上皮間葉転移の間の選択的スプライシングの検出
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
上皮間葉転換（EMT）は、胚発生の間に臓器の形態形成や組織リモデリングを駆動発生プログラムです。異常に活性化されると、EMTは腫瘍転移および臓器線維症1,2を推進しています。説得力のある研究はcobble-が失われ、接着結合タンパク質E-カドヘリンの発現を抑制するなどのツイスト、カタツムリ、およびZEBなどのいくつかの転写因子、によって定義されたEMT過程において転写調節の重要性を記載している上皮形態と紡錘形の間葉表現型3-8のゲインのような石。 RNAのゲノムワイド解析による最近の研究では、スプライシングパターン上皮または間葉表現型9,10のいずれかと関連している遺伝子群が存在することを明らかにした。私たちの研究室からの作業は、機能的に選択的スプライシングとEMTを接続。細胞表面接着分子CD44を研究することによって、私たちはCD44 alternことが実証されativeスプライシングは、しっかりとEMTの間に規制され、より重要なのは、因果的に切り替えることがCD44スプライスアイソフォームは11 EMTに貢献します。
ヒト多エキソン遺伝子の95％までは、あるいは12〜14にスプライシングされる選択的スプライシングは、遺伝子調節の普及と保存されたモデルを表す。単一の遺伝子から複数のタンパク質産物を生成することにより、選択的スプライシングは、ヒトゲノムの複雑さの別の層を追加すること、タンパク質の多様性のために必須の機構を構成する。このように、選択的スプライシングの調節不全は、潜在的にヒトの疾患の原因となる重大な生物学的効果につながる可能性があります。実際、疾患における異常な選択的スプライシングはスプライソソーム機構をコードする遺伝子の変異は、一般的に骨髄異形成症候群26-28に見られる最近の知見を含め、十年以上15-25文書化されています。従って、選択的にスプライシングされたiを検出するための信頼できる方法を開発するsoforms EMTを含む多様な生物学的プロセスの研究に非常に重要である。
ここでは、誘導性EMTモデルを用いて選択的スプライシングの変化を検出するためのプロトコルを提供する。 PCRプライマーを設計し、スプライスアイソフォームを検出するための方法は、EMTの間の選択的スプライシングの研究のためだけでなく、他の生物学的プロセスにおける選択的スプライシングの研究のためのみならず、適当である。 EMTの間に選択的スプライシングを調査することは、より良い、癌の転移を治療するための効果的な戦略の開発を促進し、EMTおよび腫瘍転移のメカニズムを理解するために不可欠である。
Protocol
Representative Results
Discussion
ここで説明する手順は、誘導性EMTモデルにおける選択的スプライシングの検出を可能にする。このように、スプライスアイソフォーム発現の動的変化は、EMTの時間経過を通して捕捉することができる。非関連の細胞株とは異なる遺伝的背景が過度選択的スプライシングに影響を与える可能性があるため、この方法は、選択的スプライシングの比較のために異なるepithelial-または間葉表現?…
Disclosures
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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