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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.
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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.
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Infecciones por Citomegalovirus , Glioma , MicroARNs , Factores de Transcripción Activadores/genética , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Humanos , MicroARNs/genéticaRESUMEN
[This corrects the article DOI: 10.3892/ol.2015.3125.].
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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.
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Evolución Molecular Dirigida/métodos , Fosfotirosina , Proteínas Proto-Oncogénicas c-fyn , Proteínas Recombinantes , Dominios Homologos src/genética , Sitios de Unión/genética , Técnicas de Visualización de Superficie Celular , Escherichia coli/genética , Humanos , Biblioteca de Péptidos , Fosfotirosina/química , Fosfotirosina/metabolismo , Unión Proteica/genética , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas c-fyn/química , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
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.
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Glioblastoma is the most aggressive intracranial tumor and diffuse migration is the leading cause of death. Recent evidence has indicated that heterogeneous nuclear ribonucleoprotein A2B1 (hnRNP A2B1) is overexpressed in human glioblastoma tissue and enhances glioblastoma invasion in vitro. We found by mass spectrometry that hnRNP A2B1 interacts with human cytomegalovirus (HCMV) immediate early 86 protein (IE86, ie2 gene-encoded) in malignant glioma cells (U87MG) infected with HCMV. However, the role of hnRNP A2 B1 in glioblastoma development remains poorly understood. Here, we report that hnRNP A2B1 is highly expressed in the HCMV·ie2 transgenic mice model. This phenomenon was confirmed in U87MG cell lines transfected with pEGFP-N3-ie2 plasmid. In addition, hnRNP A2B1 knockdown in U87MG cells inhibited tumor migration, and this effect might be mediated by hnRNP A2B1 through effects on splicing patterns of RON. Our data suggested that HCMV· ie2 promotes glioblastoma migration by regulating hnRNP A2B1 expression.
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Glioblastoma/metabolismo , Glioblastoma/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transactivadores/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Invasividad Neoplásica/patologíaRESUMEN
Although there is a high risk of mood disorders and cognitive impairment in congenital human cytomegalovirus (HCMV) infections, the molecular pathogenetic mechanisms of HCMV have not yet been fully determined. In this study, we show that immediate-early 2 (IE2) protein modulates affective and cognitive behaviors. We used a UL122 genetically-modified mice model that can continuously express IE2 protein. We used a series of animal behavior tests to determine the relationship between HCMV-encoded IE2 and psychiatric disorders. In open-field, elevated plus-maze test and tail suspension tests, we found that UL122 genetically-modified mice displayed more anxiety-depression behavior than did wild-type (WT) mice. The Morris water maze test and novel object recognition test showed that spatial learning and memory were lower in UL122 genetically-modified mice model than in WT mice. Level of fibroblast growth factor 2 (FGF2) protein in the hippocampus cornu ammonia areas (CA1, CA3) and dentate gyrus (DG) of the experimental group was significantly lower, consistent with immunohistochemical staining and western blot for neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP). Levels of SYP and PSD-95 proteins were lower in the hippocampus UL122 genetically-modified mice. These data suggest the importance of HCMV-encoded IE2 for studying anxiety and depression behaviors and for the spatial learning and memory. This would help to further explain the molecular pathological mechanism of psychiatric disorders caused by HCMV infection.
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Human cytomegalovirus (HCMV), a ubiquitous pathogen, can cause severe illness in immunocompromised individuals. Typically, glioma is one of the most common malignant primary brain tumors and originates in the central nervous system. The IE86 gene of HCMV exerts a major role in regulating virus replication. By using coimmunoprecipitation combined with mass spectrometry, the components of the IE86 complex were identified, and the heterogeneous ribonucleoprotein A2/B1 (hnRNP A2/B1) was recognized as one of the IE86 complex components. hnRNP A2/B1 is highly expressed in U251 cells, and the data suggest that IE86 can promote hnRNP A2/B1 expression. Furthermore, the knockdown of hnRNP A2/B1 significantly attenuates IE86-mediated apoptosis and cell proliferation. Importantly, IE86 can also inhibit the alternative splicing of Bcl-x by decreasing the Bcl-xS/Bcl-xL ratio, which is closely related to apoptosis. Meanwhile, the knockdown of hnRNP A2/B1 can mitigate the inhibitory effect of IE86 on the alternative splicing of Bcl-x. In conclusion, the inhibition of apoptosis and enhancement of cell proliferation by IE86 may be related to the hnRNP A2/B1-mediated alternative splicing of Bcl-x.
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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.
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Neoplasias Encefálicas/patología , Infecciones por Citomegalovirus/genética , ADN-Topoisomerasas de Tipo II/genética , Glioblastoma/patología , MicroARNs/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Regiones no Traducidas 3' , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/patología , ADN-Topoisomerasas de Tipo II/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/virología , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Proteínas de Unión a Poli-ADP-Ribosa/metabolismoRESUMEN
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.
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Factores de Transcripción Activadores/metabolismo , Centrosoma/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Ubiquitinas/metabolismo , Factores de Transcripción Activadores/química , Factores de Transcripción Activadores/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Centrosoma/enzimología , Secuencia de Consenso , Secuencia Conservada , Eliminación de Gen , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Transporte de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Ubiquitinas/antagonistas & inhibidores , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
Congenital HCMV infection has been reported to be involved in learning and memory impairment, but whether HCMV IE2 plays a key role in the process remains unknown. The purpose of this study was to study the effects of IE2 on the expression levels of NMDA receptors and CX43 in the hippocampal neurons of ul122 transgenic mice. Firstly, the ul122 genetically modified mice models that can steadily and continuously express IE2 protein were established. Then, the mice were divided into the experimental group (positive mice identified) and the control group (wild type mice. n = 24 in each group). The establishment of ul122 genetically modified mice was identified by PCR technology. The learning and memory ability were measured using the Morris water-maze test. Western blot and immunohistochemical study were performed to detect the expression level of Cx43 and NMDA receptors. The results of PCR indicated that the ul122 genetically modified model was successfully constructed. Morris water maze test result showed that in the experimental group, less platform crossings and Quadrant time (%) compared to the control group, but there was no difference in escape latency. The expression level of Cx43 in the hippocampus CA1 of the experimental group was significantly reduced in keeping with NMDA receptors in immunohistochemistry. The significant decreased expression level of Cx43 and NMDA receptors in the ul122 genetically modified mice hippocampus may be connection with the mechanism for spatial memory impairment.
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Topoisomerase (DNA) II alpha (TOP2A), an enzyme that controls and alters the topologic states of DNA during transcription, is aberrantly expressed in many cancers. However, few studies have investigated expression of TOP2A and its clinical significance in glioma. We retrieved six independent investigations from the Oncomine database and found that TOP2A is highly expressed in glioma tissues compared with corresponding normal controls. Similar results were also found in clinical specimens at the protein level. Immunohistochemical analysis indicated that TOP2A over expression was highly correlated with grade stage, KI67 positive percentage, IDH1 mutation, and age, but other clinical parameters such as sex distribution and tumor size were barely associated with high TOP2A gene expression. Meanwhile we used Prognos can to assess the prognostic value of TOP2A expression in glioma patients, and found that high expression was associated with poor prognosis of patients with glioma. Furthermore, we used the Gene-Cloud of Biotechnology Information (GCBI) bioinformatics platform predict the role of TOP2A in glioma. It was not only involved in DNA replication, chromosome condensation, and responses to DNA damage stimuli, but also promoted cancer cell mitotic cell cycle and apoptosis, and phosphatidylinositol-mediated signaling by regulating gene expression. By these approaches we demonstrate that TOP2A may be a reliable prognostic factor or therapeutic target in glioma.
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Glioblastoma, the most common primary brain tumor of adults, is characterized by poor survival rates. Programmed death ligand 1 (PD-L1, CD274) has been implicated in the immune escape of glioblastoma. The presence of human cytomegalovirus (HCMV) in glioblastoma multiforme (GBM) has sparked considerable interest and controversy. The exposure of toll-like receptor 3 (TLR3) to pathogens induces an antiviral state in cells or in animals. In the current study, the expression of PD-L1 and TLR3 in HCMV-infected glioma specimens was observed to be higher compared to the control. We therefore investigated if PD-L1 expression in glioblastoma is mediated by TLR3 triggering in HCMV infected glioblastoma. TLR3 siRNA transfections were utilized to identify the induction of PD-L1 via TLR3 triggering in HCMV infected cell lines. Also, IL-8 and TGF-ß were detected by ELISA for the antitumor role of TLR3. Thus, we propose a novel immune treatment using a combination of PD-L1 blockade with TLR3 triggering against HCMV infected glioblastoma.
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Non-alcoholic fatty liver disease (NAFLD), a liver manifestation of metabolic syndrome, is associated with considerable health and socioeconomic burdens in many populations worldwide. Recent studies suggest that human cytomegalovirus (HCMV) infection might play a role in the pathogenesis of metabolic diseases, including NAFLD, but it is still unclear whether HCMV-encoded IE2 plays an important role in this process. Interestingly, SREBP1c was recently reported to play critical roles in the development of hepatic steatosis. In this study, we aimed to study the IE2 effect on the expression levels of SREBP1c and on lipid metabolism in the liver of UL122 genetically modified mice. First, UL122 genetically modified mice models that can steadily and continuously express IE2 protein were established. Then, the mice were divided into the experimental group (positive mice identified) and the control group (wild-type mice, n=16 per group). The establishment of UL122 genetically modified mice was identified by PCR technology. The triglyceride content in their livers was measured using a colorimetric assay and oil red O-stain. Real-time PCR and immunohistochemistry were performed to detect the expression levels of SREBP1c mRNA and protein after HCMV infection. We found that SREBP1c expression was significantly elevated in the experimental group, and its overexpression in the liver cells can promote triglyceride accumulation and hepatic steatosis. Taken together, our data collectively demonstrate that HCMV infection is highly associated with NAFLD, SREBP1c overexpression promotes hepatic steatosis, and this up-regulation is most likely mediated by IE2.
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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.
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Arginasa/biosíntesis , Citomegalovirus/genética , Glioblastoma/genética , MicroARNs/genética , Adulto , Anciano , Apoptosis/genética , Arginasa/genética , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/genética , Citomegalovirus/patogenicidad , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Glioblastoma/virología , Humanos , Estimación de Kaplan-Meier , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Invasividad Neoplásica/genéticaRESUMEN
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.
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Antineoplásicos/farmacología , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Glioma/tratamiento farmacológico , Glioma/patología , MicroARNs/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Glioma/metabolismo , Humanos , Relación Estructura-ActividadRESUMEN
Human cytomegalovirus (HCMV), a widespread beta-herpes virus, infects a high percentage of gliomas. HCMV is specifically detected in human gliomas at a low level of expression raises the possibility that it may regulate the malignant phenotype in a chronic manner. Although HCMV is not recognized as an oncogenic virus, it might dysregulate signaling pathways involved in initiation and promotion of malignancy.Here, our immunohistochemical staining reveals that nucleus staining of the HCMV 86-kDa immediate-early protein (IE86) is markedly increased in GBM (58.56%) compared with that in nontumorous samples (4.20%) and low-grade glioma(19.56%). IE86 staining positively correlates with the staining of activating transcription factor 5 (ATF5) which is essential for glioma cell viability and proliferation suggesting that HCMV IE86 could have important implications in glioma biology. Moreover, we find that the IE86 overexpression enhances glioma cell's growth in vitro and in vivo. We demonstrate that IE86 protein physically interacts with, and acetylates ATF5 thereby promoting glioma cell survival. Therefore, our findings illustrate the biological significance of HCMV infection in accelerating glioma progression, and provide novel evidence that HCMV infection may serve as a therapeutic target in human glioma.
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Factores de Transcripción Activadores/metabolismo , Antígenos Virales/metabolismo , Citomegalovirus/fisiología , Glioma/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Acetilación , Factores de Transcripción Activadores/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Glioma/genética , Glioma/patología , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Clasificación del Tumor , Unión Proteica , Interferencia de ARN , Factores de Transcripción p300-CBP/metabolismoRESUMEN
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.
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Antígeno AC133/análisis , Transformación Celular Viral , Citomegalovirus/crecimiento & desarrollo , Nestina/análisis , Neuroglía/virología , Receptor Notch1/análisis , Factores de Transcripción Activadores/análisis , Línea Celular Tumoral , Proliferación Celular , Perfilación de la Expresión Génica , HumanosRESUMEN
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.
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Astrocitos/inmunología , Astrocitos/virología , Proteínas Portadoras/metabolismo , Citomegalovirus/inmunología , Citomegalovirus/fisiología , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Células Cultivadas , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Embarazo , ARN Viral/análisis , Proteínas de Unión al ARN , Carga Viral , Proteínas Virales/análisisRESUMEN
Transcription factors, which represent an important class of proteins that play key roles in controlling cellular proliferation and cell cycle modulation, are attractive targets for cancer therapy. Previous researches have shown that the expression level of activating transcription factor 5 (ATF5) was frequently increased in glioma and its acetylation level was related to glioma. The purposes of this study were to explore the methylation level of ATF5 in clinical glioma tissues and to explore the effect of ATF5 methylation on the expression of ATF5 in glioma. Methylation of the promoter region of ATF5 was assayed by bisulfite-specific polymerase chain reaction (PCR) sequencing analysis in 35 cases of glioma and 5 normal tissues. Quantitative real-time PCR (qRT-PCR) was also performed to detect ATF5 mRNA expression in 35 cases of glioma and 5 normal tissues. Clinical data were collected from the patients and analyzed. The percentages of methylation of the ATF5 gene in the promoter region in healthy control, patients with well-differentiated glioma, and those with poorly differentiated glioma were 87.78%, 73.89%, and 47.70%, respectively. Analysis of the methylation status of the promoter region of the ATF5 gene showed a gradually decreased methylation level in poorly differentiated glioma, well-differentiated glioma, and normal tissues (P<0.05). There was also a significant difference between well-differentiated glioma and poorly differentiated glioma (P<0.05). ATF5 mRNA expression in glioma was significantly higher than that in the normal tissues (P<0.05). This study provides the first evidence that the methylation level of ATF5 decreased, and its mRNA expression was evidently up-regulated in glioma.