Detectie van alternatieve splicing Tijdens-mesenchymale transitie
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
De epitheliale-mesenchymale transitie (EMT) is een ontwikkelings-programma dat orgaan morfogenese en weefselremodellering rijdt tijdens de embryogenese. Bij abnormaal geactiveerd, EMT bevordert tumor uitzaaiingen en orgaanfibrose 1,2. Overtuigend studies hebben het belang van transcriptionele regulatie beschreven tijdens het proces van EMT, bepaald door verscheidene transcriptiefactoren, zoals Twist, Slak en ZEB, die de expressie van de adherens junction eiwit E-cadherine onderdrukken, wat resulteert in verlies van klinkers steenachtige epitheliale morfologie en winst van een spoelvormige mesenchymale fenotype 3-8. Recente studies door genoom-brede analyse van RNA bleek dat er een groep genen waarvan splicing patronen tezamen met epitheliale en mesenchymale fenotypen 9,10. Werk van onze labo functioneel verbonden alternatieve splicing en EMT. Door het bestuderen van het celoppervlak adhesie molecuul CD44, we dat CD44 altern aangetoondtieve splicing wordt strak gereguleerd tijdens EMT, en nog belangrijker, dat CD44 splice isovorm causaal schakelen bijdraagt aan EMT 11.
Alternatieve splicing is een wijdverspreide en geconserveerde model van genregulatie, zoals tot 95% van menselijke multi-exon genen alternatief gesplitste 12-14. Door het genereren van meerdere eiwitproducten uit een enkel gen, alternatieve splicing een essentieel mechanisme voor diversiteit eiwit, het toevoegen van een laag van complexiteit aan het menselijk genoom. Als zodanig kan ontregeling van alternatieve splicing kunnen leiden tot ernstige biologische effecten veroorzaken menselijke ziekten. Inderdaad, heeft afwijkende alternatieve splicing in ziekten gedocumenteerd voor meer dan een decennium 15-25, met inbegrip van recente bevindingen dat mutaties in genen die coderen voor de spliceosome machines gewoonlijk worden aangetroffen in myelodysplastisch syndroom 26-28. Derhalve ontwikkeling van betrouwbare methoden voor de detectie van alternatief gesplitste isoforms is van groot belang in de studie van diverse biologische processen zoals EMT.
Hier bieden we een protocol om veranderingen in alternatieve splicing met een induceerbare EMT model detecteren. De werkwijzen voor het ontwerpen van PCR primers en het detecteren splice isovormen zijn niet alleen geschikt voor de studie van alternatieve splicing gedurende EMT, maar ook voor de studie van alternatieve splicing in andere biologische processen. Het onderzoeken van alternatieve splicing tijdens EMT is noodzakelijk met het oog op een beter begrip van de mechanismen van EMT en tumor metastase, waardoor de ontwikkeling van effectieve strategieën om de uitzaaiing van kanker te behandelen vergemakkelijken.
Protocol
Representative Results
Discussion
De hier beschreven werkwijze maakt de detectie van alternatieve splicing van een induceerbare EMT model. Als zodanig dynamische veranderingen van splice isovorm expressie kan worden ingenomen gedurende het tijdsverloop van EMT. Deze werkwijze heeft voordelen boven het gebruik van verschillende epithelial- of mesenchymale cellijnen die voor vergelijking van alternatieve splicing omdat verschillende genetische achtergronden van un-gerelateerde cellijnen onrechtmatig kunnen beïnvloeden alternatieve splicing. Wel moet de s…
Declarações
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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