Epitel-Mezenkimal Geçiş sırasında alternatif birleştirme Algılama
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
Epitel-mezenkimal geçiş (EMT) Embriyogenesis sırasında organ morfogenetiğine ve doku yenileme sürücüler gelişimsel bir programdır. Anormal aktive edildiğinde, EMT tümör metastazı ve organ fibrozis 1,2 teşvik etmektedir. Zorlayıcı çalışmalar cobble- kaybıyla sonuçlanan adherens birleşme proteini E-kaderin ifadesini bastırmaktadır örneğin Twist, salyangoz ve Zeb gibi çeşitli transkripsiyon faktörleri, tarafından tanımlanan, EMT sürecinde transkripsiyon düzenlemesi önemini tarif etmişlerdir epitel morfolojisi ve iğ biçimli mezenkimal fenotip 3-8 kazancı gibi taş. RNA'lar genom analizi ile yapılan son çalışmalar yapıştırma desenler epitel veya mezenkimal fenotiplerle 9,10 ya ile ilişkili genlerin bir grup var olduğunu ortaya koymuştur. Laboratuarımızda çalışmak işlevsel alternatif eklemeler ve EMT bağlı. Hücre yüzey adezyon molekülü CD44 inceleyerek, CD44 altern gösterdiative yapıştırma sıkı EMT sırasında düzenlenir, ve daha da önemlisi, nedensel anahtarlama bu CD44 ekleme izoformdur 11 EMT katkıda bulunmaktadır.
Alternatif 12-14 raptedilmektedir insan çoklu-ekson genlerin% 95 kadar alternatif yapıştırma, gen regülasyonu bir yaygın ve korunmuş modelini temsil eder. Tek bir genin birden fazla protein ürünlerinin üretilmesi ile beraber, alternatif birleştirme insan genomuna karmaşıklık bir katman eklenerek protein çeşitliliği için önemli bir mekanizma oluşturur. Bu nedenle, alternatif uç uca eklenmesi düzensizliği, potansiyel olarak hastalığa neden olan insan derin biyolojik etkileri yol açabilir. Nitekim, hastalıklarda anormal alternatif yapıştırma spliceosom makine kodlayan genlerde mutasyonlar sık miyelodisplastik sendromlarda 26-28 bulunan son bulgular da dahil olmak üzere, bir yılda 15-25 rapor edilmiştir. Bu nedenle, alternatif olarak eklenmiş i tespiti için güvenilir yöntemler geliştirmeksoforms EMT de dahil olmak üzere çeşitli biyolojik süreçlerin çalışmada büyük önem taşımaktadır.
Burada bir uyarılabilir EMT modeli kullanılarak alternatif ekleme değişiklikleri algılamak için bir protokol sağlar. Bağlantı izoformları PCR primerlerini tasarlama ve tespit edilmesi için yöntemler, EMT sırasında alternatif splicing çalışma için değil, aynı zamanda diğer biyolojik süreçlerde alternatif splicing çalışma için sadece uygundur. EMT sırasında alternatif eklemeler araştıran iyi böylece kanser metastazı tedavi etmek için etkili stratejilerin geliştirilmesini kolaylaştırmak, EMT ve tümör metastazı mekanizmalarını anlamak için zorunludur.
Protocol
Representative Results
Discussion
Burada tarif edilen prosedür, bir uyarılabilir EMT modelinde alternatif bağlantısının belirlenmesini sağlar. Bağlantı izoform ekspresyon örneğin dinamik değişiklik EMT zaman süreci boyunca yakalanabilir gibi. Un-bağlı hücre hatlarının farklı genetik temel gereksiz alternatif eklemeler etkileyebilir, çünkü bu yöntem alternatif splicing karşılaştırılması için farklı epithelial- da mezenkimal-temsil eden hücre çizgilerinin kullanımı üzerinde avantajlara sahiptir. Ancak, EMT başarılı …
Divulgaciones
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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