Name: detection of alternative splicing during epithelial-mesenchymal transition
Uploaded: 2014-10-09T14:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

1 Cultura de Células da EMT Indução Nota: EMT pode ser induzida por meio de tratamento de TGF-p ou expressão ectópica de factores de transcrição de torção, Snail, ou ZEB1 / 2 em células epiteliais. Descrito neste protocolo é um sistema EMT induzida via expressão da proteína de fusão Twist-ER nas células mamárias humanas imortalizadas epiteliais (HMLE / Twist-ER, um presente do Dr. J Yang, UCSD) 9,11,29. Após o 4-hidroxitamoxifeno (TAM) de tratamento, a proteína de fusão Twist-ER transloca-se para o núcleo para dirigir a transcrição e resulta numa transição EMT completo em 12-14 dias 9,11,29. Sistemas induzíveis EMT adicionais, como HMLE / Caracol-ER, HMLE / TGF e MCF10A / TGF, pode ser encontrada em nossas publicações anteriores 11,30. <ol><li> Manter células HMLE / tvist ER mamária em meio de crescimento de células epiteliais sem soro (MEGM) em uma incubadora a 37 ° C com 5% de CO 2. Passagem das células a cada 2-3 dias, quando chegar a 80% de confluência. Incubar as células com tripsina a 0,15% a 37 ° C durante 5-10 minutos, e, em seguida, inactivar a tripsina com 5% de soro de vitelo / DMEM. Gire as células e ressuspender-los em MEGM. Células da placa em uma nova placa de cultura de tecidos a uma razão de 1 a 4. </li><li> Dissolve-se em etanol a TAM para fazer uma solução 200 uM. Proteger da luz e da TAM armazenar a -20 ° C. Antes da utilização, adicionar recém TAM para os meios de MEGM a uma diluição de 1: 10000 para fazer uma concentração final de 20 nM a TAM. </li><li> Para a indução da EMT, placa células 1,6 x 10 6 HMLE / Torção-ER em um 10 centímetros de cultura de tecidos prato em duplicado. Células de cultura com 20 nM mídia TAM contendo MEGM. </li><li> Placa células 1,6 x 10 6 HMLE / Torção-ER em uma placa de 10 cm em duplicado e tratar as células com etanol 0,01% como controle tratados com não-TAM. </li><li> Dois dias após a incubação, para tirar fotos sob um microscópio de luz em aumento de 10x para gravar alterações morfológicas de células. </li><li> Aspirar o meio de um prato de controlo ou tratados com a TAMcélulas. Lavar as células com PBS frio, e as células da colheita pela demolição metade da placa em tampão de lise de ARN para o isolamento de ARN e a outra metade em tampão RIPA para análise de proteínas. </li><li> Células passagem do controle ou pratos tratados com TAM por re-chapeamento de 1,6 x 10 6 células em um prato de 10 cm em duplicado. Continuamente tratar as células com 20 nM de TAM ou veículo. </li><li> Repita os passos 5-7 em dias alternados até ao dia 14, altura em que as células tratadas Twist-ER-TAM mostram uma morfologia mesenquimal em forma de fuso, ao passo que as células de controlo tratadas com veículo a manutenção da morfologia epitelial de tipo paralelepípedo. </li></ol> 2 Caracterização de EMT Nota: A conclusão da EMT é indicado por: (1) Celulares alterações morfológicas de uma morfologia epitelial calçada semelhante a uma aparência de fibroblastos-like fusiforme; (2) Perda de localização de E-caderina nas junções célula-célula; e (3) a expressão dos marcadores de comutação EMT, definida pela perdade marcadores epiteliais e ganho de marcadores mesenquimais. Celulares alterações morfológicas são captadas por microscópio de luz em aumento de 10x durante o curso do tempo de indução EMT, descrito na Seção 1 detecção de Imuno-fluorescência de E-caderina em junções celulares é descrita nesta seção. A expressão dos marcadores de EMT é detectada por imunotransferência. Marcadores epiteliais comuns são a E-caderina, γ-catenina, e ocludina e marcadores mesenquimais incluem fibronectina, N-caderina e vimentina. Procedimentos gerais de immunoblotting estão descritos na Seção 4. <ol><li> No dia 12 do tratamento TAM, semente de 1 x 10 5 células em um de 12 milímetros de vidro lamela circular colocado num poço de uma placa de 24 poços. </li><li> 48 horas depois que as células de semeadura, médias Aspirar e lavar com pré-aquecido PBS. </li><li> Fix células com pré-aquecido a 4% de paraformaldeído durante 15 min. </li><li> Lave as células três vezes com PBS. </li><li> Incubar as células com 0,2% de Triton X-100 em PBS por 15min à temperatura ambiente. </li><li> Lave as células três vezes com PBS. </li><li> Incubar as células em 5% de BSA / PBS, durante 1 hora à temperatura ambiente para bloquear a ligação não específica. </li><li> Incubar as células com o anticorpo de E-caderina primário numa diluição 1:50 em BSA a 1% / PBS em uma câmara humidificada S / N a 4 ° C. NOTA: O intervalo para a diluição de anticorpo primário é normalmente entre 1:50 e 1: 200, e a diluição óptima para cada anticorpo deve ser determinada experimentalmente. </li><li> Decantar o anticorpo primário e lava-se as células três vezes com PBS. </li><li> Incubar as células com o anticorpo secundário marcado com fluorescência em uma diluição 1: 500, durante 1 h à TA. A partir deste passo, a proteger as células da luz. </li><li> Decantar o anticorpo secundário e lava-se três vezes com PBS. </li><li> Células mancha com 0,1-1 g / ml DAPI ou Hoechst para 10 min. </li><li> Lavar as células com PBS três vezes. </li><li> Monte lamelas com uma gota de meio de montagem, e lamelas de vedação com unha polonês. </li><li> Observar células e tomarimagens em um microscópio fluorescente 63X. </li></ol> A coloração com anticorpos adicionais podem ser realizados para confirmar o fenótipo de EMT. Por exemplo, N-caderina e actina de músculo liso-α pode ser corado por um 29,31,32 fenótipo mesenquimal. Reorganização de estrutura do citoesqueleto podem ser monitorizadas por coloração das células com faloidina para F-actina 11. Além disso, ensaios para a motilidade das células e resistência à morte celular podem ser realizados para examinar as propriedades dos EMT 11. 3 Detecção de Splice isoformas utilizando qRT-PCR Ensaios <ol><li> Projeto Primer NOTA: Os pares de primers devem ser cuidadosamente projetado para amplificar isoformas de splicing distintas. Exon pular, o acontecimento de processamento alternativo mais frequentemente observados, é mostrado como um exemplo para ilustrar a estratégia de desenho de primers. Figura 1A ilustra um exão de quatro pré-ARNm, com o terceiro exão de um exão variável. Inclusão de exão 3 resultaa produção de uma isoforma de emenda mais tempo, enquanto que o salto do exon 3 gera uma isoforma mais curta de splicing. Os iniciadores devem ser concebidos para distinguir a isoforma incluído exão-3-e isoforma do exão-3-ignorado, e para detectar o transcrito total deste ARNm. <ol><li> Para a detecção de inclusão exão, desenhar cuidadosamente um iniciador para a frente no interior do exão 3 variável, e uma sequência iniciadora reversa no interior do exão 4 constitutiva vizinhas, permitindo a amplificação específica da isoforma variável incluído-exão. Certifique-se não tanto à frente e conceber os iniciadores no seio da mesma exão variável inversa, a fim de evitar produtos de PCR amplificados a partir de ADN genómico ou de pré-ARNm. </li><li> Para amplificar especificamente os eventos ignorando exão, design um dos iniciadores, abrangendo a junção do exão 2 e constitutiva exão 4, que de outra forma seriam destruídos quando exão variável 3 está incluído. Conceber o outro iniciador no interior do exão 4 constitutiva Como tal, os produtos de PCR são gerados apenas quando o exão 3 é variável, excluied eo constitutivo exon 2 e exon 4 são posteriormente unidas. </li><li> Para detectar as transcrições totais do gene, desenhar os iniciadores de dentro de dois exons constitutivos 1 e 2 Assim, os produtos de PCR são amplificados a partir de todas as isoformas. </li><li> Certifique-se de que a temperatura de fusão (Tm) para todos os iniciadores é de aproximadamente 55 ° C, a duração de cada um dos iniciadores é aproximadamente 18-22 nucleótidos, e o tamanho dos produtos de PCR é entre 80 e 150 pares de bases. </li></ol></li><li> Extração de RNA: Extrato RNA usando um kit de isolamento total RNA (ver Tabela de Materiais). <ol><li> Recolher as células em 350 ul de tampão de lise de ARN. </li><li> Adicionar 350 mL de etanol a 70% ao ligado e misture bem. </li><li> Transfira a amostra para a coluna de purificação de ARN. </li><li> Centrifuga-se a 10000 xg durante 1 min e descarte de fluxo. </li><li> Lava-se a coluna uma vez com 500 ul de tampão de lavagem de RNA I e duas vezes com tampão de lavagem de ARN II. </li><li> Remover o tampão de lavagem residual ARNII a partir da coluna através de centrifugação à velocidade máxima durante 2 min. </li><li> Transferir a coluna para um novo tubo de 1,5 ml, adicionar 50 mL de água isenta de nuclease, no centro da matriz da coluna e eluir o ARN por centrifugação à velocidade máxima. </li></ol></li><li> Síntese da primeira cadeia de cDNA <ol><li> Determinar a concentração e a qualidade do RNA por medição da absorção de UV. NOTA: qualidade RNA bom deve ter uma relação absorbância / 280 nm 260 nm de aproximadamente 2,0. </li><li> Defina-se a transcriptase reversa (RT), de reacções que contêm 250 ng de ARN total, a 0,05 ug iniciadores de hexâmeros ao acaso, 50 pmol de MgCl2, 10 pmol de dNTPs, 2 ul de tampão 5 × GoScript, e 1 ul da enzima RT, num volume final de 20 ul. </li><li> Realizar as reacções de RT a 42 ° C durante 1 hora, seguido de incubação a 70 ° C durante 5 min, para inactivar a enzima RT. </li></ol></li><li> PCR em tempo real <ol><li> Configure 20 reacções ul que consistem em 10 mL SYBR master mix verde,0,1-1,0 modelo de cDNA ul e 5 pmol de iniciadores directos e inversos. </li><li> Executar PCR em tempo real com 40 ciclos dos seguintes passos: 95 ° C desnaturação durante 10 seg, 58 ° C, emparelhamento durante 10 segundos, e extensão a 60 ° C durante 30 seg. Realizar reações de PCR em triplicado. </li><li> Confira curvas de dissociação e certifique-se que um único pico é observado para cada conjunto de primers. </li><li> Obtenção dos valores de ciclo de quantificação (Cq), calculando os segundos valores derivados das curvas de amplificação. </li><li> Calcula-se a quantidade relativa (RQ) de mRNAs alvo em amostras induzidas, em comparação com a amostra não induzido utilizando o &quot;delta delta Cq (ΔΔCq) &#39;método, como mostrado pela seguinte fórmula. Use genes de manutenção, tais como GAPDH ou TBP, como referência. </li></ol></li></ol><img alt="Equação 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Para calcular com mais precisão os valores relativos das isoformas de splicing, tele utilizar de múltiplos genes de referência deverá ser considerada. Os resultados podem ser analisados ​​utilizando um método de quantificação de valor absoluto modificado descrito por Pfaffl et al 33,34. 4. Exame da Splice isoformas no nível de proteína <ol><li> Recolher as células em 100 de tampão RIPA arrefecido em gelo ul. </li><li> Definir lisados ​​em gelo durante cerca de 10 min. </li><li> Centrifuga-se a 14000 xg durante 10 min a 4 ° C, e recolher o sobrenadante. </li><li> Medir a concentração de proteína por meio de ensaios de Bradford. </li><li> Diluir as amostras de proteína em tampão de carga de SDS e carregar 20-40 ug de proteínas em géis de 10% SDS-PAGE. </li><li> Execute géis SDS-PAGE a 20 mA durante 1,5 horas. </li><li> Proteínas de transferência para membranas de PVDF em um sistema de transferência húmida a 4 ° C durante 3 horas a 85 V. Ajustar o tempo de transferência de acordo com o peso molecular de proteínas alvo. </li><li> Bloco de membrana em 5% de leite desnatado durante 30 minutos a 1 hora a RT. </li><li> Incubar wi membranapo um anticorpo primário a 4 ° CO / N. O intervalo para a diluição de anticorpo primário é normalmente entre 1: 200 e 1: 5000, determinar a diluição óptima para cada anticorpo experimentalmente. A diluição dos anticorpos usados ​​no presente protocolo podem ser encontrados em &quot;Tabela de reagentes e equipamentos específicos&quot;. <ol><li> Para detectar as isoformas de splicing, utilizar anticorpos específicos que reconhecem uma isoforma particular. Em alternativa, utilizar um anticorpo que reconhece o epitopo na região de codificação de exão constitutiva e detectar as isoformas de splicing, simultaneamente, com base nos seus tamanhos diferentes de proteína. </li><li> Ao mesmo tempo, controlar EMT por imunotransferência por marcadores EMT. Use E-caderina, γ-catenina, e ocludina como marcadores epiteliais, e fibronectina, N-caderina e vimentina como marcadores mesenquimais. </li></ol></li><li> Membrana de lavagem três vezes com TBS-T (50 mM Tris, 150 mM de NaCl, 0,05% de Tween 20, pH 7,4) em um intervalo de 5 minutos. </li><li> Incubar membrana com uma formiga secundário conjugado com HRPibody em uma diluição de 1: 10000 a 1 hora à temperatura ambiente. </li><li> Membrana de lavagem três vezes com TBS-T com um intervalo de 5 minutos. </li><li> Visualizar proteína com um sistema de detecção de quimioluminescência e expor a membrana a uma película de auto-radiografia. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial-mesenchymal+transition+generates+cells+with+properties+of+stem+cells.">The epithelial-mesenchymal transition generates cells with properties of stem cells.</a> Cell. 133, 704-715 (2008).</li><li>Reinke, L. M., Xu, Y., Cheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snail+represses+the+splicing+regulator+epithelial+splicing+regulatory+protein+1+to+promote+epithelial-mesenchymal+transition.">Snail represses the splicing regulator epithelial splicing regulatory protein 1 to promote epithelial-mesenchymal transition.</a> J Biol Chem. 287, 36435-36442 (2012).</li><li>Serini, G., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+myofibroblast+activity+and+phenotypic+modulation.">Mechanisms of myofibroblast activity and phenotypic modulation.</a> Exp Cell Res. 250, 273-283 (1999).</li><li>Zavadil, J., Bottinger, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+and+epithelial-to-mesenchymal+transitions.">TGF-beta and epithelial-to-mesenchymal transitions.</a> Oncogene. 24, 5764-5774 (2005).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29, e45 (2001).</li><li>Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., Vandesompele, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=qBase+relative+quantification+framework+and+software+for+management+and+automated+analysis+of+real-time+quantitative+PCR+data.">qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.</a> Genome Biol. 8, R19 (2007).</li><li>Boutz, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+post-transcriptional+regulatory+switch+in+polypyrimidine+tract-binding+proteins+reprograms+alternative+splicing+in+developing+neurons.">A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons.</a> Genes Dev. 21, 1636-1652 (2007).</li><li>Salomonis, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+regulates+mouse+embryonic+stem+cell+pluripotency+and+differentiation.">Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.</a> Proc Natl Acad Sci USA. 107, 10514-10519 (2010).</li><li>Calarco, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vertebrate+nervous+system+alternative+splicing+and+development+by+an+SR-related+protein.">Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.</a> Cell. 138, 898-910 (2009).</li><li>Grabowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+takes+shape+during+neuronal+development.">Alternative splicing takes shape during neuronal development.</a> Curr Opin Genet Dev. 21, 388-394 (2011).</li><li>Seol, D. W., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+caspase-9+variant+missing+the+catalytic+site+is+an+endogenous+inhibitor+of+apoptosis.">A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis.</a> J Biol Chem. 274, 2072-2076 (1999).</li><li>Srinivasula, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+endogenous+dominant-negative+short+isoform+of+caspase-9+that+can+regulate+apoptosis.">Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis.</a> Cancer Res. 59, 999-1002 (1999).</li><li>Akgul, C., Moulding, D. A., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+Bcl-2-related+genes+functional+consequences+and+potential+therapeutic+applications.">Alternative splicing of Bcl-2-related genes functional consequences and potential therapeutic applications.</a> Cell Mol Life Sci. 61, 2189-2199 (2004).</li><li>Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+minigene+systems+to+dissect+alternative+splicing+elements.">Use of minigene systems to dissect alternative splicing elements.</a> Methods. 37, 331-340 (2005).</li><li>Cheng, C., Sharp, P. 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The authors declare that they have no competing financial interests.

O processo aqui descrito permite a detecção de splicing alternativo de um modelo de EMT indutível. Como tal, as alterações dinâmicas de emenda de expressão da isoforma pode ser captado em todo o curso de tempo de EMT. Este método tem vantagens sobre o uso de diferentes epithelial- ou linhas de células-representando mesenquimais para a comparação de splicing alternativo, porque origens genéticas distintas de linhas celulares relacionadas un poderia influenciar indevidamente splicing alternativo. No entanto, a...

A transição epitelial-mesenquimal (EMT) é um programa de desenvolvimento que impulsiona a morfogênese de órgãos e remodelação do tecido durante a embriogênese. Quando activados anormalmente, EMT promove a metástase do tumor e órgão fibrose 1,2. Obrigando estudos descreveram a importância da regulação da transcrição durante o processo de EMT, definida por vários factores de transcrição, tais como torção, Snail, e ZEB, que reprimem a expressão da proteína de junção aderente E-caderina, que resultam na perda de um cobble- pedra, como morfologia epitelial e ganho de um fenótipo mesenquimal fusiforme 3-8. Estudos recentes por meio de análise de todo o genoma de ARN revelou que existe um grupo de genes cujos padrões de splicing estão associadas quer epiteliais ou mesenquimais fenótipos 9,10. Trabalho de nosso laboratório de funcionalmente ligados splicing alternativo e EMT. Ao estudar a molécula de adesão de superfície celular CD44, foi demonstrado que CD44 alternative splicing é rigidamente controlado durante a EMT, e mais importante, que isoforma CD44 emenda comutação causalmente contribui para EMT 11. O splicing alternativo representa um modelo disseminado e conservada da regulação de genes, tal como até 95% de genes de multi-exões humanos são de splicing alternativo 12-14. Através da produção de vários produtos de proteínas a partir de um único gene, o splicing alternativo constitui um mecanismo essencial para a diversidade de proteínas, a adição de uma outra camada de complexidade para o genoma humano. Como tal, a desregulação de splicing alternativo pode potencialmente levar a profundos efeitos biológicos que causam doenças humanas. Na verdade, o splicing alternativo aberrante em doenças tem sido documentada por mais de uma década de 15-25, incluindo as recentes descobertas de que as mutações em genes que codificam a maquinaria spliceosome são comumente encontrados em síndromes mielodisplásicas 26-28. Portanto, o desenvolvimento de métodos confiáveis ​​para a detecção de i de splicing alternativosoforms é de grande importância no estudo de processos biológicos diversos, incluindo EMT. Aqui nós fornecemos um protocolo para detectar mudanças no splicing alternativo utilizando um modelo EMT induzida. Os métodos para a concepção de iniciadores de PCR e detectar as isoformas de splicing são adequadas não apenas para o estudo de splicing alternativo durante EMT, mas também para o estudo de splicing alternativo em outros processos biológicos. Investigando splicing alternativo durante EMT é imprescindível, a fim de compreender melhor os mecanismos da EMT ea metástase do tumor, facilitando, assim, o desenvolvimento de estratégias eficazes para tratar a metástase do câncer.

<table><tbody><tr><td>4-hydroxytamoxifen</td><td>Sigma</td><td>H7904</td><td>Make stock solution by dissolving 4-hydroxytamoxifen in ethanol to 200 &amp;#956;M. Keep the solution at -20 &amp;#176;C protected from light.</td></tr><tr><td>E.Z.N.A. Total RNA Isolation kit</td><td>Omega Bio-Tek</td><td>R6731</td><td>Total RNA isolation kit</td></tr><tr><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5001</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>LightCycler 480 Real-Time PCR System</td><td>Roche</td><td></td><td>Equipment for qRT-PCR assay</td></tr><tr><td>CD44 antibody</td><td>R&amp;amp;D Systems</td><td>BBA10</td><td>1:1,000 dilution</td></tr><tr><td>E-cadherin antibody</td><td>Cell Signaling Technology</td><td>4065</td><td>1:2,500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td></tr><tr><td>&amp;gamma;-Catenin antibody</td><td>Cell Signaling Technology</td><td>2309</td><td>1:1,000 dilution</td></tr><tr><td>Occludin antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-5562</td><td>1:500 dilution</td></tr><tr><td>Fibronectin antibody</td><td>BD Transduction Laboratories</td><td>610077</td><td>1:5,000 dilution</td></tr><tr><td>N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>610920</td><td>1:2,000 dilution</td></tr><tr><td>Vimentin antibody</td><td>NeoMarkers</td><td>MS-129-p1</td><td>1:500 dilution</td></tr><tr><td>GAPDH antibody</td><td>Millipore Corporation</td><td>MAB374</td><td>1:10,000 dilution</td></tr><tr><td>Amasham ECL Western blotting detection reagent</td><td>GE Health Life Science</td><td>RPN2209</td><td>Chemiluminescence system</td></tr></tbody></table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Os procedimentos descritos acima fornecem um método robusto para detectar splicing alternativo durante EMT. Os resultados representativos de CD44 splice isoforma de comutação durante EMT induzida por torção são dadas abaixo como um exemplo. EMT induzida por torção em células HMLE / twist-ER foi caracterizado por uma transição de uma calçada de pedra como fenótipo epitelial para um fenótipo fibroblástica alongada (Figura 2A), a ausência de marcadores epiteliai...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Detecção de splicing alternativo Durante epitelial-mesenquimal Transição

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.9K visualizações.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Vídeo: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Usando RNA-sequenciação para detectar Novel Splice variantes relacionadas à resistência aos medicamentos em<em&gt; In Vitro</em&gt; modelos de cancro

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Pesquisa do câncer

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Engenharia de fatores artificiais para manipular Especificamente splicing alternativo em células humanas

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genética

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Análise quantitativa de alternativo Pre-mRNA emendar nas seções de cérebro de rato usando o RNA In Situ da hibridação do ensaio

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

A indução de transições mesenquimais-epiteliais em células de sarcoma

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Biologia do Desenvolvimento

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Estudando a sinalização TGF-β e a transição epitelial-mesenquimal induzida pelo TGF-β em câncer de mama e células normais

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

Usando a ferramenta E1A Minigene para estudar alterações de emenda mRNA

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

Um ensaio celular baseado em repórter para monitorar a eficiência do splicing

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Bioquímica

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Biologia

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

Identificação de Splicing Alternativo e Poliadenilação em Dados de RNA-seq

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.
Splicing is Mediated by the Spliceosome
Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5&#39; end of the intron and to a branch point sequence containing an A towards the 3&#39; end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5&#39; splice site. Subsequently, a chemical reaction cleaves the 5&#39; end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5&#39; end of the downstream exon reacts with the 3&#39; end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.
Splicing Allows the Expression of Several Proteins from a Single Gene
Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.
Different patterns of alternative splicing include exon skipping, alternative 5&#39; or 3&#39; splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.
Abnormal Splicing Can Cause Diseases
Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform&#8212;BCL-XL and BCL-XS, respectively&#8212; through the use of alternative 5&#39; splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

RNA Splicing, Lariat and the Spliceososme

Splicing do RNA

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called ...