Påvisning af alternativ splejsning løbet Epithelial-Mesenchymal 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
Epitel-mesenkymale overgang (EMT) er en udviklingsmæssig program, der driver orgel morfogenese og vævsombygning under embryogenese. Når unormalt aktiveret, EMT fremmer tumormetastase og orgel fibrose 1,2. Overbevisende undersøgelser har beskrevet betydningen af transkriptionel regulering under processen med EMT defineret af flere transkriptionsfaktorer, såsom Twist, sneglen, og ZEB, der undertrykker ekspressionen af adherens krydset protein E-cadherin, hvilket resulterer i tab af en cobble- sten ligesom epitelial morfologi og gevinst på en spindel-formet mesenkymale fænotype 3-8. Nylige undersøgelser gennem hele genomet analyse af RNA'er viste, at der findes en gruppe af gener, hvis splejsning mønstre er forbundet med enten epiteliale eller mesenkyme fænotyper 9,10. Arbejde fra vores laboratorium funktionelt forbundet alternativ splejsning og EMT. Ved at studere celleoverfladen adhæsionsmolekyle CD44 vi vist, at CD44 ALTERNtiv splejsning reguleres stramt under EMT, og endnu vigtigere, at CD44 splejsningsisoform skifte kausalt bidrager til EMT 11.
Alternativ splejsning er en udbredt og bevaret model af genregulering, som op til 95% af humane multi-exon gener alternativt splejsede 12-14. Ved at generere flere protein produkter fra et enkelt gen, alternativ splejsning udgør en væsentlig mekanisme for protein mangfoldighed, tilføjer endnu et lag af kompleksitet til det humane genom. Som sådan kunne dysregulering af alternativ splejsning potentielt føre til dybtgående biologiske effekter, der forårsager sygdomme hos mennesker. Faktisk har afvigende alternativ splejsning i sygdomme, er dokumenteret i over et årti 15-25, herunder de seneste resultater, at mutationer i gener, der koder for spliceosome maskiner er almindeligt forekommende i myelodysplastisk syndrom 26-28. Derfor udvikler pålidelige metoder til påvisning af alternativt splejsede Isoforms er af stor betydning i studiet af forskellige biologiske processer, herunder EMT.
Her giver vi en protokol for at konstatere ændringer i alternativ splejsning ved hjælp af en inducerbar EMT-model. Metoderne til at designe PCR-primere og detektion splejsningsisoformer ikke blot er egnede til undersøgelse af alternativ splejsning i EMT, men også til undersøgelse af alternativ splejsning i andre biologiske processer. Undersøgelse af alternativ splejsning i EMT er afgørende for bedre at forstå de mekanismer, EMT og tumormetastase, og dermed fremme udviklingen af effektive strategier til behandling af kræft metastaser.
Protocol
Representative Results
Discussion
Den her beskrevne procedure giver påvisning af alternativ splejsning i en inducerbar EMT-model. Som sådan, dynamiske ændringer i splejsningsisoform ekspression kan indfanges i hele tidsforløbet af EMT. Denne metode har fordele i forhold til brugen af forskellige epithelial- eller mesenkymale-repræsenterer cellelinjer til sammenligning af alternativ splejsning, fordi forskellige genetiske baggrunde fra un-relaterede cellelinier uretmæssigt kan påvirke alternativ splejsning. Dog skal en vellykket induktion af…
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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