Nachweis von Alternative Splicing Während Epithelial-mesenchymale Transition
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
Die epitheliale-mesenchymale Transition (EMT) ist ein Entwicklungsprogramm, das Organ Morphogenese und Gewebeumbau während der Embryogenese steuert. Wenn abnorm aktiviert, fördert die EMT Tumormetastasierung und Organfibrose 1,2. Zwingende Studien haben die Bedeutung der transkriptionellen Regulierung während des Prozesses der EMT beschrieben, durch mehrere Transkriptionsfaktoren, wie Verdrehen, Schnecke, und ZEB, die die Expression des Zonula-junction-Protein E-Cadherin-Repression, was zum Verlust eines Pflaster definiert Stein wie epitheliale Morphologie und Verstärkung eines spindelförmigen mesenchymalen Phänotyp 3-8. Jüngste Studien durch genomweiten Analyse von RNAs aufgedeckt, daß es eine Gruppe von Genen, deren Klebemuster entweder mit epithelialen oder mesenchymalen Phänotypen 9,10 zugeordnet. Arbeiten aus unserem Labor alternatives Spleißen und EMT funktionell verbunden. Durch das Studium der Zelloberfläche Adhäsionsmolekül CD44 wir gezeigt, dass CD44 alterntiven Spleißen dicht bei EMT geregelt, und noch wichtiger, dass CD44 Spleiß-Isoform Schalt kausal trägt EMT 11.
Alternatives Spleißen ist ein weit verbreitet und konservierten Modell der Genregulation, wie bis zu 95% der menschlichen Multi-Exon-Gene sind alternativ gespleißte 12-14. Durch die Erzeugung von mehreren Protein-Produkte von einem einzigen Gen, stellt alternative Spleißen einen wesentlichen Mechanismus für die Proteinvielfalt, indem eine weitere Schicht von Komplexität des menschlichen Genoms. Als solche könnte eine Fehlregulation von alternativem Spleißen möglicherweise tiefgreifende biologische Wirkungen verursachen Erkrankungen des Menschen führen. Tatsächlich hat abweichende alternative Spleißen in Krankheiten, für mehr als ein Jahrzehnt 15-25 dokumentiert, einschließlich der jüngsten Erkenntnisse, die Mutationen in Genen die Spleißosom Maschinen codieren, werden häufig in myelodysplastischen Syndromen 26-28 gefunden. Daher ist die Entwicklung zuverlässiger Verfahren zum Nachweis von alternativ gespleißten isoforms ist von großer Bedeutung in der Erforschung der vielfältigen biologischen Prozesse, einschließlich EMT.
Hier bieten wir ein Protokoll, um Veränderungen in alternative Spleißen unter Verwendung eines induzierbaren EMT-Modell zu erkennen. Die Verfahren zum Entwerfen von PCR-Primern und das Nachweisen Spleiß-Isoformen sind nicht nur für die Untersuchung von alternativem Spleißen während EMT, sondern auch zur Untersuchung des alternativen Spleißens in anderen biologischen Prozessen. Untersuchung alternatives Spleißen während EMT ist zwingend notwendig, um ein besseres Verständnis der Mechanismen der EMT und Metastasierung von Tumoren, so die Entwicklung von Strategien, um die Krebsmetastasen zu behandeln.
Protocol
Representative Results
Discussion
Das hier beschriebene Verfahren ermöglicht den Nachweis des alternativen Spleißens in einem induzierbaren EMT-Modell. Als solche dynamische Veränderungen der Spleiß-Isoform-Expression im gesamten zeitlichen Verlauf der EMT erfasst werden. Diese Methode hat Vorteile gegenüber der Verwendung von unterschiedlichen epithelial- oder mesenchymalen-Zelllinien, die für den Vergleich von alternativen Spleißen, weil verschiedene genetische Hintergründe aus un-verwandten Zelllinien konnte mäßig beeinflussen alternative S…
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 |
Referencias
- Thiery, J. P., Sleeman, J. P. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol. 7, 131-142 (2006).
- Yang, J., Weinberg, R. A. Epithelial-mesenchymal transition at the crossroads of development and tumor metastasis. Dev Cell. 14, 818-829 (2008).
- Yang, J., et al. a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 117, 927-939 (2004).
- Batlle, E., et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2, 84-89 (2000).
- Cano, A., et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2, 76-83 (2000).
- Comijn, J., et al. The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell. 7, 1267-1278 (2001).
- Bolos, V., et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci. 116, 499-511 (2003).
- Eger, A., et al. DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene. 24, 2375-2385 (2005).
- Shapiro, I. M., et al. An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genet. 7, e1002218 (2011).
- Warzecha, C. C., et al. An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition. EMBO J. 29, 3286-3300 (2010).
- Brown, R. L., et al. CD44 splice isoform switching in human and mouse epithelium is essential for epithelial-mesenchymal transition and breast cancer progression. J Clin Invest. 121, 1064-1074 (2011).
- Wang, E. T., et al. Alternative isoform regulation in human tissue transcriptomes. Nature. 456, 470-476 (2008).
- Pan, Q., Shai, O., Lee, L. J., Frey, B. J., Blencowe, B. J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 40, 1413-1415 (2008).
- Liu, S., Cheng, C. Alternative RNA splicing and cancer. Wiley Interdiscip Rev RNA. 4, 547-566 (2013).
- Shtivelman, E., Lifshitz, B., Gale, R. P., Roe, B. A., Canaani, E. Alternative splicing of RNAs transcribed from the human abl gene and from the bcr-abl fused gene. Cell. 47, 277-284 (1986).
- Krangel, M. S. Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway. J Exp Med. 163, 1173-1190 (1986).
- Brickell, P. M., Latchman, D. S., Murphy, D., Willison, K., Rigby, P. W. Activation of a Qa/Tla class I major histocompatibility antigen gene is a general feature of oncogenesis in the mouse. Nature. 306, 756-760 (1983).
- Barbaux, S., et al. Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nat Genet. 17, 467-470 (1997).
- Charlet, B. N., et al. Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell. 10, 45-53 (2002).
- Hutton, M., et al. Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17. Nature. 393, 702-705 (1998).
- Kohsaka, T., et al. Exon 9 mutations in the WT1 gene, without influencing KTS splice isoforms, are also responsible for Frasier syndrome. Hum Mutat. 14, 466-470 (1999).
- Philips, A. V., Timchenko, L. T., Cooper, T. A. Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science. 280, 737-741 (1998).
- Savkur, R. S., Philips, A. V., Cooper, T. A. Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet. 29, 40-47 (2001).
- Spillantini, M. G., et al. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc Natl Acad Sci USA. 95, 7737-7741 (1998).
- Wang, J., et al. Myotonic dystrophy evidence for a possible dominant-negative RNA mutation. Hum Mol Genet. 4, 599-606 (1995).
- Graubert, T. A., et al. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 44, 53-57 (2012).
- Papaemmanuil, E., et al. Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N Engl J Med. 365, 1384-1395 (2011).
- Yoshida, K., et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 478, 64-69 (2011).
- Mani, S. A., et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 133, 704-715 (2008).
- Reinke, L. M., Xu, Y., Cheng, C. Snail represses the splicing regulator epithelial splicing regulatory protein 1 to promote epithelial-mesenchymal transition. J Biol Chem. 287, 36435-36442 (2012).
- Serini, G., Gabbiani, G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res. 250, 273-283 (1999).
- Zavadil, J., Bottinger, E. P. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene. 24, 5764-5774 (2005).
- Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).
- Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., Vandesompele, J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19 (2007).
- Boutz, P. L., et al. A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons. Genes Dev. 21, 1636-1652 (2007).
- Salomonis, N., et al. Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation. Proc Natl Acad Sci USA. 107, 10514-10519 (2010).
- Calarco, J. A., et al. Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein. Cell. 138, 898-910 (2009).
- Grabowski, P. Alternative splicing takes shape during neuronal development. Curr Opin Genet Dev. 21, 388-394 (2011).
- Seol, D. W., Billiar, T. R. A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis. J Biol Chem. 274, 2072-2076 (1999).
- Srinivasula, S. M., et al. Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis. Cancer Res. 59, 999-1002 (1999).
- Akgul, C., Moulding, D. A., Edwards, S. W. Alternative splicing of Bcl-2-related genes functional consequences and potential therapeutic applications. Cell Mol Life Sci. 61, 2189-2199 (2004).
- Cooper, T. A. Use of minigene systems to dissect alternative splicing elements. Methods. 37, 331-340 (2005).
- Cheng, C., Sharp, P. A. Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol. 26, 362-370 (2006).
