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.
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.
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。
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?…
The authors have nothing to disclose.
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.
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 |