Name: detection of alternative splicing during epithelial-mesenchymal transition
Uploaded: 2014-10-09T14:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

EMT İndüksiyon 1. Hücre Kültürü Not: EMT TGFβ tedavisi veya epitel hücrelerinde transkripsiyon faktörleri Twist, salyangoz veya Zeb1 / 2 ektopik sentezlenmesi ile indüklenebilir. Ölümsüzleştirilmiş insan meme epitelyum hücreleri (HMLE / döndürme-ER, Dr. J. Yang bir hediye UCSD) 9,11,29 yılında Twist-ER, füzyon proteininin sentezlenmesi yoluyla uyarılabilir bir EMT sistemi bu protokolde tarif edilmektedir. 4-OH (TAM) İşleme üzerine, füzyon proteini Bükülme ER 12-14 gün 9,11,29 tam EMT geçiş transkripsiyon ve sonuçları elde etmek için çekirdeğe doğru yer değiştirir. Bu HMLE / Salyangoz-ER, HMLE / TGFβ ve MCF10A / TGFβ gibi ek EMT uyarılabilir sistemler, önceki yayınlarda 11,30 bulunabilir. <ol><li> % 5 CO2 içeren bir 37 ° C kuluçka makinesi içinde, serumsuz meme epitelyal hücre büyüme ortamında (MEGM) olarak HMLE / Bükülme ER hücreleri korumak. Passage hücreler her 2-3 gün% 80 izdiham ulaştığınızda. % 5 buzağı serumu / DMEM ile tripsin inaktive daha sonra 5-10 dakika boyunca 37 ° C&#39;de% 0.15 tripsin ile inkübe hücreleri, vb. Hücreleri aşağı Spin ve MEGM onları tekrar süspansiyon. 1-4 arasında bir oranda yeni bir doku kültürü çanağı içinde plakası hücreleri. </li><li> 200 uM çözelti yapmak için etanol içinde TAM çözülür. -20 ° C&#39;de ışık ve mağaza TAM koruyun. 20 nM TAM bir son konsantrasyon yapmak için 10,000 seyreltmenin kullanımdan önce yeni bir 1 de MEGM ortamına TAM ekleyin. </li><li> EMT uyarılması için, iki nüsha halinde 10 cm doku kültürü çanak içine 1,6 x 10 6 HMLE / Twist ER hücreleri plaka. 20 nM TAM-ihtiva eden ortam ile MEGM Kültür hücreleri. </li><li> Nüsha halinde 10 cm çanak içine Plaka 1,6 x 10 6 HMLE / Twist ER hücreleri ve olmayan TAM muamele kontrol olarak% 0.01 etanol ile tedavi hücreleri. </li><li> İki gün kuluçkadan sonra, hücrelerin morfolojik değişikliklerini kaydetmek için 10X büyütmede ışık mikroskobu altında fotoğraf çekmek. </li><li> Kontrol veya TAM ile muamele edilmiş bir çanak ortamı basınçla emiliphücreler. RNA izolasyonu ve protein analizi için RIPA tamponunda diğer yarısı için RNA, liziz tamponu içinde plakanın yarısını hurdaya soğuk PBS ile hücreleri ve hasat hücreleri yıkayın. </li><li> Nüsha halinde 10 cm çanak yeniden kaplama 1,6 x 10 6 hücre tarafından kontrol veya TAM-işlenmiş yemekler Passage hücreleri. Sürekli 20 nM TAM veya araç ile hücre tedavisi. </li><li> Tekrar, araçla tedavi edilmiş kontrol hücreleri, parke taşı gibi epitel morfolojisi muhafaza ise, bu sürenin sonunda, TAM ile muamele edilmiş Bükülme ER hücreler bir iğ şeklinde mezenkimal morfolojisini, 14 gün kadar 5 kez her gün ila 7. </li></ol> EMT 2. Karakterizasyonu Not: EMT A tamamlanması ile gösterilir: bir iğ şeklinde fibroblastik gibi görünümü için bir Arnavut kaldırımı gibi epitel morfolojiden (1) Cep morfolojik değişimler; (2) hücre-hücre kavşaklarda e-kaderin lokalizasyon kaybı; kaybıyla tanımlanan EMT belirteçleri ve (3) açık ifadesi,epitel belirteçleri ve mezenkimal belirteçlerinin kazanç. Hücre morfolojik değişiklikler bu bölümde anlatılan hücre kavşaklarda E-kaderin Bölüm 1. immüno-floresans algılama tarif EMT indüksiyon zamanı sırasında 10X büyütmede ışık mikroskobu tarafından yakalanır. EMT markerlerin ifadesi immünolojik işaretleme ile tespit edilir. Ortak epitelial yapıcıları E-kadherin, γ-katenin ve occludin, ve mezenkimal belirteçler, fibronektin, N-kaderin ve vimentin içerir. Imünoblotlarını genel prosedür Bölüm 4&#39;te açıklanmıştır. <ol><li> TAM işlemden geçirilmesi, tohumların, bir 24-yuvalı plakanın bir çukuruna yerleştirilmiş bir 12 mm&#39;lik dairesel bir örtücü cam üzerine 1 x 10 5 hücre bölgesinin 12. günde. </li><li> Önceden ısıtılmış PBS ile tohumlama, aspirat orta ve yıkama hücrelerin 48 saat sonra. </li><li> 15 dakika boyunca ön ısıtılmış% 4 paraformaldehid, hücreler. </li><li> PBS ile hücreler üç kez yıkayın. </li><li> 15 için PBS içinde% 0.2 Triton X-100 ile inkübe hücrelerioda sıcaklığında Min. </li><li> PBS ile hücreler üç kez yıkayın. </li><li> Spesifik olmayan bağlanmayı bloke etmek için oda sıcaklığında 1 saat süreyle% 5 BSA / PBS içinde hücreleri inkübe edin. </li><li> 4 ° C&#39;de 1:50 seyreltme,% 1 BSA / nemli bir oda O PBS / H de birincil antikor E-kaderin hücreleri inkübe edin. Not: Primer antikor seyreltmesi için aralık 1:50 ile 1 arasında, normal olarak: 200, ve her bir antikor için optimal dilüsyon deneysel olarak tespit edilmelidir. </li><li> Birincil antikor süzün ve PBS ile hücreler üç kez yıkayın. </li><li> Oda sıcaklığında 1 saat boyunca 500 oranında seyreltilmiş bir 1: floresan-etiketli sekonder antikor ile inkübe hücreleri. Bu aşama, ışıktan hücreleri korumak. </li><li> İkincil antikor süzün ve üç kez PBS ile yıkayın. </li><li> 10 dakika boyunca 0.1-1 ug / ml DAPI veya Hoechst ile Leke hücreleri. </li><li> PBS ile üç kez hücreleri durulayın. </li><li> Dağı oje ile bir montaj orta damla ve mühür lamelleri ile lamelleri. </li><li> Hücreleri gözlemlemek ve almak63X floresan mikroskop altında görüntüler. </li></ol> Ek antikorlar ile lekeleme EMT fenotipi teyit etmek için yapılabilir. Örneğin, N-kaderin ve α-düz kas aktini mezenkimal fenotip 29,31,32 için boyanmış olabilir. Iskelet yapısı yeniden düzenlenmesi, F-aktin için 11 falloidinle hücrelerin boyanması ile izlenebilir. Buna ek olarak, hücre motilitesi ve hücre ölümü direnç için deneyler, EMT 11 özelliklerini incelemek için gerçekleştirilebilir. Mah-PCR Tahliller kullanma Algılama Splice izoformlannın 3. <ol><li> Primer Tasarım Not: Primer çiftleri dikkatlice nokta bağlantı izoformları amplifiye etmek için tasarlanmış olmalıdır. Atlama ekson, en sık görülen alternatif bağlama olayı, birincil tasarım stratejisini açıklamak için bir örnek olarak gösterilmektedir. Şekil 1A değişken ekson olarak üçüncü ekson, bir dört-ekson ön-mRNA göstermektedir. Ekson İçerme 3 sonuçlarıekson 3&#39;ün atlama ise daha uzun bir bağlantı izoformunun üretimi, daha kısa bir bağlantı izoformunu oluşturur. Primerler, ekson-3 dahil olmak üzere iki izoformu ve ekson-3-atlanan izoformunu ayırt etmek ve bu mRNA&#39;nın toplam transkript tespit etmek için tasarlanmış olması gerekir. <ol><li> Ekson dahil belirlenmesi için, dikkatli bir şekilde değişken ekson dahil izoformunun spesifik amplifikasyonu sağlayan, değişken ekson 3 ve ekson 4 yakın kurucu içinde bir ters primer içinde bir ileri primer tasarımı. Hem ileri hem de tasarım ve genomik DNA veya ön-mRNA &#39;dan çoğaltılmış PCR ürünleri önlemek amacıyla aynı değişken ekson içinde ters primerlerli değil emin olun. </li><li> Özellikle ekson atlama olayları, tasarım amplifiye etmek için, değişken ekson 3 dahil edildiğinde, aksi imha edilecek kurucu ekson 2 ve ekson 4 birleşim yerini kapsayarak tek primer. Bu nedenle yapısal ekson 4&#39;ün içine, diğer birincil tasarım, PCR ürünleri değişken ekson 3 exclud olduğu zaman oluşturulacaked ve ekson 2 ve ekson 4 sonradan birleştirilir kurucu. </li><li> , Genin toplam transkriptlerini algılamak iki kurucu ekson 1 ve 2 içinde primerler Böylece tasarlamak için, PCR ürünleri tüm izoformları amplifiye edilmiştir. </li><li> Emin, tüm primerler için erime sıcaklığı (Tm) yaklaşık olarak 55 ° C arasında, her bir primer uzunluğu yaklaşık 18-22 nükleotid olduğunu olun ve PCR ürünlerinin boyutunu 80 ile 150 baz çiftidir. </li></ol></li><li> RNA ekstraksiyonu: (Malzeme Tabloya bakınız), toplam RNA izolasyon kiti kullanılarak RNA ekstrakte edin. <ol><li> 350 ul RNA lizis tampon hücreleri toplayın. </li><li> Lizat% 70 etanol içinde 350 ul ekleyin ve iyice karıştırın. </li><li> RNA saflaştırma Numuneyi kolona aktarın. </li><li> 1 dakika ve atma flow-through için 10.000 x g&#39;de santrifüj. </li><li> RNA, yıkama tamponu II ile bir kez RNA, yıkama tamponu I 500 ul ile ve iki kez de sütun yıkayın. </li><li> RNA, kalıntı yıkama tamponunu çıkarın2 dakika süreyle maksimum hızda santrifüj ile kolondan II. </li><li> , Yeni bir 1.5 ml&#39;lik bir tüp içine transfer sütun sütun matrisine merkezinde 50 ul nükleaz içermeyen su ilave edin ve en yüksek hızda santrifüj ile RNA aynlır. </li></ol></li><li> Birinci-şerit cDNA sentezi <ol><li> UV absorpsiyon ölçümü ile konsantrasyon ve RNA kalitesini belirler. NOT: Kaliteli RNA yaklaşık 2,0 260 nm / 280 nm absorbans oranı olmalıdır. </li><li> 20 ul nihai hacim içinde 250 ng toplam RNA, 0.05 ug tesadüfi heksamer öncüllerin, 50 pmol MgCl2, 10 pmol dNTP, 2 ul 5 x GoScript tamponu ve 1 ul RT enzimi ters transkriptaz (RT) reaksiyon ayarlayın. </li><li> RT enzimin inaktivasyonu için 5 dakika boyunca 70 ° C &#39;de inkübasyon, ardından 1 saat için 42 ° C RT reaksiyonları gerçekleştirmek. </li></ol></li><li> Gerçek zamanlı PCR <ol><li> 10 ul SYBR yeşil ana karışımı oluşur 20 ul reaksiyonları kurmak,0.1-1.0 ul cDNA şablon ve 5 ileri pmol ve geri primerler. </li><li> Aşağıdaki adımlardan 40 döngü ile gerçek-zamanlı PCR çalıştırın: 95 ° C&#39;de 10 saniye denatürasyon, 10 saniye için 58 ° C tavlama, 30 saniye için 60 ° C uzatma. PCR reaksiyonları, üç kopya halinde gerçekleştirin. </li><li> Ayrışma eğrileri kontrol edin ve bir tek tepe her astar kümesi için gözlenen emin olun. </li><li> Amplifikasyon eğrileri ikinci türev değerleri hesaplanarak ölçüm döngüsü (Cq) değerlerini elde edin. </li><li> &quot;Delta Cq (ΔΔCq &#39;), aşağıdaki formül ile gösterildiği gibi, yöntem kullanılarak indüklenmemiş numunesine kıyasla neden örneklerdeki hedef mRNA nispi miktarı (RQ) hesaplayın. Bir referans olarak, örneğin GAPDH veya TBP olarak temizlik genleri kullanın. </li></ol></li></ol><img alt="Denklem 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Daha doğru bağlantı izoformlannın göreli değerlerini hesaplamak için to dikkate alınmalıdır çoklu referans gen kullanır. Sonuçlar Pfaffl ve ark 33,34 tarafından tarif edilen modifiye mutlak değeri, ölçüm yöntemi kullanılarak analiz edilebilir. Protein Düzeyinde Splice izoformlannın 4. incelenmesi <ol><li> 100 ul buz gibi soğuk RIPA tampon maddesi içinde hücreler toplanır. </li><li> Yaklaşık 10 dakika boyunca buz üzerinde lizatları ayarlayın. </li><li> 4 ° C&#39;de 10 dakika boyunca 14,000 x g&#39;de santrifüje ve süpernatan toplamak. </li><li> Bradford protein konsantrasyonu tahlilleri ile ölçülür. </li><li> SDS yükleme tamponu içinde protein örnekleri seyreltin ve 10% SDS-PAGE jelleri içine proteinlerin 20-40 ug yükleyin. </li><li> 1.5 saat için 20 mA&#39;da SDS-PAGE jelleri çalıştırın. </li><li> 85 V, 3 saat boyunca 4 ° C&#39;de nemli bir transfer sistemi olarak PVDF membranlarına transfer proteinleri hedef proteinlerin moleküler ağırlığa göre transfer süresi ayarlayın. </li><li> 30 dakika oda sıcaklığında 1 saat için% 5 yağsız süt içinde Blok membran. </li><li> Membran wi inkübe4 ° CO / K bir birincil antikor th. 200 ve 1: 5000, deneysel olarak her bir antikor için optimum seyreltme oranını belirleyen birincil antikor seyreltmesi için aralık normalde 1 arasındadır. Bu protokolde kullanılan antikorların seyreltme &quot;özel reaktifler ve ekipman Tablo&quot; bulunabilir. <ol><li> Bağlantı izoform tespit etmek için, belirli bir izoformunu tanıyan spesifik antikorlar kullanılır. Seçenek olarak ise, kurucu ekson kodlama bölgesi epitopunu tanıyan bir antikorun kullanılması ve bunların, protein farklı boyutlarına göre eş zamanlı olarak bağlantı izoform algılar. </li><li> Aynı zamanda, EMT belirteçleri için immüno-lekeleme EMT izler. Mezenkimal belirteçler olarak e-kaderin, γ-katenin ve epitel belirteç olarak occludin ve fibronektin, N-kaderin ve vimentin. </li></ol></li><li> 5 dakikalık bir aralıkla TBS-T (50 mM Tris, 150 mM NaCI,% 0.05 Tween 20, pH 7.4) ile yıkayın zar üç kez. </li><li> HRP-konjuge sekonder ant ile membran inkübeoda sıcaklığında 1 saat için 10,000 seyreltmede bir 1: ibody. </li><li> 5 dk aralıklarla TBS-T ile yıkayın membran üç kez. </li><li> Bir kimyasal ışıldama tespit sistemi ile proteinin görsel ve otoradyografi filmine maruz membran. </li></ol>

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The authors declare that they have no competing financial interests.

Burada tarif edilen prosedür, bir uyarılabilir EMT modelinde alternatif bağlantısının belirlenmesini sağlar. Bağlantı izoform ekspresyon örneğin dinamik değişiklik EMT zaman süreci boyunca yakalanabilir gibi. Un-bağlı hücre hatlarının farklı genetik temel gereksiz alternatif eklemeler etkileyebilir, çünkü bu yöntem alternatif splicing karşılaştırılması için farklı epithelial- da mezenkimal-temsil eden hücre çizgilerinin kullanımı üzerinde avantajlara sahiptir. Ancak, EMT başarılı ...

Epitel-mezenkimal geçiş (EMT) Embriyogenesis sırasında organ morfogenetiğine ve doku yenileme sürücüler gelişimsel bir programdır. Anormal aktive edildiğinde, EMT tümör metastazı ve organ fibrozis 1,2 teşvik etmektedir. Zorlayıcı çalışmalar cobble- kaybıyla sonuçlanan adherens birleşme proteini E-kaderin ifadesini bastırmaktadır örneğin Twist, salyangoz ve Zeb gibi çeşitli transkripsiyon faktörleri, tarafından tanımlanan, EMT sürecinde transkripsiyon düzenlemesi önemini tarif etmişlerdir epitel morfolojisi ve iğ biçimli mezenkimal fenotip 3-8 kazancı gibi taş. RNA&#39;lar genom analizi ile yapılan son çalışmalar yapıştırma desenler epitel veya mezenkimal fenotiplerle 9,10 ya ile ilişkili genlerin bir grup var olduğunu ortaya koymuştur. Laboratuarımızda çalışmak işlevsel alternatif eklemeler ve EMT bağlı. Hücre yüzey adezyon molekülü CD44 inceleyerek, CD44 altern gösterdiative yapıştırma sıkı EMT sırasında düzenlenir, ve daha da önemlisi, nedensel anahtarlama bu CD44 ekleme izoformdur 11 EMT katkıda bulunmaktadır. Alternatif 12-14 raptedilmektedir insan çoklu-ekson genlerin% 95 kadar alternatif yapıştırma, gen regülasyonu bir yaygın ve korunmuş modelini temsil eder. Tek bir genin birden fazla protein ürünlerinin üretilmesi ile beraber, alternatif birleştirme insan genomuna karmaşıklık bir katman eklenerek protein çeşitliliği için önemli bir mekanizma oluşturur. Bu nedenle, alternatif uç uca eklenmesi düzensizliği, potansiyel olarak hastalığa neden olan insan derin biyolojik etkileri yol açabilir. Nitekim, hastalıklarda anormal alternatif yapıştırma spliceosom makine kodlayan genlerde mutasyonlar sık miyelodisplastik sendromlarda 26-28 bulunan son bulgular da dahil olmak üzere, bir yılda 15-25 rapor edilmiştir. Bu nedenle, alternatif olarak eklenmiş i tespiti için güvenilir yöntemler geliştirmeksoforms EMT de dahil olmak üzere çeşitli biyolojik süreçlerin çalışmada büyük önem taşımaktadır. Burada bir uyarılabilir EMT modeli kullanılarak alternatif ekleme değişiklikleri algılamak için bir protokol sağlar. Bağlantı izoformları PCR primerlerini tasarlama ve tespit edilmesi için yöntemler, EMT sırasında alternatif splicing çalışma için değil, aynı zamanda diğer biyolojik süreçlerde alternatif splicing çalışma için sadece uygundur. EMT sırasında alternatif eklemeler araştıran iyi böylece kanser metastazı tedavi etmek için etkili stratejilerin geliştirilmesini kolaylaştırmak, EMT ve tümör metastazı mekanizmalarını anlamak için zorunludur.

<table><tbody><tr><td>4-hydroxytamoxifen</td><td>Sigma</td><td>H7904</td><td>Make stock solution by dissolving 4-hydroxytamoxifen in ethanol to 200 &amp;#956;M. Keep the solution at -20 &amp;#176;C protected from light.</td></tr><tr><td>E.Z.N.A. Total RNA Isolation kit</td><td>Omega Bio-Tek</td><td>R6731</td><td>Total RNA isolation kit</td></tr><tr><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5001</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>LightCycler 480 Real-Time PCR System</td><td>Roche</td><td></td><td>Equipment for qRT-PCR assay</td></tr><tr><td>CD44 antibody</td><td>R&amp;amp;D Systems</td><td>BBA10</td><td>1:1,000 dilution</td></tr><tr><td>E-cadherin antibody</td><td>Cell Signaling Technology</td><td>4065</td><td>1:2,500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td></tr><tr><td>&amp;gamma;-Catenin antibody</td><td>Cell Signaling Technology</td><td>2309</td><td>1:1,000 dilution</td></tr><tr><td>Occludin antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-5562</td><td>1:500 dilution</td></tr><tr><td>Fibronectin antibody</td><td>BD Transduction Laboratories</td><td>610077</td><td>1:5,000 dilution</td></tr><tr><td>N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>610920</td><td>1:2,000 dilution</td></tr><tr><td>Vimentin antibody</td><td>NeoMarkers</td><td>MS-129-p1</td><td>1:500 dilution</td></tr><tr><td>GAPDH antibody</td><td>Millipore Corporation</td><td>MAB374</td><td>1:10,000 dilution</td></tr><tr><td>Amasham ECL Western blotting detection reagent</td><td>GE Health Life Science</td><td>RPN2209</td><td>Chemiluminescence system</td></tr></tbody></table>

detection of alternative splicing during epithelial-mesenchymal transition

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 &#947;-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.

Yukarıda tarif edilen prosedürler sırasında EMT alternatif eklemeler tespit etmek için güçlü bir yöntem sağlar. Twist kaynaklı EMT sırasında CD44 ekleme izoformu anahtarlama Temsilcisi sonuçları bir örnek olarak aşağıda verilmiştir. HMLE / Büküm-ER hücrelerinde Bükülme kaynaklı EMT uzatılmış bir fibroblastik fenotipe epitel fenotip gibi çakıl taşı bir geçiş (Şekil 2A), epitelyal işaretleri E-kadherin, γ-katenin ve occludin ve yokluğu il...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

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.

Epitel-Mezenkimal Geçiş sırasında alternatif birleştirme Algılama

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.9K Görüntüleme.  Northwestern University Feinberg School of Medicine. 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.

Video: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

İlaç Direnci İlgili Roman Splice türevlerini algılamak için RNA sıralamayı kullanarak<em&gt; İn Vitro</em&gt; Kanser Modelleri

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Kanser Araştırmaları

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Yapay Faktörler Mühendislik Özellikle İnsan Hücreleri alternatif bağlantı işleyin için

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genetik

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Kantitatif analiz alternatif Pre-mRNA RNA In Situ hibridizasyon tahlil kullanarak fare beyin bölümlerde Uçbirleştirme

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Sarkoma Hücreler Mezenkimal-Epitel Geçişler endüklenmesi

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Gelişim Biyolojisi

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Meme Kanseri ve Normal Hücrelerde TGF-β Sinyalizasyon ve TGF-β kaynaklı Epitel-mezenkimal Geçişin Incelenmesi

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA Birleştirme Değişikliklerini incelemek için E1A Minigene Aracını Kullanma

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

Birleştirme Verimliliğini İzlemek için Muhabir Tabanlı Hücresel Test

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biyokimya

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Biyoloji

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

RNA-seq Verilerinde Alternatif Ekleme ve Poliadenilasyonun Tanımlanması

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.
Splicing is Mediated by the Spliceosome
Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5&#39; end of the intron and to a branch point sequence containing an A towards the 3&#39; end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5&#39; splice site. Subsequently, a chemical reaction cleaves the 5&#39; end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5&#39; end of the downstream exon reacts with the 3&#39; end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.
Splicing Allows the Expression of Several Proteins from a Single Gene
Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.
Different patterns of alternative splicing include exon skipping, alternative 5&#39; or 3&#39; splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.
Abnormal Splicing Can Cause Diseases
Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform&#8212;BCL-XL and BCL-XS, respectively&#8212; through the use of alternative 5&#39; splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

RNA Splicing, Lariat and the Spliceososme

RNA Uçbirleştirme

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called ...