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1.
DNA Cell Biol ; 38(10): 1155-1165, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31433201

RESUMO

Our previous genome-wide association study has identified a suggestive association at rs11264799 within FCRL3 (Fc receptor-like 3) locus on 1q23.1 for IgA nephropathy (IgAN) in a Chinese Han population. This study aims to investigate the association of FCRL3 variants with the susceptibility, clinicopathological phenotypes and prognosis of IgAN. Eleven FCRL3 single-nucleotide polymorphisms (SNPs) were selected and analyzed in this two-stage case/control study with a total of 1750 IgAN cases and 2500 healthy controls in a Chinese Han population. Unconditional logistic regression models were used to estimate odds ratios and 95% confidence intervals (CIs) as implemented in the PLINK software. Luciferase assays were applied to detect the allelic effect of rs11264794 on gene expression regulation. We found that four SNPs (rs11264794, rs7865684, rs11264799, and rs6691569) were significantly associated with IgAN susceptibility after Bonferroni correction in the combined samples. Genotype/phenotype association analysis observed that two SNPs (rs11264794 and rs11264793) were associated with less disease severity. After adjusting for confounders, rs11264794 was independently correlated with renal outcome in IgAN patients (hazard ratio = 0.64, 95% CI = 0.43-0.97, p = 0.033). In addition, the protective allele A of rs11264794 was significantly associated with higher FCRL3 gene expression. Furthermore, luciferase reporter gene assays demonstrated that the minor allele of rs11264794 obviously reduced the specific binding between miR-183-5p.1 and FCRL3 3'-untranslated region. Our results indicate that FCRL3 gene polymorphisms are associated with the development and progression of IgAN, and the rs11264794-A allele showed a protective role for IgAN.


Assuntos
Predisposição Genética para Doença , Glomerulonefrite por IGA/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Receptores Imunológicos/genética , Regiões 3' não Traduzidas , Alelos , Grupo com Ancestrais do Continente Asiático , Estudos de Casos e Controles , Feminino , Expressão Gênica , Genes Reporter , Estudos de Associação Genética , Glomerulonefrite por IGA/diagnóstico , Glomerulonefrite por IGA/etnologia , Glomerulonefrite por IGA/patologia , Humanos , Modelos Logísticos , Luciferases/genética , Luciferases/metabolismo , Masculino , MicroRNAs/metabolismo , Razão de Chances , Fenótipo , Prognóstico , Receptores Imunológicos/metabolismo , Índice de Gravidade de Doença
2.
Dokl Biochem Biophys ; 486(1): 209-212, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367823

RESUMO

This paper presents the preliminary results of the separation of the Chaetopterus variopedatus bioluminescent system into luciferin and luciferase and a brief description of some of their properties.


Assuntos
Benzotiazóis/metabolismo , Luciferases/metabolismo , Poliquetos/metabolismo , Animais , Medições Luminescentes
3.
Gene ; 712: 143958, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278963

RESUMO

The Wnt signaling pathway has been identified for its function in carcinogenesis and embryonic development. It is known to play a vital role in the initiation and development of colorectal cancer (CRC). Therefore, it is of great importance for CRC research to illuminate the mechanisms which regulate Wnt pathway activity. Here, we intended to examine the effect of hsa-miR-942 (miR-942) on the Wnt signaling activity, cell cycle progression, and its expression in CRC tissues. RT-qPCR results indicated that miR-942 is significantly upregulated in colorectal cancer. Then, overexpression of miR-942 promoted, whereas its inhibition decreased the Wnt signaling activity, detected by RT-qPCR and Top/Fop flash assay. Inhibition of Wnt signaling by using PNU-74654 or IWP-2 small molecules indicated that miR-942 applies its effect to the ß-catenin degradation complex level. Then, RT-qPCR and dual luciferase assay showed that miR-942 upregulated Wnt signaling through direct targeting of APC, which is a tumor suppressor in Wnt signaling pathway. Furthermore, the western blotting analysis indicated that ß.catenin, as a main member of Wnt signaling pathway is upregulated following the overexpression of miR-942. Finally, miR-942 overexpression resulted in cell cycle progression in SW480 cells. Taken together, our findings established an oncogenic role for miR-942 in CRC and indicated that this miRNA might be a crucial target for CRC therapy.


Assuntos
Neoplasias Colorretais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Via de Sinalização Wnt , Regiões 3' não Traduzidas , Carcinogênese/genética , Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Biologia Computacional , Ciclina D1/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Masculino , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , Transdução de Sinais , Regulação para Cima , beta Catenina/metabolismo
4.
Biol Res ; 52(1): 38, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349873

RESUMO

BACKGROUND: Breast cancer is the second common malignant cancer among females worldwide. Accumulating studies have indicated that deregulation of miRNA expression in breast cancer will contribute to tumorigenesis and form different cancer subtypes. However, the reported studies on miR-29b-3p-regulated breast cancer are limited so far. Herein, we investigated the role and mechanism of miR-29b-3p in the triple negative breast cancer cell line MDA-MB-231. METHODS: The relative miR-29b-3p expression in different breast cancer cell lines were determined by qRT-PCR. CCK8 and colony formation assay were used to determine the influence of miR-29b-3p on cell proliferation. Migration assay and invasion assay were performed for cell migration and invasion respectively. To study the cell integrity immunofluorescence was performed. TUNEL assay, flow cytometry assay, hoechst staining and western blot were conducted to determine the influence of miR-29b-3p inhibitor on cell apoptosis. TRAF3 was found to be the target gene of miR-29b-3p using bioinformatics predictions. Dual-luciferase assay was performed to determine the relative luciferase activity in NC, miR-29b-3p mimic, miR-29b-3p inhibitor with TRAF3 3'-UTR wt or TRAF3 3'-UTR mt reporter plasmids. The proteins expression of NF-κB signaling pathway in MDA-MB-231 after transfection with NC, miR-29b-3p mimic, miR-29b-3p inhibitor were determined by western blot. RESULTS: The miR-29b-3p expression was significantly increased in MDA-MB-231 compare with MCF-10A. miR-29b-3p inhibitor reduced the cell viability of MDA-MB-231 and inhibited cell migration and invasion. Cell cytoskeleton integrity destroyed after miR-29b-3p inhibitor treatment. Furthermore, we identified the mechanism and found miR-29b-3p targets the TRAF3 and activates NF-κB signaling pathway. CONCLUSIONS: From the above studies, our results indicated that miR-29b-3p acts as a promoter for the development of MDA-MB-231.


Assuntos
Apoptose/efeitos dos fármacos , Regulação para Baixo/genética , MicroRNAs/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Luciferases/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Neoplasias de Mama Triplo Negativas/patologia
5.
Anim Sci J ; 90(8): 1042-1049, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31237073

RESUMO

Glycogen synthase kinase beta (GSK3ß) plays an important role in skeletal muscle growth, regeneration, and repair. However, the mechanism of GSK3ß regulating MyHC2a expression is currently not clear. In this study, GSK3ß inhibition promoted skeletal muscle satellite cells (SMSCs) differentiation and increased expression of MyoD, MyoG, MyHC1, and MyHC2a genes. Then we cloned approximately 1.1 kb of goat MyHC2a gene promoter. The deletion fragment (-514/+55) of MyHC2a promoter exhibited the highest level of promoter activity, and a NFATc2 element in this region was responsible for MyHC2a promoter activity. Treatment of SB216713 significantly decreased the transcriptional activity of the fragment (-514/+55). Furthermore, GSK3ß inhibition had no effect on the luciferase activity of MyHC2a promoter after mutating the NFATc2-binding site. These results demonstrated that GSK3ß inhibition promoted SMSCs differentiation and regulated the MyHC2a gene expression through NFATc2 in goat-differentiated SMSCs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica/genética , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/fisiologia , Músculo Esquelético/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Feminino , Cabras , Luciferases/metabolismo , Fatores de Transcrição NFATC
6.
Dokl Biochem Biophys ; 485(1): 157-161, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201640

RESUMO

The results of a comparative study of the luciferin-luciferase systems of seven species of bioluminescing oligochaetes-Henlea petushkovi, Henlea rodionovae, Fridericia heliota (Enchytraeidae), Microscolex phosphoreus (Acanthodrilidae), Pontodrilus litoralis (Megascolecidae), Eisenia lucens, and Avelona ligra (Lumbricidae)-are presented.


Assuntos
Luciferases , Oligoquetos , Animais , Luciferases/genética , Luciferases/metabolismo , Oligoquetos/enzimologia , Oligoquetos/metabolismo
7.
Cell Biol Int ; 43(7): 789-798, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31042002

RESUMO

As a cationic non-viral gene delivery vector, poly(agmatine/ N, N'-cystamine-bis-acrylamide) (AGM-CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM-CBA/pDNA polyplexes was found to have a non-linear relationship with AGM-CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM-CBA), we used pGL3-control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM-CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate-limiting step for pLUC transfection expression. Further optimization of the non-viral gene delivery system can be focused on the improvement of gene intracellular availability.


Assuntos
Núcleo Celular/metabolismo , Genes Reporter/genética , Luciferases/genética , Luciferases/metabolismo , Plasmídeos/genética , Transfecção/métodos , Acrilamidas/química , Agmatina/química , Animais , Camundongos , Células NIH 3T3 , Polietilenoimina/química
8.
Cell Mol Biol Lett ; 24: 28, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061665

RESUMO

Background: Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor with a pivotal role in physiological and pathological responses to hypoxia. While HIF-1α is known to be involved in hypoxia-induced upregulation of microRNA (miRNA) expression, HIF-1α is also targeted by miRNAs. In this study, miRNAs targeting HIF-1α were identified and their effects on its expression and downstream target genes under hypoxic conditions were investigated. Cell migration under the same conditions was also assessed. Methods: microRNAs that target HIF-1α were screened using 3'-untranslated region luciferase (3'-UTR-luciferase) reporter assays. The expression levels of HIF-1α and its downstream target genes after transfection with miRNA were assessed using quantitative RT-PCR and western blot analyses. The effect of the miRNAs on the transcriptional activity of HIF-1α was determined using hypoxia-responsive element luciferase (HRE-luciferase) assays. Cell migration under hypoxia was examined using the wound-healing assay. Results: Several of the 19 screened miRNAs considerably decreased the luciferase activity. Transfection with miR-200c had substantial impact on the expression level and transcription activity of HIF-1α. The mRNA level of HIF-1α downstream genes decreased in response to miR-200c overexpression. MiR-200c inhibited cell migration in normoxia and, to a greater extent, in hypoxia. These effects were partly reversed by HIF-1α expression under hypoxic conditions. Conclusion: miR-200c negatively affects hypoxia-induced responses by downregulating HIF-1α, a key regulator of hypoxia. Therefore, overexpression of miR-200c might have therapeutic potential as an anticancer agent that inhibits tumor hypoxia.


Assuntos
Movimento Celular/genética , Regulação para Baixo/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Hipóxia Celular/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Luciferases/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Regulação para Cima/genética , Cicatrização
9.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083458

RESUMO

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.


Assuntos
Elasmobrânquios/classificação , Elasmobrânquios/genética , Evolução Molecular , Redes Reguladoras de Genes , Filogenia , Receptor de Pregnano X/genética , Animais , Células COS , Cercopithecus aethiops , Genes Reporter , Inativação Metabólica/genética , Luciferases/metabolismo , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Sintenia/genética , Ativação Transcricional/genética
10.
Nat Methods ; 16(6): 526-532, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086341

RESUMO

Glucose is a major source of energy for most living organisms, and its aberrant uptake is linked to many pathological conditions. However, our understanding of disease-associated glucose flux is limited owing to the lack of robust tools. To date, positron-emission tomography imaging remains the gold standard for measuring glucose uptake, and no optical tools exist for non-invasive longitudinal imaging of this important metabolite in in vivo settings. Here, we report the development of a bioluminescent glucose-uptake probe for real-time, non-invasive longitudinal imaging of glucose absorption both in vitro and in vivo. In addition, we demonstrate that the sensitivity of our method is comparable with that of commonly used 18F-FDG-positron-emission-tomography tracers and validate the bioluminescent glucose-uptake probe as a tool for the identification of new glucose transport inhibitors. The new imaging reagent enables a wide range of applications in the fields of metabolism and drug development.


Assuntos
Transportador de Glucose Tipo 1/fisiologia , Glucose/metabolismo , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Animais , Transporte Biológico , Feminino , Fluordesoxiglucose F18/metabolismo , Humanos , Luciferases/metabolismo , Camundongos Knockout , Camundongos Nus , Neoplasias Experimentais/patologia , Compostos Radiofarmacêuticos/metabolismo , Células Tumorais Cultivadas
11.
Plant Mol Biol ; 100(1-2): 19-32, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31001712

RESUMO

KEY MESSAGE: BcMAF2 plays a key role in flowering regulation by controlling BcTEM1, BcSOC1 and BCSPL15 in Pak-choi. Flowering is a key event in the life cycle of plants. Flowering time shows an extensive variation from different Pak-choi (Brassica rapa ssp. chinensis) cultivars. However, the regulation mechanism of flowering in Pak-choi remains rarely known. In this study, a systematic identification and functional analysis of a Pak-choi MADS Affecting Flowering (MAF) gene, BcMAF2, was carried out. BcMAF2 encoded a protein containing a conserved MADS-box domain, which was localized in the nucleus. QPCR analysis indicated that the expression of BcMAF2 was higher in the leaves and flowers. Overexpression of BcMAF2 in Arabidopsis showed that BcMAF2 repressed flowering, which was further confirmed by silencing endogenous BcMAF2 in Pak-choi. In addition, Tempranillo 1 (TEM1) expression was up-regulated and MAF2 expression was down-regulated in the BcMAF2-overexpressing Arabidopsis. The expression of BcMAF2 and BcTEM1 was down-regulated in BcMAF2-silencing Pak-choi plants. The yeast one-hybrid, dual luciferase and qPCR results revealed that BcMAF2 protein could directly bind to BcTEM1 promoter and activate its expression, which was not reported in Arabidopsis. Meanwhile, a self-inhibition was found in BcMAF2. Taken together, this work suggested that BcMAF2 could repress flowering by directly activating BcTEM1.


Assuntos
Brassica rapa/metabolismo , Flores/fisiologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Luciferases/metabolismo , Modelos Biológicos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/genética
12.
Methods Mol Biol ; 1970: 315-330, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30963500

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs of 22-25 nucleotides that control gene expression at the posttranscriptional level through the degradation of mRNAs or translational repression. In the last 15 years, the study of these small molecules helped elucidate their role in the regulation of many cellular processes and the onset and development of several diseases. Therefore, many computational tools based on algorithms for target prediction have been developed to identify potential miRNA-target interactions. The improvement of experimental approaches to more easily and quickly confirm in silico predictions has become essential for the study of these small RNAs and their molecular functions. In this chapter, we summarized the principal steps of one of the most used techniques for the validation of microRNA targets, the Luciferase assay, thus explaining the underlying principles and the procedures to apply it best.


Assuntos
Biologia Computacional/métodos , Luciferases/metabolismo , MicroRNAs/genética , RNA Mensageiro/análise , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos de Validação como Assunto
13.
Methods Mol Biol ; 1970: 341-353, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30963502

RESUMO

MicroRNAs are a class of small noncoding RNA involved in the mechanism of RNA silencing and regulation of gene expression at a posttranscriptional level. Recently, the discovery of their targets led to the understanding of the molecular role of these small molecules and their involvement in the pathogenesis of numerous diseases, including cancer. Not long ago, the improvement of several informatics tools for microRNA target prediction has supported the experimental research through the selection of potential mRNA as microRNA target candidates. Since the regulation mediated by microRNA affects gene expression at a posttranscriptional level, the analysis of the proteins encoded by the gene targets is essential in understanding the involvement of these small molecules in biological processes and their role in several diseases. In this chapter, we describe the experimental procedure of Western blotting applied to the validation of microRNA targets. Western blotting is one of the most common and largest know technique for protein analysis. This method, coupled with the luciferase assay, represents the standard procedure for the experimental confirmation of microRNA targeting.


Assuntos
Western Blotting/métodos , Biologia Computacional/métodos , Luciferases/metabolismo , MicroRNAs/genética , RNA Mensageiro/análise , Regulação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos de Validação como Assunto
14.
Mol Biotechnol ; 61(6): 400-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30945164

RESUMO

Transgenic chickens are of great interest for the production of recombinant proteins in their eggs. However, the use of constitutive strong promoters or the tissue-specific ovalbumin promoter for the generation of the transgenic chickens have different drawbacks that have to be overcome in order to make chicken bioreactor an efficient production system. This prompted us to investigate the use of an alternative tissue-specific promoter, the vitellogenin promoter, which could overcome the difficulties currently found in the generation of chicken bioreactors. In the present work we establish and characterize a DNA construct consisting of a fragment of the 5´-flanking region of the chicken vitellogenin II gene cloned in a reporter vector. This construct is capable of showing the ability of the promoter to drive expression of a reporting gene in a tissue-specific manner and in a way that closely resembles physiologic regulation of vitellogenin, making it an ideal candidate to be used in the future for generation of avian bioreactors. Besides, we validate an in vitro culture system to test the performance of the DNA construct under study that could be used as a practical tool before generating any transgenic chicken. These results are important since they provide the proof of concept for the use of the vitellogenin promoter for future genetic modification of chickens bioreactors with improved characteristics in terms of quality of the recombinant protein produced.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Vetores Genéticos/química , Proteínas Recombinantes de Fusão/genética , Vitelogeninas/genética , Região 5'-Flanqueadora , Animais , Animais Geneticamente Modificados , Proteínas Aviárias/metabolismo , Reatores Biológicos , Embrião de Galinha , Galinhas/metabolismo , Clonagem Molecular , Estradiol/farmacologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Luciferases/genética , Luciferases/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores Estrogênicos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Vitelogeninas/metabolismo , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
15.
Biomed Res ; 40(2): 67-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30982802

RESUMO

T1R1 and T1R3 are receptors expressed in taste buds that detect L-amino acids. These receptors are also expressed throughout diverse organ systems, such as the digestive system and muscle tissue, and are thought to function as amino acid sensors. The mechanism of transcriptional regulation of the mouse T1R1 gene (Tas1r1) has not been determined; therefore, in this study, we examined the function of Tas1r1 promoter in the mouse myoblast cell line, C2C12. Luciferase reporter assays showed that a 148-bp region upstream of the ATG start codon of Tas1r1 had a promoter activity. The GT box in the Tas1r1 promoter was conserved in the dog, human, mouse, and pig. Site-directed mutagenesis of this GT box significantly reduced the promoter activation. The GT box in promoters is a recurring motif for Sp/KLF family members. RNAi-mediated depletion of Sp4 and Klf5 decreased Tas1r1 expression, while overexpression of Klf5, but not Sp4, significantly increased Tas1r1 expression. The ENCODE data of chromatin immunoprecipitation and sequencing (ChIP-seq) showed that Klf5 bound to the GT box during the myogenic differentiation. Furthermore, the Klf5 knockout cell lines led to a considerable decrease in the levels of Tas1r1 expression. Collectively, these results showed that Klf5 binds to the GT box in the Tas1r1 promoter and regulates Tas1r1 expression in C2C12 cells.


Assuntos
Fatores de Transcrição Kruppel-Like/genética , Mioblastos/metabolismo , Regiões Promotoras Genéticas , Receptores Acoplados a Proteínas-G/genética , Fator de Transcrição Sp4/genética , Sítio de Iniciação de Transcrição , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Sequência Conservada , Cães , Regulação da Expressão Gênica , Genes Reporter , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Desenvolvimento Muscular/genética , Mioblastos/citologia , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Fator de Transcrição Sp4/antagonistas & inibidores , Fator de Transcrição Sp4/metabolismo , Suínos
16.
Bioengineered ; 10(1): 98-107, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31023186

RESUMO

Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Efeito Fundador , Técnicas de Introdução de Genes/métodos , Luciferases/genética , Progranulinas/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genoma Humano , Células HEK293 , Humanos , Luciferases/metabolismo , Progranulinas/agonistas , Progranulinas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
17.
Mol Vis ; 25: 60-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820142

RESUMO

Purpose: To identify novel mutations in FZD4 and to investigate their pathogenicity in a cohort of Chinese patients with familial exudative vitreoretinopathy (FEVR). Methods: Next-generation sequencing was performed in patients with a clinical diagnosis of FEVR. Wide-field angiography was performed in probands and family members if available. Clinical data were collected from patient charts. The effect of the mutations in FZD4 on its biologic activity in the Norrin/ß-catenin signaling pathway was analyzed with the luciferase reporter assay. Results: Four novel mutations in FZD4 (c.1188_1192del/p.F396fs, c.1220delC/p.A407Vfs*24, c.905G>A/p.C302Y, c.1325T>A/p.V442E) were identified in four unrelated families. The mutations were not detected in 200 healthy individuals. The variability of the ocular phenotypes was not only observed in the probands and parents harboring the same mutation but also between two eyes in one individual. All four novel mutations introduced reduction in luciferase activity. Compared with the wild-type, the FZD4 level of the four mutants also decreased variably. Conclusions: Four novel mutations in FZD4 were identified in Chinese patients with FEVR. No correlation in the reduced luciferase activity and the ocular phenotype was observed in this study. This study further emphasized the complexity of the FEVR-causing machinery.


Assuntos
Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Receptores Frizzled/genética , Mutação , Proteínas do Tecido Nervoso/genética , Doenças Retinianas/genética , beta Catenina/genética , Adulto , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , Estudos de Casos e Controles , Pré-Escolar , Oftalmopatias Hereditárias/diagnóstico por imagem , Oftalmopatias Hereditárias/etnologia , Oftalmopatias Hereditárias/patologia , Proteínas do Olho/metabolismo , Feminino , Angiofluoresceinografia , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Fenótipo , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/etnologia , Doenças Retinianas/patologia , Transdução de Sinais , beta Catenina/metabolismo
18.
Mol Vis ; 25: 165-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820152

RESUMO

Purpose: The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function. Methods: N-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene. Results: Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation. Conclusions: The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.


Assuntos
Sequência de Bases , Proteínas do Olho/genética , Proteínas Recombinantes de Fusão/genética , Retina/metabolismo , Deleção de Sequência , Ativação Transcricional , Proteínas de Xenopus/genética , Motivos de Aminoácidos , Animais , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis
19.
Methods Mol Biol ; 1957: 393-406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919368

RESUMO

Endothelin-1 (ET-1), which acts through the endothelin A receptor (ETAR) or ETBR, belonging to the large family of G-protein coupled receptors (GPCR), is involved in physiopathological processes, such as cancer. In epithelial ovarian cancer, a pervasively activated ET-1/ETAR axis drives different steps of tumor progression and confers drug resistance. In this malignancy, one major aspect associated with the ETAR signaling machinery resides in the fact that this receptor may use ß-arrestin-1 (ß-arr1) function to spatially and temporally activate key oncogenic pathways. This results in specificity of ET-1/ETAR signal transduction mechanisms and downstream signaling pathways. As such, ß-arr1 has been recognized as an important signal transducer involved in multiple cross talks with other signaling pathways, including those activated by tyrosine kinase receptors. The interaction with diverse sets of partners positions ß-arr1 as a critical regulator of GPCR signal transduction and permits the integration of ETAR-mediated signals with other cytoplasmic or nuclear inputs. In particular, the scaffolding function of ß-arr1 provides an essential link in translating ETAR function by altering ß-catenin localization and function, promoting ß-catenin-related transcriptional activity and gene transcription relevant to tumor progression. This chapter outlines the methodologies for the measurement of ß-arr1/ß-catenin protein interactions and functional activity in tumor cells.


Assuntos
Biologia Molecular/métodos , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , beta-Arrestina 1/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , DNA/metabolismo , Feminino , Genes Reporter , Humanos , Luciferases/metabolismo
20.
Methods Mol Biol ; 1942: 61-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900175

RESUMO

Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.


Assuntos
Sistemas CRISPR-Cas , Proteína do X Frágil de Retardo Mental/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Luciferases/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Síndrome do Cromossomo X Frágil/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
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