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1.
Philos Trans R Soc Lond B Biol Sci ; 369(1652)2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25135963

RESUMO

Exosomes are tiny vesicles (30-150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.


Assuntos
Exossomos/genética , RNA/genética , Soro/química , Urina/química , Sequência de Bases , Biomarcadores/sangue , Biomarcadores/urina , Exossomos/ultraestrutura , Fluorescência , Células HeLa , Humanos , Dados de Sequência Molecular , Análise de Sequência de RNA
2.
Methods Mol Biol ; 764: 183-97, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21748641

RESUMO

RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (post-transfection) and in animals (post- injection). We have adopted the Applied Biosystems TaqMan(®) based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. In this chapter, application protocols are described, which enable robust quantification of siRNA, including chemically modified molecules, in vitro and in vivo.


Assuntos
Bioensaio , MicroRNAs/análise , RNA Interferente Pequeno/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Bioensaio/instrumentação , Bioensaio/métodos , Inativação Gênica , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/isolamento & purificação , Interferência de RNA , Estabilidade de RNA , RNA Interferente Pequeno/isolamento & purificação , Transfecção
3.
Methods Mol Biol ; 764: 199-213, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21748642

RESUMO

RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome). A properly designed and selected siRNA sequence, siRNA modification format, choice of transfection reagent/technique, optimized protocols of siRNA in vitro delivery, and an appropriate and optimized readout are all critical for ensuring a successful experiment. In this chapter, we describe a typical in vitro siRNA experiment with optimization of transfection conditions and analysis of siRNA potency, i.e., mRNA knockdown with quantitative real-time PCR.


Assuntos
Bioensaio , Técnicas de Silenciamento de Genes/métodos , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Bioensaio/instrumentação , Bioensaio/métodos , Feminino , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Células HeLa , Humanos , Lipídeos/farmacologia , Interferência de RNA/efeitos dos fármacos , Estabilidade de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/isolamento & purificação , Transfecção
4.
Oligonucleotides ; 19(2): 203-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19284311

RESUMO

RNA interference (RNAi) is a mechanism in which the introduction of small interfering RNAs (siRNAs) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. Applications of RNAi include gene function and pathway analysis, target identification and validation, and therapeutics. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (posttransfection) and in animals (postinjection). We have leveraged the Applied Biosystems TaqMan-based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. The application protocols developed enable robust quantification of siRNA, including chemically modified siRNA molecules, in vitro and in vivo.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Camundongos , RNA Interferente Pequeno/metabolismo , Transfecção
5.
Cell Cycle ; 7(6): 759-64, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18344688

RESUMO

MicroRNAs have been increasingly implicated in human cancer and interest has grown about the potential to use microRNAs to combat cancer. Lung cancer is the most prevalent form of cancer worldwide and lacks effective therapies. Here we have used both in vitro and in vivo approaches to show that the let-7 microRNA directly represses cancer growth in the lung. We find that let-7 inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts in immunodeficient mice. Using an established orthotopic mouse lung cancer model, we show that intranasal let-7 administration reduces tumor formation in vivo in the lungs of animals expressing a G12D activating mutation for the K-ras oncogene. These findings provide direct evidence that let-7 acts as a tumor suppressor gene in the lung and indicate that this miRNA may be useful as a novel therapeutic agent in lung cancer.


Assuntos
Genes Supressores de Tumor , Terapia Genética/métodos , Neoplasias Pulmonares/terapia , MicroRNAs/uso terapêutico , Adenoviridae , Administração Intranasal , Animais , Linhagem Celular Tumoral , Proliferação de Células , Vetores Genéticos , Humanos , Neoplasias Pulmonares/genética , Camundongos , MicroRNAs/administração & dosagem
6.
Methods Mol Biol ; 419: 289-301, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18369991

RESUMO

Although the majority of gene function studies center themselves around protein-encoding RNAs, the study of non-protein-encoding RNAs is becoming more widespread because of the discovery of hundreds of small RNA termed micro (mi) RNA that have regulator functions within cells. Currently, over 470 human miRNA genes are predicted to exist and are annotated within the "miRBase" public miRNA database ( http://microrna.sanger.ac.uk/ ). There is no denying that short interfering (si) and short hairpin (sh) RNAs have revolutionized how scientists approach understanding gene function; however, si and shRNAs are not effective for analyzing the function of miRNAs given that miRNAs are typically short (17-24 bases). In turn, new sets of agents that allow for the expression of miRNA above endogenous levels and inhibition of miRNAs have become a valuable technology for the study of these small regulatory RNAs. In this chapter, we provide step-by-step methods on how to utilize synthetic precursor and antisense inhibitor molecules for understanding miRNA function.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Ciclo Celular/genética , Clonagem Molecular , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Células HeLa , Humanos , MicroRNAs/síntese química , Biologia Molecular/métodos , Precursores de RNA/síntese química , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Antissenso/síntese química , RNA Antissenso/genética , RNA Antissenso/metabolismo , Transfecção
7.
Methods Mol Biol ; 419: 303-16, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18369992

RESUMO

Knockdown of cellular RNA using short interfering RNA has enabled researchers to perform loss-of-function (LOF) experiments in a wide variety of cell types and model systems. RNA interference techniques and reagents have made possible experiments that test everything from the analysis of function of single genes to screening for genes that are involved in critical biological pathways on a genome-wide scale. Although siRNA experiments are generally common practice in research laboratories, it is still important to keep in mind that many factors can influence efficacy of knockdown. A properly designed siRNA, optimized protocols of siRNA delivery, and an appropriate and well-optimized readout are all critical parameters for ensuring the success of your experiment. In this chapter, we provide step-by-step procedures for performing an siRNA knockdown experiment from cell culture to analysis of knockdown using quantitative real-time PCR.


Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Interferente Pequeno/genética , Células HeLa , Humanos , Biologia Molecular/métodos , Interferência de RNA , Transcrição Gênica
8.
Bioorg Med Chem Lett ; 17(19): 5295-9, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17728131

RESUMO

Design, synthesis, and biological evaluation of 2'-fluoro-substituted cap analogs, i.e., m(7,2'F)G[5']ppp[5']G and m(7,2'F)G[5']ppp[5']m(7)G are described. Structures were confirmed by (1)H, (31)P, (19)F NMR and MS data. The effects of the 2'-fluoro-substituted moiety from the normal and N(7) double methylated mCAP were evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. Luciferase fusion protein production was monitored by measuring the luciferase activity. mRNA poly(A) capped with 2'-fluoro-substituted cap analogs, (m(7,2'F)G[5']ppp[5']G) and (m(7,2'F)G[5']ppp[5']m(7)G), were translated approximately 2.4- and 2.5-fold more efficiently, respectively, than mRNA capped with conventional m(7)G[5']ppp[5']G.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Guanosina/análogos & derivados , Guanosina/síntese química , Guanosina/farmacologia , Proteínas Virais/antagonistas & inibidores , Actinas/biossíntese , Actinas/genética , Desenho de Fármacos , Genes Reporter/genética , Guanosina/química , Células HeLa , Humanos , Luciferases/genética , Plasmídeos/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Relação Estrutura-Atividade
9.
Cell ; 120(5): 635-47, 2005 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-15766527

RESUMO

MicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , Proteínas ras/genética , Regiões 3' não Traduzidas/genética , Animais , Caenorhabditis elegans , Carcinoma/genética , Carcinoma/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Transformação Celular Neoplásica/genética , Genes Reporter/genética , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Dados de Sequência Molecular
10.
Nucleic Acids Res ; 33(4): 1290-7, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15741182

RESUMO

Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3'-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.


Assuntos
Apoptose , Proliferação de Células , MicroRNAs/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Genes Reporter , Células HeLa , Humanos , Luciferases/análise , Luciferases/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética
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