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Biología

检测选择性剪接的过程中上皮 - 间质转化

Published: October 09, 2014
doi:
10.3791/51845
, ,
1Division of Hematology/Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center,Northwestern University Feinberg School of Medicine

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

上皮 – 间质转化（EMT）是推动胚胎发育过程中的器官形态和组织重塑一个发展计划。当异常激活，EMT促进肿瘤转移和器官纤维化1,2。 EMT过程中引人注目的研究已经描述转录调控的重要性，通过几个转录因子，如扭曲，蜗牛和ZEB，这抑制了粘着连接蛋白的E-钙粘蛋白的表达，从而导致cobble-的损失定义石样上皮形态学和纺锤状的间充质表型3-8的增益。通过RNA的全基因组分析，最近的研究表明，存在一组基因的剪接方式有两种上皮或间质表型9,10相关联的。从我们的实验室工作功能连接选择性剪接和EMT。通过研究细胞表面粘附分子CD44，我们证明了CD44 ALTERNative剪接EMT过程中紧密调节，并且更重要的是，CD44剪接同种型转换因果有助于EMT 11。

选择性剪接表示基因调控的一种普遍的和保守的模型，如高达95％的人的多外显子基因可变剪接12-14。通过从单个基因产生多个蛋白的产品，选择性剪接构成蛋白多样性的重要机制，增加了另一层复杂性，以人类基因组。因此，选择性剪接的失调可能会导致引起人类疾病的深刻的生物学效应。事实上，在疾病的异常剪接都记载了十多年15-25，其中包括在基因编码的剪接突变机械骨髓增生异常综合征26-28通常发现最近的调查结果。因此，开发可靠的方法的选择性剪接í检测soforms是在不同的生物过程，包括EMT的研究非常重要的。

在这里，我们提供了一个协议来检测使用可诱导的EMT模型的变化剪接。该方法用于设计PCR引物和检测剪接异构体是合适的，不仅用于选择性剪接的EMT过程中的研究，而且对选择性剪接在其他生物过程的研究。 EMT过程中调查剪接是必须为了更好地理解EMT和肿瘤转移的机制，从而促进有效的策略的开发来治疗癌症转移。

Protocol

EMT诱导1细胞培养注：EMT可以通过治疗TGFβ或转录因子扭，蜗牛，或ZEB1 / 2在上皮细胞异常表达所诱导。另外，在本协议是通过扭ER融合蛋白在人永生化乳腺上皮细胞（HMLE /扭ER，J杨博士的礼物，加州大学圣地亚哥分校）9,11,29表达可诱导的EMT系统。当4-羟基他莫昔（TAM）的治疗中，融合蛋白的扭ER转位到细胞核中以驱动转录并产生一个完整的EMT的过渡在12-14天9,11,29。</s…

Representative Results

上述过程提供了一个可靠的方法EMT过程中检测可变剪接。的CD44剪接同种型转换时扭诱导的EMT代表性的结果在下面给出的一个例子。 在HMLE /扭ER细胞扭诱导的EMT的特点是从鹅卵石状上皮细胞表型的细长成纤维细胞表型的转变（ 图2A），无上皮标志物E-钙粘蛋白，γ-catenin和occludin和中上调的间质标记物纤连蛋白，N-钙粘蛋白，波形蛋白（ 图2B）。此外，EMT…

Discussion

这里描述的方法使得能够选择性剪接的检测中可诱导的EMT模型。如剪接同种型的表达，例如，动态改变可以在整个EMT的时间过程被捕获。这种方法比使用不同的上皮 – 间质或，代表细胞系的选择性剪接的比较优势，因为从联合国有关的细胞不同的遗传背景，可能不适当地影响选择性剪接。然而，成功诱导的EMT必须谨慎使用上的变化剪接任何结论之前，各种EMT的标志，可以得出证实。此外，除了此?…

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
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Referencias

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Tags

Alternative SplicingEpithelial-mesenchymal TransitionEMTTwistE-cadherinN-cadherinVimentinIsoform-specific PrimersQuantitative RT-PCRImmunoblotting
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Huang, H., Xu, Y., Cheng, C. Detection of Alternative Splicing During Epithelial-Mesenchymal Transition. J. Vis. Exp. (92), e51845, doi:10.3791/51845 (2014).
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