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1.
J Cancer Res Clin Oncol ; 145(10): 2433-2444, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485767

RESUMO

PURPOSE: The clinical importance of cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC) is well recognized. However, a reliable method for the detection of functioning CSC has not yet been established. We hypothesized that YAP1, a transcriptional coactivator, and SOX2, a master transcription factor of SCC, may cooperatively induce stemness through transcriptional reprogramming. METHODS: We immunohistochemically examined the expression of SOX2 and YAP1 in the CD44 variant 9 (CD44v9)-positive invasion front. A CSC-inducible module was identified through a combination of siRNAs and sphere formation assays. YAP1 and SOX2 interactions were analyzed in vitro. RESULTS: The triple overexpression of SOX2, YAP1, and CD44v9 was significantly associated with poor prognosis. TCGA data revealed that the CSC-inducible module, which was related to EMT and angiogenesis, was significantly correlated with poor prognosis. The KLF7 expression, representatively chosen from the module, also correlated with poor prognosis and was essential for sphere formation and CSC propagation. Sphere stress-activated YAP1 enhanced SOX2 activity. CONCLUSIONS: The stress-triggered activation of YAP1/SOX2 transcriptionally reprograms HNSCC for the acquisition of stemness. Triple SOX2, YAP1, and CD44v9 immunostaining assays may be useful for the selection of high-risk patients with functioning CSCs, and YAP1 targeting may lead to the development of a CSC-targeting therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Estresse Fisiológico , Ativação Transcricional , Biomarcadores , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Estresse Fisiológico/genética
2.
Braz J Med Biol Res ; 52(10): e8631, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31531526

RESUMO

The long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3), a tumor suppressor, is critical for the carcinogenesis and progression of different cancers, including hepatocellular carcinoma (HCC). To date, the roles of lncRNA MEG3 in HCC are not well illustrated. Therefore, this study used western blot and qRT-PCR to evaluate the expression of MEG3, miR-9-5p, and Sex determining Region Y-related HMG-box 11 (SOX11) in HCC tissues and cell lines. RNA pull-down and luciferase reporter assay were used to evaluate these molecular interactions. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry detected the viability and apoptosis of HCC cells, respectively. The results showed that MEG3 and SOX11 were poorly expressed but miR-9-5p was highly expressed in HCC. The expression levels of these molecules suggested a negative correlation between MEG3 and miR-9-5p and a positive correlation with SOX11, confirmed by Pearson's correlation analysis and biology experiments. Furthermore, MEG3 could combine with miR-9-5p, and SOX11 was a direct target of miR-9-5p. Moreover, MEG3 over-expression promoted cell apoptosis and growth inhibition in HCC cells through sponging miR-9-5p to up-regulate SOX11. Therefore, the interactions among MEG3, miR-9-5p, and SOX11 might offer a novel insight for understanding HCC pathogeny and provide potential diagnostic markers and therapeutic targets for HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Fatores de Transcrição SOXC/genética , Apoptose/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXC/metabolismo , Ativação Transcricional , Transfecção , Regulação para Cima
4.
Nat Methods ; 16(9): 887-893, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406383

RESUMO

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes , Redes Reguladoras de Genes , Engenharia Genética , Genoma Humano , RNA Guia/genética , Acidaminococcus/enzimologia , Endonucleases/genética , Células HEK293 , Humanos , Plasmídeos/genética , Ativação Transcricional
5.
Cancer Sci ; 110(10): 3328-3339, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31429167

RESUMO

Apatinib, an antiangiogenic agent, shows efficient antitumor activity in a broad range of malignancies. Considering tumor is a type of metabolic disease, we investigated the metabolomics changes in serum and tumor after apatinib treatment and the molecular mechanism of characteristic changes associated with its antitumor efficacy. Molecules in serum and tumor tissue were extracted and analyzed by a gas chromatography-mass spectrometry metabolic platform. Apatinib significantly inhibited e tumor growth and alleviated metabolic rearrangement in both serum and tumor of A549 xenograft mice. Among these endogenous metabolites, 3-hydroxybutyric acid (3-HB) was significantly increased in serum, tumor and liver after apatinib treatment. Interestingly, giving exogenous 3-HB also inhibited tumor growth. Gene expression, dual luciferase reporter gene assay and molecular docking analysis all indicated that apatinib could induce 3-HB production through the dependent activation of peroxisome proliferator-activated receptor α (PPARα) and promotion of fatty acid utilization in the liver. Therefore, increased content of 3-HB induced by PPARα activation in the liver partially contributed to the antitumor effect of apatinib. It may provide clues to another potential mechanism underlying the antitumor effect of apatinib besides its antiangiogenic effect through inhibiting vascular endothelial growth factor receptor 2.


Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Fígado/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , PPAR alfa/genética , Piridinas/administração & dosagem , Células A549 , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Metabolômica/métodos , Camundongos , Piridinas/farmacologia , Ativação Transcricional , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Life Sci ; 233: 116731, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31394128

RESUMO

AIMS: Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by widespread inflammation. LncRNA taurine-up-regulated gene 1 (TUG1) has been reported to be involved in multiple biological processes and human diseases. The aim of this study was to investigate the role of lncRNA TUG1 in MS and the underlying mechanism. MAIN METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55). Lentiviral vectors encoding sh-TUG1 was constructed to silence TUG1 in MOG-EAE mice by intracerebroventricular (ICV) injection. The effect of TUG1 on inflammation in MS was evaluated by real-time PCR, Western blot, ELISA and Hematoxylin-eosin staining. To further study the mechanism of TUG1 in MS, TUG1 knockdown and miR-9-5p overexpression were performed in LPS-induced BV2 cells. KEY FINDINGS: Down-regulation of TUG1 improved mice behavior, reduced granulocyte-macrophage colony stimulating factor (GM-CSF) level, decreased the levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-6 and IL-17, and increased IL-10 in EAE mice. Notably, TUG1 expression was negatively correlated with miR-9-5p expression, while positively correlated with NF-κB1/p50. Knockdown of TUG1 or enforced expression of miR-9-5p inhibited LPS-induced inflammation in BV2 cells, while these effects were abolished by inhibition of miR-9-5p. We further verified that TUG1 negatively regulated miR-9-5p expression and NF-κB1/p50 is a direct target of miR-9-5p. SIGNIFICANCE: Down-regulation of TUG1 attenuates MS through inhibition of inflammation by sponging miR-9-5p via targeting NF-κB1/p50, suggesting that TUG1 is a potential therapeutic target for MS treatment.


Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Regulação da Expressão Gênica , Inflamação/prevenção & controle , MicroRNAs/genética , Esclerose Múltipla/prevenção & controle , Subunidade p50 de NF-kappa B/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Animais , Apoptose , Proliferação de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Inflamação/induzido quimicamente , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subunidade p50 de NF-kappa B/genética , RNA Longo não Codificante/genética , Ativação Transcricional
7.
J Agric Food Chem ; 67(33): 9286-9294, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31339733

RESUMO

Natural aryl hydrocarbon (AHR) ligands have been identified in food and herbal medicines, and they may exhibit beneficial activity in humans. In this study, white button (WB) feeding significantly decreased AHR target gene expression in the small intestine of both conventional and germ-free mice. High-performance liquid chromatography (HPLC) fractionation and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) combined with an AHR-responsive cell-based luciferase gene reporter assay were used to isolate and characterize benzothiazole (BT) derivatives and 6-methylisoquinoline (6-MIQ) as AHR modulators from WB mushrooms. The study showed dose-dependent changes of AHR transformation determined by the cell-based luciferase gene reporter assay and transcription of CYP1A1 in human Caco-2 cells by BT derivatives and 6-MIQ. These findings suggested that WB mushroom contains new classes of natural AHR modulators and demonstrated HPLC fractionation and UHPLC-MS/MS combined with a cell-based luciferase gene reporter assay as a useful approach for isolation and characterization of the previously unidentifed AHR modulators from natural products.


Assuntos
Agaricus/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Receptores de Hidrocarboneto Arílico/genética , Animais , Benzotiazóis/química , Benzotiazóis/isolamento & purificação , Benzotiazóis/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Genes Reporter , Humanos , Isoquinolinas/química , Isoquinolinas/isolamento & purificação , Isoquinolinas/farmacologia , Ligantes , Camundongos , Extratos Vegetais/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Espectrometria de Massas em Tandem , Ativação Transcricional/efeitos dos fármacos , Verduras/química
8.
Gene ; 714: 143985, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31330236

RESUMO

In all eukaryotes, the response to heat stress (HS) is dependent on the activity of HS transcription factors (Hsfs). Plants contain a large number of Hsfs, however, only members of the HsfA1 subfamily are considered as master regulators of stress response and thermotolerance. In Solanum lycopersicum, among the four HsfA1 members, only HsfA1a has been proposed to possess a master regulator function. We performed a comparative analysis of HsfA1a, HsfA1b, HsfA1c and HsfA1e at different levels of regulation and function. HsfA1a is constitutively expressed under control and stress conditions, while the other members are induced in specific tissues and stages of HS response. Despite that all members are localized in the nucleus when expressed in protoplasts, only HsfA1a shows a wide range of basal activity on several HS-induced genes. In contrast, HsfA1b, HsfA1c, and HsfA1e show only high activity for specific subsets of genes. Domain swapping mutants between HsfA1a and HsfA1c revealed that the variation in that transcriptional transactivation activity is due to differences in the DNA binding domain (DBD). Specifically, we identified a conserved arginine (R107) residue in the turn of ß3 and ß4 sheet in the C-terminus of the DBD of HsfA1a that is highly conserved in plant HsfA1 proteins, but is replaced by leucine and cysteine in tomato HsfA1c and HsfA1e, respectively. Although not directly involved in DNA interaction, R107 contributes to DNA binding and consequently the activity of HsfA1a. Thus, we demonstrate that this variation in DBD in part explains the functional diversification of tomato HsfA1 members.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico/genética , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Temperatura Alta , Domínios Proteicos/genética , Protoplastos/fisiologia , Temperatura Ambiente , Termotolerância/genética , Transcrição Genética/genética , Ativação Transcricional/genética
9.
J Agric Food Chem ; 67(31): 8649-8659, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31283213

RESUMO

Spent coffee grounds (SCG) are the most abundant coffee byproduct and are generally discarded as waste. The horticultural use of SCG and SCG compost (SCGC) has become popular due to a growing interest in environmentally friendly measures for waste disposal. Estrogen-like endocrine disrupting chemicals in the soil can be absorbed by plants and subsequently by humans who consume these plants. The objectives of this study are to determine the phytochemical profiles of extracts of SCG and SCGC and to evaluate the estrogen-like activities of SCG, SCGC, and the major coffee phenolic acids, specifically, 5-O-caffeoylquinic acid (CQA), caffeic acid, and ferulic acid. Their inductive effects on estrogen receptor (ER)-mediated gene transcription have been examined in cultured cell lines. CQA was the most abundant phenolic acid in SCG and SCGC and was further examined for its ER-mediated estrogen-like activity using various assays. This is the first study to report the estrogen-like signaling activities of coffee byproducts and their major constituents.


Assuntos
Coffea/química , Hidroxibenzoatos/metabolismo , Fitoestrógenos/metabolismo , Extratos Vegetais/metabolismo , Receptores Estrogênicos/genética , Ativação Transcricional , Resíduos/análise , Animais , Ácidos Cafeicos/análise , Ácidos Cafeicos/metabolismo , Linhagem Celular , Compostagem , Feminino , Genes Reporter , Humanos , Hidroxibenzoatos/química , Fitoestrógenos/química , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Receptores Estrogênicos/metabolismo , Sementes/química
10.
Nat Commun ; 10(1): 2939, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270324

RESUMO

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.


Assuntos
Fator de Transcrição E2F4/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteína do Retinoblastoma/metabolismo , Ativação Transcricional , Animais , Ciclo Celular , Divisão Celular , Fator de Transcrição E2F4/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Família Multigênica , Proteína do Retinoblastoma/genética
11.
Plant Mol Biol ; 101(1-2): 113-127, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300998

RESUMO

Transcriptional regulation is an essential molecular machinery in controlling gene expression in diverse plant developmental processes including fruit ripening. This involves the interaction of transcription factors (TFs) and promoters of target genes. In banana, although a number of fruit ripening-associated TFs have been characterized, their number is relatively small. Here we identified a nuclear-localized basic leucine zipper (bZIP) TF, MabZIP93, associated with banana ripening. MabZIP93 activated cell wall modifying genes MaPL2, MaPE1, MaXTH23 and MaXGT1 by directly binding to their promoters. Transient over-expression of MabZIP93 in banana fruit resulted in the increased expression of MaPL2, MaPE1, MaXTH23 and MaXGT1. Moreover, a mitogen-activated protein kinase MaMPK2 and MabZIP93 were found to interact with MabZIP93. The interaction of MabZIP93 with MaMPK2 enhanced MabZIP93 activation of cell wall modifying genes, which was likely due to the phosphorylation of MabZIP93 mediated by MaMPK2. Overall, this study shows that MaMPK2 interacts with and phosphorylates MabZIP93 to promote MabZIP93-mediated transcriptional activation of cell wall modifying genes, thereby expanding our understanding of gene networks associated with banana fruit ripening.


Assuntos
Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes/genética , Musa/genética , Proteínas de Plantas/metabolismo , Ativação Transcricional , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Núcleo Celular/metabolismo , Parede Celular/metabolismo , Frutas/genética , Musa/fisiologia , Fosforilação , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética
12.
Cancer Sci ; 110(10): 3340-3349, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31342590

RESUMO

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion-metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET-knockout cells were prepared and reconstituted with WT-MET or V370D-MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT-MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D-MET cells. The V370D mutation abrogated HGF-dependent drug resistance of lung cancer cells to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI). Compared with WT-MET cells, V370D-MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth-stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D-MET and HGF was reduced compared with that for WT-MET. Further analysis of the association between V370D-MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D-MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss-of-function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genética , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Resistencia a Medicamentos Antineoplásicos , Técnicas de Inativação de Genes , Humanos , Mutação com Perda de Função , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas c-met/metabolismo , Ativação Transcricional
13.
Chemosphere ; 233: 786-795, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31340409

RESUMO

Microbial volatile organic compounds (mVCs) are formed in the metabolism of microorganisms and widely distributed in nature and pose threats to human health. However, the air pollution by microorganisms is a situation which is poorly understood. In this study, the cytotoxicity of E. aerogenes VCs was evaluated in the model organism Saccharomyces cerevisiae. E. aerogenes VCs inhibited the survival of yeast and triggered the formation of intracellular reactive oxygen species (ROS). The hypersensitive of MAP kinase mpk1/slt2 and 19S regulatory assembly chaperone adc17 mutants to the E. aerogenes VCs indicated cell wall integrity (CWI) pathway together with stress-inducible proteasome assembly regulation are essentially involved in mVCs tolerance mechanism. Furthermore, exposure to the mVCs resulted in the transcriptional upregulation of the CWI pathway, the regulatory particle assembly chaperones, and genes involved in proteasome regulations. Our research suggested that the ROS/MAPK signaling and proteasome regulatory pathway play pivotal roles in the integration and fine-tuning of the mVCs stress response. This study provides a molecular framework for future study of the effects of mVCs on more complex organisms, such as humans.


Assuntos
Enterobacter aerogenes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Compostos Orgânicos Voláteis/farmacologia , Poluentes Atmosféricos/análise , Poluição do Ar/análise , Parede Celular/metabolismo , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional
14.
Genome Biol ; 20(1): 145, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349852

RESUMO

The CRISPR/Cas9 system is unable to edit all targetable genomic sites with full efficiency in vivo. We show that Cas9-mediated editing is more efficient in open chromatin regions than in closed chromatin regions in rice. A construct (Cas9-TV) formed by fusing a synthetic transcription activation domain to Cas9 edits target sites more efficiently, even in closed chromatin regions. Moreover, combining Cas9-TV with a proximally binding dead sgRNA (dsgRNA) further improves editing efficiency up to several folds. The use of Cas9-TV/dsgRNA thus provides a novel strategy for obtaining efficient genome editing in vivo, especially at nuclease-refractory target sites.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Cromatina/química , Edição de Genes , Ativação Transcricional , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Oryza/genética , RNA/genética , Transativadores/genética
15.
Nature ; 571(7763): 107-111, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31217582

RESUMO

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.


Assuntos
Diarreia/congênito , Diarreia/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes , Intestinos/fisiologia , Deleção de Sequência/genética , Animais , Cromossomos Humanos Par 16/genética , Modelos Animais de Doenças , Feminino , Genes Reporter , Loci Gênicos/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Linhagem , Fenótipo , Ativação Transcricional , Transcriptoma/genética , Transgenes/genética
16.
Food Chem Toxicol ; 131: 110532, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31154085

RESUMO

Pyrrolizidine alkaloids (PAs) are secondary metabolites from plants that have been found in substantial amounts in herbal supplements, infusions and teas. Several PAs cause cancer in animal bioassays, mediated via a genotoxic mode of action, but for the majority of the PAs, carcinogenicity data are lacking. It is assumed in the risk assessment that all PAs have the same potency as riddelliine, which is considered to be one of the most potent carcinogenic PAs in rats. This may overestimate the risks, since many PAs are expected to have lower potencies. In this study we determined the concentration-dependent genotoxicity of 37 PAs representing different chemical classes using the γH2AX in cell western assay in HepaRG human liver cells. Based on these in vitro data, PAs were grouped into different potency classes. The group with the highest potency consists particularly of open diester PAs and cyclic diester PAs (including riddelliine). The group of the least potent or non-active PAs includes the monoester PAs, non-esterified necine bases, PA N-oxides, and the unsaturated PA trachelanthamine. This study reveals differences in in vitro genotoxic potencies of PAs, supporting that the assumption that all PAs have a similar potency as riddelliine is rather conservative.


Assuntos
Histonas/metabolismo , Mutagênicos/toxicidade , Alcaloides de Pirrolizidina/toxicidade , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Histonas/genética , Humanos , Internet , Modelos Biológicos , Alcaloides de Pirrolizidina/classificação , Ativação Transcricional/efeitos dos fármacos
17.
Sheng Wu Gong Cheng Xue Bao ; 35(6): 1126-1134, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31232009

RESUMO

Human bocavirus 1 (HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293T cells using transfection reagent. The HEK 293T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline. The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.


Assuntos
Bocavirus Humano , Ativação Transcricional , Regiões Promotoras Genéticas , Proteínas não Estruturais Virais , Replicação Viral
18.
Plant Physiol Biochem ; 141: 231-239, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195253

RESUMO

Anther/pollen development is a highly programmed process in flowering plants. However, the molecular mechanism of regulating anther/pollen development is still largely unclear so far. Here, we report a cotton WRKY transcription factor (GhWRKY22) that functions in anther/pollen development. Quantitative RT-PCR and GUS activity analyses revealed that GhWRKY22 is predominantly expressed in the late developing anther/pollen of cotton. The transgenic Arabidopsis plants expressing GhWRKY22 displayed the male fertility defect with the fewer viable pollen grains. Expression of the genes involved in jasmonate (JA) biosynthesis was up-regulated, whereas expression of the JA-repressors (JAZ1 and JAZ8) was down-regulated in the transgenic Arabidopsis plants expressing GhWRKY22, compared with those in wild type. Yeast one-hybrid and ChIP-qPCR assays demonstrated that GhWRKY22 modulated the expression of JAZ genes by directly binding to their promoters for regulating anther/pollen development. Yeast two-hybrid assay indicated that GhMYB24 could interact with GhJAZ8-A and GhJAZ13-A. Furthermore, expression of AtMYB24, AtPAL2 and AtANS2 was enhanced in the transgenic Arabidopsis plants, owing to GhWRKY22 overexpression. Taking the data together, our results suggest that GhWRKY22 acts as a transcriptional repressor to regulate anther/pollen development possibly by modulating the expression of the JAZ genes.


Assuntos
Gossypium/metabolismo , Pólen/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hipocótilo/metabolismo , Oxilipinas/metabolismo , Fenótipo , Infertilidade das Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sementes/metabolismo , Ativação Transcricional , Transgenes , Técnicas do Sistema de Duplo-Híbrido
19.
Plant Physiol Biochem ; 141: 300-305, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31202194

RESUMO

MYB-type transcription factors are known to participate in the response of plants to a number of stress agents. MsMYB2L is an alfalfa member of this large gene family. Its transcription in alfalfa seedlings was found to be rapidly and strongly induced by salinity, moisture deficiency and exogenously supplied abscisic acid. An analysis based on a yeast one hybrid assay indicated that its product is able to activate transcription, consistent with its function as a transcription factor. When the gene was constitutively expressed in Arabidopsis thaliana, both germination and seedling growth were more sensitive to ABA treatment than wild type, and growth was less strongly compromised by salinity and moisture deficiency stress, presumably as a result of the induction of certain stress-related genes active in ABA-dependent pathways. The transgenic seedlings' enhanced the synthesis of many osmotic regulatory substances such as proline and soluble sugar, and decreased the lipid peroxidation. In all, MsMYB2L represents a potential candidate gene for manipulating the salinity and drought tolerance of alfalfa.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/fisiologia , Proteínas de Ligação a DNA/genética , Secas , Medicago sativa/genética , Proteínas de Plantas/genética , Salinidade , Fatores de Transcrição/genética , Regulação da Expressão Gênica de Plantas , Técnicas Genéticas , Germinação , Filogenia , Plantas Geneticamente Modificadas/fisiologia , Polietilenoglicóis/química , Plântula/fisiologia , Estresse Fisiológico , Açúcares/química , Transcrição Genética , Ativação Transcricional
20.
Life Sci ; 231: 116559, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31200001

RESUMO

AIM: Previously, we reported that mice deficient in most of the Zfp521 coding region (Zfp521Δ/Δ mice) displayed abnormal behaviors, including hyperlocomotion and lower anxiety. In this study, we aimed to elucidate the involvement and mechanisms of monoamine variation. MAIN METHODS: First, we compared the levels of dopamine (DA), noradrenaline (NA), and serotonin in the brains of Zfp521Δ/Δ and Zfp521+/+ mice using enzyme-linked immunosorbent assay. Next, we elucidated the mechanisms using quantitative PCR and Western Blotting. Additionally, we administered inhibitory drug to the mice and performed behavioral tests. KEY FINDINGS: Our results showed that the DA level decreased and the NA level increased in Zfp521Δ/Δ mice. We found that ZFP521 suppresses the expression of dopamine ß-hydroxylase (DBH), which converts DA into NA. We also demonstrated that paired homeodomain transcription factor 2 and early growth response protein-1, which are the transcription factors for Dbh, were involved in the upregulation of Dbh by ZFP521. The administration of nepicastat, a specific inhibitor of DBH, attenuated the abnormal behaviors of Zfp521Δ/Δ mice. SIGNIFICANCE: These results suggest that the lack of ZFP521 upregulates the expression of DBH, which leads to a decrease in the DA level and an increase in the NA level in the brain, resulting in abnormal behaviors.


Assuntos
Comportamento Animal/fisiologia , Encéfalo/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Fatores de Transcrição/deficiência , Animais , Encéfalo/enzimologia , Linhagem Celular Tumoral , Dopamina/metabolismo , Imidazóis/farmacologia , Locomoção/efeitos dos fármacos , Locus Cerúleo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Norepinefrina/metabolismo , Serotonina/metabolismo , Tionas/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Dedos de Zinco
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