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.

<ol>
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The authors declare that they have no competing financial interests.

الإجراء الموضح هنا يمكن الكشف عن الربط البديلة في نموذج EMT محرض. على هذا النحو، التغيرات الديناميكية للصق شكل الإسوي التعبير يمكن التقاط طوال الوقت من EMT. هذا الأسلوب له مزايا على استخدام epithelial- مختلفة أو خطوط الخلايا التي تمثل الوسيطة للمقارنة بين الربط البديلة لخلفيات وراثية مختلفة عن خطوط الخلايا ذات الصلة للأمم المتحدة يمكن أن تؤثر على نحو غير ملائم الربط البديلة. ومع ذلك، فإن الحث الناجح لEMT يجب أكدت بعناية باستخدام مختلف علامات EMT قبل أي استنتاجات بشأن التغييرات في الربط البديلة التي يمكن استخلاصها. وعلاوة على ذلك، بالإضافة إلى نظام EMT تويست ER-محرض وصفها هنا، ينصح EMT أنظمة أخرى مثل تلك الناجمة عن الحلزون-ER أو TGFβ للتأكد من صحة النتائج التجريبية 11،30. ويمكن الكشف عن التحول من الأشكال الإسوية لصق خلال EMT في كل مستويات RNA والبروتين. لصق ISOويفضل تكوين أجسام مضادة محددة لتحليل البروتين. عندما الأجسام المضادة شكل الإسوي محددة ليست متوفرة، يمكن تحديد مستويات البروتين من الأشكال الإسوية لصق على أساس الوزن الجزيئي على SDS-PAGE بواسطة immunoblotting. يمكن قياس مستوى RNA من كل شكل الإسوي من قبل الاشعال شكل الإسوي محددة. استخدام المقايسات QRT-PCR، التغيير أضعاف من كل شكل الإسوي يمكن تحديد بحساسية ودقة. على وجه الخصوص، عندما المواد التجريبية محدودة، مثل تحليل تعبير عن شكل الإسوي لصق معين في العينات السريرية، يتيح تحليل QRT-PCR مقياس كمي التي من شأنها أن تعوض عن عدم وجود الأجسام المضادة شكل الإسوي محددة لالمناعية. الطريقة المذكورة هنا هي مناسبة للكشف عن التغييرات لصق شكل الإسوي المعروف خلال EMT. بالإضافة إلى ذلك، وهذه الطريقة يمكن توسيع لدراسة الجينوم على نطاق الربط البديلة وللتعرف على الرواية لصق شكل الإسوي التبديل خلال EMT. لتحقيق ذلك،استخدام تقنية على نطاق واسع، مثل RNA العميقة التسلسل أو الربط منصات ميكروأري حساسة، يمكن أن يؤديها باستخدام الرنا معزولة عن نموذج EMT محرض المذكورة أعلاه. ومع ذلك، مطلوب التحقق من صحة QRT-PCR للتغيرات شكل الإسوي التالية أي تحليل على نطاق واسع. في الختام، وينظم الربط البديلة ديناميكيا اثناء EMT، وهي العملية التي أمر بالغ الأهمية لتحقيق التنمية الجنينية وورم خبيث. النظر في وتيرة عالية من الربط البديلة في الجينوم البشري، فمن المرجح أن تنظيم الربط البديلة تلعب دورا هاما في العديد من العمليات البيولوجية والمرضية الأخرى بما في ذلك تمايز الخلايا، وتطوير الأنسجة، وموت الخلايا المبرمجة 35-41. سوف بروتوكول الموصوفة هنا للكشف عن الأشكال الإسوية لصق تكون قابلة للتطبيق لهذه الأنظمة الأخرى كذلك. طرائق تجريبية إضافية، مثل المقايسات مراسل الربط minigene، يمكن بالبريد تستخدم لمواصلة التحقيق في الآليات التنظيمية من العناصر المفعول رابطة الدول المستقلة والعوامل التي تعمل العابرة التي تتحكم الربط البديلة 30،42،43. وعلاوة على ذلك، فإن الدور الوظيفي للشكل الإسوي لصق خاص خلال EMT يمكن التحقيق من قبل إسكات أو ectopically معربا عن شكل الإسوي محددة ومراقبة EMT الحث في نظام EMT-محرض وصفه 11.

الانتقال طلائي الوسيطة (EMT) هو برنامج التنموي الذي يدفع التشكل الجهاز وإعادة عرض الأنسجة أثناء مرحلة التطور الجنيني. عند تفعيلها بشكل غير طبيعي، EMT تعزز الانبثاث ورم وتليف الجهاز 1،2. وقد وصفت دراسات مقنعة على أهمية تنظيم النسخي خلال عملية EMT، التي تحددها عدة عوامل النسخ، مثل تويست، الحلزون، وZEB، الذي قمع التعبير عن الملتصقة بروتين تقاطع E-كادهيرين، مما أدى إلى فقدان cobble- حجر مثل مورفولوجيا الظهارية ومكاسب على شكل مغزل الوسيطة النمط الظاهري 3-8. وكشفت الدراسات الحديثة عن طريق تحليل الجينوم على نطاق من الرنا أن هناك مجموعة من الجينات التي ترتبط إما الظهارية أو الوسيطة الظواهر 9،10 أنماط الربط. العمل من مختبرنا توصيل ظيفيا الربط البديلة وEMT. من خلال دراسة سطح الخلية جزيء التصاق CD44، أثبتنا أن CD44 alternوينظم سالبة الربط بإحكام أثناء EMT، والأهم من ذلك، أن شكل الإسوي CD44 لصق التحول سببيا يسهم في EMT 11. ويمثل الربط البديلة نموذج على نطاق واسع والحفظ من تنظيم الجينات، ما يصل إلى 95٪ من الجينات متعددة اكسون الإنسان وتقسم بدلا من 12-14. عن طريق توليد منتجات البروتين متعددة من جين واحد، يشكل الربط البديلة آلية أساسية للتنوع البروتين، مضيفا طبقة أخرى من التعقيد إلى الجينوم البشري. على هذا النحو، يمكن ديسريغولاتيون من الربط بديل يحتمل أن يؤدي إلى تأثيرات بيولوجية عميقة تسبب الأمراض التي تصيب الإنسان. في الواقع، وقد تم توثيق الربط البديلة الشاذة في أمراض لأكثر من عقد 15-25، بما في ذلك النتائج الأخيرة أن الطفرات في الجينات التي تشفر الآلات جسيم التضفير توجد عادة في متلازمات خلل التنسج النقوي 26-28. لذلك، وتطوير طرق موثوقة للكشف عن ط تقسم بدلا من ذلكsoforms هو من أهمية كبيرة في دراسة العمليات البيولوجية المختلفة بما في ذلك EMT. هنا نقدم بروتوكول للكشف عن تغيرات في الربط البديلة باستخدام نموذج EMT محرض. أساليب لتصميم الاشعال PCR وكشف الإسوية لصق هي مناسبة ليس فقط لدراسة الربط البديلة خلال EMT، ولكن أيضا لدراسة الربط البديلة في العمليات البيولوجية الأخرى. التحقيق الربط البديلة خلال EMT الضروري من أجل فهم أفضل للآليات EMT وورم خبيث، مما يسهل وضع استراتيجيات فعالة لعلاج ورم خبيث السرطان.

<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.

الإجراءات المذكورة أعلاه توفر وسيلة قوية للكشف عن الربط البديلة خلال EMT. وتعطى نتائج ممثلة من CD44 لصق شكل الإسوي التبديل خلال الناجم تويست EMT أدناه كمثال. تميزت EMT الناجم عن تطور في الخلايا HMLE / تويست-ER من الانتقال من رصف حجر مثل النمط الظاهري الظهارية إلى النمط الظاهري أرومي ليفي ممدود (الشكل 2A)، وغياب علامات الظهارية E-كادهيرين، γ-catenin وoccludin و upregulation من علامات الوسيطة فبرونيكتين، N-كادهيرين، وvimentin (الشكل 2B). وعلاوة على ذلك، تم تقييم EMT عن فقدان E-كادهيرين التعريب في تقاطعات خلية خلية (الشكل 2C). تم فحص التعبير تحولت من CD44 الإسوية لصق خلال EMT بواسطة QRT-PCR وimmunoblotting. ويتكون الجين البشري CD44 تسعة الإكسونات المتغيرة. الربط بديل للCD44 يسمح للخلايا الموالية لالمتغيرات ديوس CD44 (CD44v) التي تشمل اكسون متغير واحد على الأقل، ومعيار CD44 (CD44s) أن يخلو من كل الإكسونات المتغيرة. يصور الشكل 3A استراتيجية التصميم التمهيدي للكشف عن مختلف الأشكال الإسوية لصق CD44. للكشف عن CD44s منتج اكسون تخطي، ويقع التمهيدي إلى الأمام في اكسون التأسيسي 5، في حين يمتد التمهيدي العكسي عبر تقاطع بين الإكسونات التأسيسية 5 و6. للكشف عن CD44v الإسوية لصق تحتوي على V5 و V6 متغير تم تصميم الإكسونات، وإلى الأمام وعكس الاشعال داخل V5 وV6، على التوالي. الاشعال للكشف عن الأخرى CD44 الإسوية التي تحتوي على اكسون متغير يمكن تصميمها باستخدام نفس الاستراتيجية. بالإضافة إلى ذلك، تم الكشف عن نسخة إجمالي CD44 باستخدام الأمام والاشعال تقع في التأسيسي اكسون 2 واكسون 3، على التوالي (الشكل 3A) عكس. كما هو مبين في الشكل 3B، QRT-PCR التحليلات باستخدام هذه المجموعات التمهيدي أشارت إلى حدوث انخفاض كبير في CD44V مرنا وزيادة في CD44s مرنا بعد 14 يوما من العلاج TAM في الخلايا HMLE التعبير تويست-ER. على النقيض من ذلك، ظل النص الكلي للCD44 دون تغيير خلال EMT (الشكل 3C). تتفق مع نتائج QRT-PCR، ومستوى البروتين من CD44v انخفض بشكل ملحوظ، في حين أن التعبير عن بروتين CD44s كان upregulated كثيرا (الشكل 3D). ولذلك، كان تحول شكل الإسوي كبير من CD44 من CD44v إلى CD44s أثناء عملية EMT. <img alt="الشكل 1" fo:content-width="3.5in" src="/files/ftp_upload/51845/51845fig1highres.jpg" width="600" /> تصميم الشكل 1. التمهيدي. المخططات تخطيطي يوضح موقع الاشعال للكشف عن الأشكال الإسوية الربط في نموذج تخطي اكسون. وتمثل المربعات الرمادية الإكسونات التأسيسية، تمثل صناديق البرتقال الإكسونات متغيرة، وخطوط رقيقة صناديق ربط دلالة الإنترونات. المواقع التمهيدي هي indicatإد من السهام: تشير السهام السوداء مجموعات التمهيدي للكشف عن مجموع مرنا، الأسهم البرتقالية تشير مجموعات التمهيدي لتضخيم الإسوية تضمين اكسون، وتشير الأسهم الزرقاء مجموعات التمهيدي للكشف عن الأشكال الإسوية-تخطي اكسون. <img alt="الشكل 2" fo:content-width="6in" src="/files/ftp_upload/51845/51845fig2highres.jpg" width="600" /> 2. الرقم التعريفي للEMT. (A) وصور المرحلة النقيض (10X) توضح تغيرات شكلية في الخلايا HMLE / تويست-ER قبل (غير المعالجة) وبعد 14 يوما من العلاج TAM. (B) تحليل طخة مناعية من علامات EMT في الخلايا HMLE / تويست-ER قبل (غير المعالجة) وبعد 14 يوما من العلاج TAM. على TAM العلاج، علامات الظهارية-E كادهيرين، γ-catenin، وoccludin تم downregulated، والوسيطة علامات فبرونيكتين، N-كادهيرين، وvimentin تم upregulated. (C) الصور مضان المناعية (63X) تشير إلى فقدان E-كادهيرينالتعريب في تقاطعات خلية خلية بعد 14 يوما من العلاج TAM. تلطيخ الأخضر يشير E-كادهيرين، وتلطيخ دابي (الأزرق) إلى نوى. شريط النطاق = 10 ميكرومتر. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51845/51845fig2highres.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <img alt="الرقم 3" fo:content-width="5in" src="/files/ftp_upload/51845/51845fig3highres.jpg" width="500" /> الرقم 3. التبديل من الأشكال الإسوية لصق CD44 خلال EMT. (A) تصميم التمهيدي للكشف عن الأشكال الإسوية لصق CD44. وتمثل المربعات الرمادية الإكسونات التأسيسية، تمثل صناديق البرتقال الإكسونات متغيرة، وخطوط رقيقة صناديق ربط دلالة الإنترونات. يشار إلى المواقع التمهيدي من السهام: تشير السهام السوداء مجموعات التمهيدي للكشف عن CD44 إجمالي مرنا، الأسهم البرتقالية تشير مجموعات التمهيدي لتضخيم CD44v5-6، وتشير الأسهم الزرقاء مجموعات التمهيدي لكشف CD44s. (B، C) تحليل QRT-PCR لمستويات الإسوية CD44 خلال EMT باستخدام بادئات التي تكشف على وجه التحديد CD44v تحتوي الإكسونات V5 المتغيرة وV6 (V5 / 6) وCD44s (B)، وCD44 مرنا الكلي (C) . مستويات التعبير النسبية للمرنا في يوم 14 تم تطبيع الخلايا ليوم المقابلة 0 الخلايا في TAM تعامل والجماعات غير المعالجة. أشرطة الخطأ تشير SEM. ن = 3. (D) تحليل طخة مناعية من الأشكال الإسوية CD44 خلال EMT TAM التي يسببها في خلايا HMLE / تويست-ER. تم رصد مستويات E-كادهيرين وN-كادهيرين لتحديد الوضع EMT.

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.

الكشف عن البديل خلال الربط طلائي-الوسيطة الانتقالية

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

Universidad San Sebastian

Research

JoVE Journal

Biology

12.9K Views.  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.

باستخدام الحمض النووي الريبي لكشف التسلسل رواية لصق المتغيرات ذات الصلة لمقاومة المخدرات في<em&gt; في المختبر</em&gt; نماذج السرطان

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

Cancer Research

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.

هندسة العوامل الاصطناعي للتلاعب على وجه التحديد الربط البديل في الخلايا البشرية

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

Genetics

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.

التحليل الكمي للربط مرناً قبل البديلة في أقسام الدماغ الماوس باستخدام الحمض النووي الريبي في الموقع التهجين المقايسة

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.

تحريض الوسيطة-طلائي التحولات في خلايا الورم اللحمي

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

Developmental Biology

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.

دراسة TGF-β الإشارة و TGF-β الناجمة عن الظهارية إلى mesenchymal التحول في سرطان الثدي والخلايا الطبيعية

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.

استخدام أداة E1A Minigene لدراسة تغييرات الربط مرنا

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.

فحص خلوي قائم على المراسل لمراقبة كفاءة الربط

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

Biochemistry

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.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

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

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.

تحديد الربط البديل والبولي أدينيل في بيانات RNA-seq

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

توصيل (إزالة انترونات) RNA

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