Detección de splicing alternativo Durante epitelial-mesenquimal de transición
Summary
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.
Abstract
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 γ-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.
Introduction
La transición epitelial-mesenquimal (EMT) es un programa de desarrollo que impulsa la morfogénesis de órganos y la remodelación de tejidos durante la embriogénesis. Cuando se activa de forma anormal, EMT promueve la metástasis tumoral y la fibrosis de órganos 1,2. Estudios convincentes han descrito la importancia de la regulación transcripcional durante el proceso de la EMT, que se define por varios factores de transcripción, como Twist, Caracol, y ZEB, que reprimen la expresión de la proteína de unión adherens E-cadherina, lo que resulta en la pérdida de un cobble- piedra, como la morfología del epitelio y la ganancia de un fenotipo mesenquimal fusiforme 3-8. Estudios recientes a través del análisis de todo el genoma de ARN revelaron que existe un grupo de genes cuyos patrones de empalme están asociados ya sea con fenotipos epiteliales o mesenquimales 9,10. El trabajo de nuestro laboratorio funcionalmente conectado splicing alternativo y EMT. Mediante el estudio de la superficie celular molécula de adhesión CD44, hemos demostrado que CD44 alternativa empalme está estrechamente regulada durante EMT, y lo más importante, que la isoforma de empalme CD44 conmutación causalmente contribuye a EMT 11.
Splicing alternativo representa un modelo generalizado y conservado de la regulación de genes, ya que hasta el 95% de los genes de varios exones humanos están empalmados alternativamente 12-14. Mediante la generación de múltiples productos de proteína a partir de un único gen, corte y empalme alternativo constituye un mecanismo esencial para la diversidad de proteínas, la adición de otra capa de complejidad al genoma humano. Como tal, la desregulación de splicing alternativo podría dar lugar a profundos efectos biológicos que causan enfermedades humanas. De hecho, el splicing alternativo aberrante en enfermedades se ha documentado desde hace más de una década 15-25, incluyendo los recientes hallazgos de que las mutaciones en los genes que codifican la maquinaria spliceosome se encuentran comúnmente en los síndromes mielodisplásicos 26-28. Por lo tanto, el desarrollo de métodos fiables para la detección de i empalmados alternativamentesoforms es de gran importancia en el estudio de diversos procesos biológicos incluyendo la EMT.
Aquí proporcionamos un protocolo para detectar cambios en el splicing alternativo utilizando un modelo EMT inducible. Los métodos para diseñar cebadores de PCR y la detección de isoformas de empalme son adecuados no sólo para el estudio de corte y empalme alternativo durante la EMT, sino también para el estudio de splicing alternativo en otros procesos biológicos. La investigación de corte y empalme alternativo durante EMT es imperativo con el fin de comprender mejor los mecanismos de la EMT y la metástasis del tumor, facilitando así el desarrollo de estrategias eficaces para tratar la metástasis del cáncer.
Protocol
Representative Results
Discussion
El procedimiento descrito aquí permite la detección de corte y empalme alternativo en un modelo inducible EMT. Como tales, las alteraciones dinámicas de empalme isoforma expresión pueden ser capturados durante todo el curso de tiempo de EMT. Este método tiene ventajas sobre el uso de diferentes epithelial- o líneas de células mesenquimales-que representa para la comparación de splicing alternativo debido a antecedentes genéticos distintos de líneas celulares no relacionados, podrían influir indebidamente en e…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister’s Hope Foundation.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| 4-hydroxytamoxifen | Sigma | H7904 | Make stock solution by dissolving in ethanol to 200μM, and keep at -20℃ protected from light. |
| E.Z.N.A. Total RNA Isolation kit | Omega Bio-Tek | R6731 | Total RNA isolation kit |
| GoScript Reverse Transcription System | Promega | A5001 | Reagent for qRT-PCR assay |
| GoTaq qPCR Master Mix | Promega | A6002 | Reagent for qRT-PCR assay |
| LightCycler 480 Real-Time PCR System | Roche | Equipment for qRT-PCR assay | |
| CD44 antibody | R&D Systems | BBA10 | 1:1000 dilution |
| E-cadherin antibody | Cell Signaling Technology | 4065 | 1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence |
| γ-catenin antibody | Cell Signaling Technology | 2309 | 1:1000 dilution |
| occludin antibody | Santa Cruz Biotechnology Inc. | sc-5562 | 1:500 dilution |
| fibronectin antibody | BD Transduction Laboratories | 610077 | 1:5000 dilution |
| N-cadherin antibody | BD Transduction Laboratories | 610920 | 1:2000 dilution |
| vimentin antibody | NeoMarkers | MS-129-p1 | 1:500 dilution |
| GAPDH antibody | Millipore Corporation | MAB374 | 1:10000 dilution |
| Amasham ECL Western blotting detection reagent | GE Health Life Science | RPN2209 | Chemiluminescence system |
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