Detection of Alternative Splicing During 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

The epithelial-mesenchymal transition (EMT) is a developmental program that drives organ morphogenesis and tissue remodeling during embryogenesis. When abnormally activated, EMT promotes tumor metastasis and organ fibrosis^1,2. Compelling studies have described the importance of transcriptional regulation during the process of EMT, defined by several transcription factors, such as Twist, Snail, and ZEB, which repress the expression of the adherens junction protein E-cadherin, resulting in loss of a cobble-stone like epithelial morphology and gain of a spindle-shaped mesenchymal phenotype^3-8. Recent studies through genome-wide analysis of RNAs reve....

Protocol

1. Cell Culture of EMT Induction

Note: EMT can be induced by treatment of TGFβ or ectopic expression of transcription factors Twist, Snail, or Zeb1/2 in epithelial cells. Described in this protocol is an inducible EMT system via expression of the Twist-ER fusion protein in immortalized human mammary epithelial cells (HMLE/Twist-ER, a gift of Dr. J Yang, UCSD)^9,11,29. Upon 4-hydroxytamoxifen (TAM) treatment, the fusion protein Twist-ER translocates to the nucleus to drive transcription and results in a full EMT transition in 12-14 days^9,11,29. Additional EMT inducible systems, such as HMLE/Snail-ER, HMLE/TGFβ, and MCF10A/....

Results

The procedures described above provide a robust method to detect alternative splicing during EMT. Representative results of CD44 splice isoform switching during Twist-induced EMT are given below as an example.

Twist-induced EMT in HMLE/Twist-ER cells was characterized by a transition from a cobble-stone like epithelial phenotype to an elongated fibroblastic phenotype (Figure 2A), the absence of epithelial markers E-cadherin, γ-catenin and occludin and the upregulation of mesen.......

Discussion

The procedure described here enables the detection of alternative splicing in an inducible EMT model. As such, dynamic alterations of splice isoform expression can be captured throughout the time course of EMT. This method has advantages over the use of different epithelial- or mesenchymal-representing cell lines for comparison of alternative splicing because distinct genetic backgrounds from un-related cell lines could unduly influence alternative splicing. However, the successful induction of EMT must be carefully conf.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-hydroxytamoxifen	Sigma	H7904	Make stock solution by dissolving 4-hydroxytamoxifen in ethanol to 200 μM. Keep the solution at -20 °C 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	1:1,000 dilution
E-cadherin antibody	Cell Signaling Technology	4065	1:2,500 dilution for immunoblotting; 1:50 dilution for immunofluorescence
γ-Catenin antibody	Cell Signaling Technology	2309	1:1,000 dilution
Occludin antibody	Santa Cruz Biotechnology Inc.	sc-5562	1:500 dilution
Fibronectin antibody	BD Transduction Laboratories	610077	1:5,000 dilution
N-cadherin antibody	BD Transduction Laboratories	610920	1:2,000 dilution
Vimentin antibody	NeoMarkers	MS-129-p1	1:500 dilution
GAPDH antibody	Millipore Corporation	MAB374	1:10,000 dilution
Amasham ECL Western blotting detection reagent	GE Health Life Science	RPN2209	Chemiluminescence system