Påvisning av alternativ spleising Under 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
Epiteliale-mesenchymale overgang (EMT) er en utviklings program som driver organ morphogenesis og vev remodeling under embryogenesen. Når unormalt aktivert, fremmer EMT tumometastaser og orgel fibrose 1,2. Overbevisende studier har beskrevet viktigheten av transcriptional regulering under prosessen med EMT, definert av flere transkripsjonsfaktorer som Twist, sneglen, og ZEB, som undertrykker uttrykk for adherens krysset protein E-cadherin, som resulterer i tap av en cobble- stein som epithelial morfologi og gevinst på en spindel-formet mesenchymale fenotype 3-8. Nyere studier gjennom genom-bredt analyse av RNAer avslørte at det eksisterer en gruppe av gener hvis spleising mønstre er forbundet med enten epiteliale eller mesenkymale fenotyper 9,10. Arbeid fra vår lab funksjonelt koblet alternativ spleising og EMT. Ved å studere celleoverflaten adhesjonsmolekylet CD44, vi viste at CD44 alternAtive spleising er strengt regulert i EMT, og enda viktigere, at CD44 spleise isoform bytte årsaks bidrar til EMT 11.
Alternativ spleising representerer en utbredt og konservert modell av genregulering, som opp til 95% av menneskelige multi-ekson gener er alternativt skjøtes 12-14. Ved å generere multiple proteinprodukter fra en enkelt gen, utgjør alternativ spleising en viktig mekanisme for protein diversitet, og legger et lag av kompleksiteten til det humane genom. Som sådan, kan feilregulering av alternativ spleising potensielt føre til dyptgripende biologiske effekter forårsaker menneskelige sykdommer. Faktisk er dokumentert avvikende alternativ spleising i sykdommer i over et tiår 15-25, inkludert nylige funn at mutasjoner i gener som koder for spliceosome maskiner er ofte funnet i myelodysplastisk syndrom 26-28. Derfor er utvikling av pålitelige fremgangsmåter for påvisning av alternativt spleisede isoforms er av stor betydning i studiet av ulike biologiske prosesser, inkludert EMT.
Her gir vi en protokoll for å oppdage endringer i alternativ spleising ved hjelp av en induserbar EMT modell. Metodene for å designe PCR primere og oppdager skjøte isoformer er egnet ikke bare for studier av alternativ spleising under EMT, men også for studier av alternativ spleising i andre biologiske prosesser. Gransker alternativ spleising under EMT er avgjørende for å bedre forstå mekanismene for EMT og tumor metastasering, og dermed legge til rette for utvikling av effektive strategier for å behandle kreft metastasering.
Protocol
Representative Results
Discussion
Fremgangsmåten som beskrives her muliggjør påvisning av alternativ spleising i en induserbar EMT modell. Som sådan, dynamiske endringer Skjøt isoform uttrykk kan fanges hele tiden løpet av EMT. Denne metoden har fordeler fremfor bruk av ulike epithelial- eller mesenchymale-representerer cellelinjer for sammenligning av alternativ spleising fordi distinkte genetiske bakgrunn fra un-relaterte cellelinjer kan utilbørlig påvirke alternativ spleising. Imidlertid må den vellykkede induksjon av EMT nøye bekreftet ved…
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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