data provide compelling evidence that human cytomegalovirus reduced the level of microRNA-613 which functions as an anti-onco-miRNA in glioblastoma, primarily by downregulating the expression of arginase-2.
Assuntos
Arginase/biossíntese , Citomegalovirus/genética , Glioblastoma/genética , MicroRNAs/genética , Adulto , Idoso , Apoptose/genética , Arginase/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Citomegalovirus/patogenicidade , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Glioblastoma/virologia , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Invasividade Neoplásica/genéticaRESUMO
BACKGROUND: Survivin, an inhibitor of apoptosis, is overexpressed in pancreatic ductal adenocarcinoma (PDAC). Its expression is known to be associated with poor clinical outcome. However, to our knowledge, there has been no study to characterize its usefulness as a serum marker for human pancreatic cancer. Furthermore, the relation between survivin expression and the serum level of survivin has not been widely studied in PDAC. We performed this study to investigate the expression and serum level of survivin in PDAC and its clinical significance as a prognostic factor. METHODS: We performed immunohistochemical staining for survivin in formalin-fixed, paraffin-embedded blocks from 80 PDAC tissues. The serum level of survivin from the patients (n = 80) and age-matched healthy volunteers (n = 80) were analyzed by enzyme-linked immunosorbent assays (ELISAs) prior to surgical resection. Levels of expression were correlated with clinicopathological parameters. RESULTS: Serum survivin concentrations were significantly elevated in patients with PDAC when compared with healthy sera (P < 0.001). High serum survivin levels were significantly associated with perineural invasion, venous invasion, lymph node status (N stage), cell differentiation, and recurrence but not with the tumor size, age, gender of the patients, or tumor location. The median overall survival time of the group with normal serum survivin levels was longer than that of the group with elevated serum survivin. The independent factors associated with overall survival were advanced pancreatic cancer and elevated serum survivin level. Of the 80 cases of PDAC, 65 (81.25 %) were positive for survivin expression. There were significant associations between survivin expression and perineural invasion, venous invasion, and lymph node status. A significant difference in overall survival was associated with survivin expression. CONCLUSIONS: Patients with elevated serum survivin level and high survivin expression at diagnosis demonstrated a poor outcome. Detection of serum survivin or tissue survivin expression may predict the prognosis of patients with PDAC.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/sangue , Proteínas Inibidoras de Apoptose/sangue , Recidiva Local de Neoplasia/sangue , Neoplasias Pancreáticas/sangue , Adulto , Idoso , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Estudos de Casos e Controles , Terapia Combinada , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Estadiamento de Neoplasias , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Prognóstico , Taxa de Sobrevida , Survivina , Neoplasias PancreáticasRESUMO
Human cytomegalovirus (HCMV) infections are the leading cause of viral induced birth defects, affecting the central nervous system (CNS) primarily. Fetal CNS is especially vulnerable to CMV induced injury. As HCMV permissive cells, astrocytes are responsible for major glutamate transport and regulate extracellular levels of glutamate avoiding its accumulation which is implicated in neurodegenerative disorders. In this study, highly purified astrocytes isolated from human first trimester aborted fetal brain were infected with HCMV AD169, glutamate uptake function was detected by (3)H labeling technic, and the expression level alterations of glutamate transporters (GLAST, GLT-1), glutamine synthetase (GS) and its activity were also investigated. Protein kinases C (PKC) inhibitor treatment was to identify whether PKC signalling involved in regulating glutamate uptake, protein expression of GLAST, GLT-1, GS and GS activity. Results indicated HCMV AD169 infection could modulate glutamate uptake, expression levels of GLAST, GLT-1, GS and it activity through PKC signalling, suggesting a great susceptibility of human fetal astrocytes to HCMV infection, which significantly alters the uptake and metabolism of an important excitatory amino acid, glutamate, may be a potential mechanism for HCMV associated neurological disease, and an effective therapeutic target in neural diseases.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Citomegalovirus/fisiologia , Feto/metabolismo , Ácido Glutâmico/metabolismo , Feto/citologia , HumanosRESUMO
M-IL-2((88)Arg, (125)Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2((88)Arg, (125)Ala). In this study, we constructed an expression system of M-IL-2((88)Arg, (125)Ala) in Pichia pastoris: GS115/pPICZα A/M-IL-2((88)Arg, (125)Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2((88)Arg, (125)Ala) reached up to 814.5mg/L, higher than the system in Escherichiacoli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26kDa, conforming the theoretical value. And M-IL-2((88)Arg, (125)Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2((88)Arg, (125)Ala) using P. pastoris as an expression host and paves the way to clinical practice.
Assuntos
Interleucina-2/genética , Meliteno/genética , Neoplasias Ovarianas/tratamento farmacológico , Pichia/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Nucleophosmin (NPM1/B23) and the activating transcription factor 5 (ATF5) are both known to subject to cell type-dependent regulation. NPM1 is expressed weakly in hepatocytes and highly expressed in hepatocellular carcinomas (HCC) with a clear correlation between enhanced NPM1 expression and increased tumor grading and poor prognosis, whereas in contrast, ATF5 is expressed abundantly in hepatocytes and down-regulated in HCC. Re-expression of ATF5 in HCC inhibits cell proliferation. We report here that using an unbiased approach, tandem affinity purification (TAP) followed with mass spectrometry (MS), we identified NPM1 as a novel ATF5-interacting protein. Unlike many other NPM1-interacting proteins that interact with the N-terminal oligomerization domain of NPM1, ATF5 binds via its basic leucine zipper to the C-terminal region of NPM1 where its nucleolar localization signal is located. NPM1 association with ATF5, whose staining patterns partially overlap in the nucleoli, promotes ATF5 protein degradation through proteasome-dependent and caspase-dependent pathways. NPM1-c, a mutant NPM1 that is defective in nucleolar localization, failed to stimulate ATF5 polyubiquitination and was unable to down-regulate ATF5. NPM1 interaction with ATF5 displaces HSP70, a known ATF5-interacting protein, from ATF5 protein complexes and antagonizes its role in stabilization of ATF5 protein. NPM1-promoted ATF5 down-regulation diminished ATF5-mediated repression of cAMP-responsive element-dependent gene transcription and abrogates ATF5-induced G(2)/M cell cycle blockade and inhibition of cell proliferation in HCC cells. Our study establishes a mechanistic link between elevated NPM1 expression and depressed ATF5 in HCC and suggests that regulation of ATF5 by NPM1 plays an important role in the proliferation and survival of HCC.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Carcinoma Hepatocelular/metabolismo , Caspases/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fatores Ativadores da Transcrição/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Caspases/genética , Pontos de Checagem do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mutação , Proteínas de Neoplasias/genética , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/genética , Nucleofosmina , Complexo de Endopeptidases do Proteassoma/genética , Elementos de Resposta/genética , Ubiquitinação/genéticaRESUMO
BACKGROUND: To understand the prevalence and characteristics of human cytomegalovirus (HCMV) infection in children in the Weifang area, and to provide information for its prevention and treatment. METHODS: A comprehensive survey was performed from 2009 to 2012 in 7582 children from birth to 6 years of age hospitalized in the Maternity and Child Health Hospital of Weifang. ELISA HCMV serology results and survey data were analyzed by age group and socio-economic level. The infection rates were based on IgG and IgM serology. RESULTS AND CONCLUSIONS: The overall infection rate from IgG and IgM in the Weifang area from 2009 to 2012 was 42.5% (3496/7582), among which 34.2% were HCMV-IgG positive, suggesting past infection. Overall, the probability of active HCMV infection showed no gender difference in any age group (P >0.05). Recent infections centered on the first 6 months of life, presumably due to breastfeeding. Among the 654 children hospitalized for active HCMV infection, 379 (57.9%) were from rural areas and 275 (42.1%) from urban areas, showing that active infection in the countryside was higher than that in the city (χ2 = 32.65, p <0.01).
Assuntos
Anticorpos Antivirais/sangue , Infecções por Citomegalovirus/epidemiologia , Citomegalovirus/imunologia , Fatores Etários , Criança , Pré-Escolar , China/epidemiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Recém-Nascido , Masculino , Estudos Soroepidemiológicos , Fatores SocioeconômicosRESUMO
Background: We have previously reported that human cytomegalovirus (HCMV) infection could promote the progression of glioma. Here we discovered a stress-induced nuclear protein ZC3H11A (ZC3) through high-throughput sequencing after HCMV infection, which has been reported recently by our research group in regulating mRNA export under stress conditions. And also, a thorough analysis of ZC3 in pan-cancer and the omics data of ZC3 are yet to be conducted. Methods: The transcriptomes of glioma cells after HCMV infection were assessed by RNA sequencing. ZC3 mRNA and protein level following HCMV infection were validated and measured by qRT-PCR and Western-blot. The RNA sequencing and protein expression information of ZC3 across pan-cancer were analyzed and visualized by R packages. The localization of ZC3 protein was assessed by IHC images from HPA. The ZC3 proteomics and transcriptomics data in different cancers were extracted through the CPTAC data portal, and comparisons were conducted with a Python script. The genetic alteration, survival prognosis, immune infiltration analysis of ZC3 in pan-cancer were analyzed by cBioPortal, TCGA, and TIMER2 databases. The protein interaction networks were revealed by STRING, GEPIA2 and TCGA. Results: Genes in mRNA processing pathways were upregulated after HCMV infection and ZC3 expression in mRNA and protein level was validated. We also discovered that the status of ZC3 were generally at high levels in cancers, although varied among different cancer types. ZC3 protein in tumor cells localized to the nuclear whereas in normal cells it was mainly found in cytoplasmic/membranous. However, from ZC3 proteomics and transcriptomics data in some cancer types, the increase in ZC3 protein was not accompanied by a significant elevation in mRNA level. Additionally, our analysis indicated that elevated ZC3 expression was primarily linked to a negative prognosis in majority cancers but still varied depending on the cancer types. Our annotation analysis suggested that ZC3-related proteins are mainly involved in mRNA processing clusters. Conclusion: We demonstrated that ZC3 significantly impacted by HCMV infection in gliomas. Furthermore, we identified a set of genes exhibiting analogous expression patterns to ZC3H11A in TCGA pan-cancer cohorts, implying a potential functional role for ZC3H11A in mRNA processing. Our study provided valuable insights into the role of a new mRNA export protein ZC3 in HCMV infection and pan-cancer progression. These results lay the foundation for our next research on the regulatory mechanism of ZC3 in virus-infected tumors.
RESUMO
The activating transcription factor 5 (ATF5) is highly expressed in many kinds of tumors including glioblastoma and breast cancers, but its expression in epithelial ovarian neoplasms has not been investigated. Here, we show that ATF5 is highly expressed in the majority of epithelial ovarian cancer samples (43/60) as compared with benign ovarian tumor tissues (4/13) and normal ovarian tissues (1/10). Furthermore, we found that ATF5 expression significantly correlated with advanced clinical stage (P<0.05) and poor differentiation of epithelial ovarian carcinomas (P<0.05). Previous studies suggested that ATF5 is required for the survival of cancer cells, but the mechanisms by which ATF5 regulates genes and promotes cell survival are not clear. Our data additionally demonstrated that interference with the function of ATF5 could markedly increase the apoptosis of ovarian cancer cells and identified B-cell leukemia lymphoma-2 as an ATF5-targeted apoptosis-related gene. These findings may provide potential therapeutic application in epithelial ovarian cancer.
Assuntos
Fatores Ativadores da Transcrição/fisiologia , Apoptose , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Fatores Ativadores da Transcrição/análise , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/química , Neoplasias Ovarianas/química , Proteínas Proto-Oncogênicas c-bcl-2/análiseRESUMO
[This corrects the article DOI: 10.3892/ol.2015.3125.].
RESUMO
Human cytomegalovirus (HCMV) harnesses a cell-specific manner to infect human nervous system cancer cells, establishes a life-long persistent infection without cell death, and modulates signaling pathways associated with cancer. We previously identified that the HCMV immediate-early 2 (IE2-86) protein binds and activates activating transcription factor 5 (ATF5), a survival factor in many tumor cells. In this study, we investigated a new mechanism of stress-induced miRNA regulation at the ATF5 3' UTR under the HCMV infection and other cellular stress conditions. We employed RNA-Seq and in silico analysis to screen stress response gene sets and identify miRNA candidates as potential regulators of ATF5 following HCMV infection. We found that ATF5 and cellular stress response genes were significantly upregulated under HCMV infection and diverse stress conditions. Three downregulated miRNAs were filtrated based on our threshold, and their binding sites for 3' UTR of ATF5 were predicted. Then, luciferase reporter assays were carried out to verify the binding sites for all three miRNA candidates targeting ATF5. However, in vitro validation has shown that miR-134-5p is the only candidate that can reverse the ATF5 protein upregulation under infection and other cell stresses. Additionally, miR-134-5p levels were significantly reduced and inversely related to ATF5 mRNA under HCMV infection. These results provide new evidence that quiescent HCMV infection can trigger a stress response in glioma cells and modulate ATF5 levels by downregulating specific miRNA.
Assuntos
Infecções por Citomegalovirus , Glioma , MicroRNAs , Fatores Ativadores da Transcrição/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Humanos , MicroRNAs/genéticaRESUMO
Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple-mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two-orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross-reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr-containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.
Assuntos
Evolução Molecular Direcionada/métodos , Fosfotirosina , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes , Domínios de Homologia de src/genética , Sítios de Ligação/genética , Técnicas de Visualização da Superfície Celular , Escherichia coli/genética , Humanos , Biblioteca de Peptídeos , Fosfotirosina/química , Fosfotirosina/metabolismo , Ligação Proteica/genética , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-fyn/química , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
To develop a gene therapeutic method for human cytomegalovirus (HCMV), the IE86 specific short hairpin (sh) RNA expressing vector was constructed and subsequently transfected into MRC-5 cells. After infection of these cells with HCMV AD169, expression of IE86 was reduced strikingly as compared to the control. In addition, the inhibitory effect corresponded to a decrease in viral DNA replication and the virus-induced cytopathic effect. Measurement of the virus yield demonstrated that infection of cells expressing IE86-specific shRNA resulted in suppression of the formation of infectious viral progeny. These observations indicate that IE86 can be used as an effective target against HCMV infection using RNA interference (RNAi) technology, which provides new possibilities for anti-HCMV studies.
Assuntos
Infecções por Citomegalovirus/genética , Citomegalovirus/fisiologia , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Sequências Repetidas Invertidas , Interferência de RNA , RNA Interferente Pequeno/genética , Transativadores/deficiência , Transativadores/genética , Linhagem Celular , Citomegalovirus/metabolismo , Efeito Citopatogênico Viral/genética , Replicação do DNA/genética , DNA Viral/biossíntese , Terapia Genética , Vetores Genéticos/genética , Humanos , RNA Mensageiro/genética , Transfecção , Replicação Viral/genéticaRESUMO
OBJECTIVE: This study aims to investigate the effect of human cytomegalovirus (HCMV) infection on epithelial-to-mesenchymal transition (EMT) in glioblastoma cells and the possible underlying molecular mechanism. METHODS: We established primary cell cultures and measured the expression of the HCMV immediate early protein (IE1) to determine HCMV infection by immunohistochemical assays. Human glioma cells were divided into four groups: primary HCMV-positive, primary HCMV-negative, HCMV-positive U87, and HCMV-negative U87 cells. Cells were treated with transforming growth factor (TGF-ß1, 5 ng/ml) to induce EMT. Morphologic changes of the cells were observed microscopically at 0, 24, 48, and 72 h post TGF-ß1 treatment. Following EMT induction, E-cadherin and vimentin were detected using RT-PCR. Expression of MMP-2, E-cadherin, and vimentin was measured by western blotting. The invasiveness of glioma cells was also measured using the Transwell migration assay and a wound-healing assay. RESULTS: Morphologic changes in primary glioblastoma cells and U87 cells were observed at different times after exposure to TGF-ß1, and the extent of these changes was greater in HCMV-positive compared with HCMV-negative cells. Following exposure to TGF-ß1, the transcription of E-cadherin was significantly lower in HCMV-positive primary cells and U87 cells compared with HCMV-negative cells (P<0.01), which was consistent with the results of western blotting. The expression levels of vimentin were also elevated in HCMV-positive cells at 48 and 72 h. HCMV-positive U87 cells were significantly more invasive and migratory than HCMV-positive primary cells. TGF-ß1 and HCMV were observed to accelerate EMT and cell invasion by the Jun N-terminal kinase (JNK) pathway. Collectively, our findings indicate that HCMV and TGF-ß1 promoted cell invasion and migration in glioma cells by the JNK pathway. CONCLUSION: HCMV infection can promote EMT and strengthen the invasiveness of glioma cells.
RESUMO
Human cytomegalovirus is a common human pathogen that can cause life-threatening disease under certain conditions. During infection of host cells, the virus expresses regulatory proteins such as IE72 and IE86 that are important for viral propagation. IE86 plays a critical role in the modulation of viral replication as well as host cell cycle control and apoptosis. In this study, a Tet-On system was used to quantify the effect of IE86 on apoptosis and p53 expression. Our results indicate that IE86 inhibits tumor necrosis factor (TNF)-alpha induced apoptosis and that the anti-apoptotic activity of this viral protein correlates with its expression levels. In addition, IE86 did not alter the mRNA level of p53. The system developed should provide a method for functional analysis of human cytomegalovirus (HCMV) IE86 protein.
Assuntos
Apoptose/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Transativadores/fisiologia , Sequência de Bases , Ciclo Celular/fisiologia , Linhagem Celular , Primers do DNA , Doxiciclina/farmacologia , Citometria de Fluxo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). METHODS: U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. RESULTS: The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P < 0.05). CONCLUSION: HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.