RESUMEN
ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.
Asunto(s)
Animales , Ratas , Tejido Adiposo/citología , Diferenciación Celular/genética , Desmina/metabolismo , Proteína MioD/genética , Mioblastos/citología , ARN Bicatenario , Células Madre/citología , Western Blotting , Supervivencia Celular , Citometría de Flujo , Expresión Génica , Inmunohistoquímica , Proteína MioD/metabolismo , Mioblastos/metabolismo , Cultivo Primario de Células , Regiones Promotoras Genéticas/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Madre/metabolismo , Transfección , Activación Transcripcional/fisiología , Uretra/patología , Incontinencia Urinaria de Esfuerzo/genética , Incontinencia Urinaria de Esfuerzo/metabolismoRESUMEN
A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.
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Animales , Transfección/métodos , Factores de Tiempo , Proteínas Recombinantes de Fusión/análisis , Regiones Promotoras Genéticas/fisiología , Plásmidos , Luciferasas/genética , Estadios del Ciclo de Vida/fisiología , Giardia lamblia/genética , Ingeniería Genética/métodos , Genes Reporteros/genética , Genes Protozoarios/genética , Orden Génico , Expresión Génica/genética , Proteínas Activadoras de GTPasa/genética , Southern Blotting/métodosRESUMEN
The expression of the GLUT2 glucose transporter gene in liver is suppressed in cultured hepatoma cell lines and primary cultured hepatocytes. Earlier report showed that CCAAT/enhancer binding protein (C/EBP) regulates the promoter activity of the rat GLUT2 glucose transporter gene in liver cells. C/EBPa and C/EBPb activated the promoter activity by binding to at least two regions of the promoter and one of the C/EBP binding sites, named as site F, also has the AP-1 binding consensus. In this study, we investigated whether the AP-1 can influence on C/EBP binding to this site. The addition of recombinant c-Jun protein with liver extract caused the attenuation of C/EBP binding to site F with the appearance of a new shifted band. The shifted band was competed out with the addition of unlabeled AP-1 consensus oligonucleotide, indicating that c-Jun also can bind to site F. Another C/EBP site on GLUT2 promoter, site H, did not bind AP-1. Analysis of the DNA-protein complex revealed that C/EBP and c-Jun bind to site F in mutually exclusive manner rather than form heterodimeric complex with each other. From these results, it is suggested that the transcriptional activation of C/EBP may be influenced by c-Jun protein in certain status of the liver cells, such as acute phase response, as well as hepatocarcinogenesis.
Asunto(s)
Animales , Masculino , Ratas , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Hígado/citología , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas c-jun/genética , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Factor de Transcripción AP-1/genéticaRESUMEN
We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines
Asunto(s)
Animales , Ratones , Células 3T3 , Actinas/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Echinococcus/genética , Regiones Promotoras Genéticas/fisiología , Secuencia de Bases , Técnicas de Cultivo de Célula , Clonación Molecular , Expresión Génica , Genes Reporteros , Glicocálix , Regiones Promotoras Genéticas/genética , Transfección/genéticaRESUMEN
In previous studies we reported that the expression of HLA-DR on melanoma cell lines was differentially modulated by IFN- gamma and that the transcription rate was responsible for this differential modulation. We have also reported the nucleotide sequence variations in the promoter region of HLA-DR genes, and proposed that differences in the promoter activity by the sequence variations of the HLA-DR promoters might contribute to such a differential transcriptional regulation at the promoter level. In this study, in order to assess whether the sequence variations of the HLA-DR promoters affect the factor binding and exert influence on the promoter activity, nuclear factor binding to our previous six HLA-DRA and fourteen HLA-DRB promoter clones was evaluated with the nuclear protein extracted from a B-lymphoblastoid cell line (BLCL), BH, together with the chloramphenicol acetyltransferase (CAT) reporter assay. In the HLA-DRA promoters, clone 35 containing one bp nucleotide sequence variation at the octamer binding site (OCT) (GATTTGC to GATCTGC) showed relatively weak factor binding. In the HLA-DRB promoters, clusters I, III, and IV of our previous HLA-DRB promoter homologues, containing one bp nucleotide sequence variation (GATTCG) in their Y boxes exhibited weak factor binding and CAT activity compared to other clusters (GATTGG) that showed strong factor binding and CAT activity. This data suggests chat the binding patterns of transcription factors influenced by the nucleotide sequence variations of the HLA-DR promoter could affect the promoter activity and the DNA sequence elements in the HLA-DR promoter could mediate transcriptional regulation.