उपकला-Mesenchymal संक्रमण के दौरान वैकल्पिक Splicing की जांच
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
उपकला-mesenchymal संक्रमण (EMT) embryogenesis दौरान अंग morphogenesis और ऊतक remodeling ड्राइव कि एक विकास कार्यक्रम है. असामान्य रूप से जब सक्रिय, EMT ट्यूमर मेटास्टेसिस और अंग फाइब्रोसिस 1,2 को बढ़ावा देता है. सम्मोहक पढ़ाई एक cobble- के नुकसान में जिसके परिणामस्वरूप adherens जंक्शन प्रोटीन ई cadherin की अभिव्यक्ति को दबाने जो ऐसे ट्विस्ट, घोंघा, और Zeb के रूप में कई प्रतिलेखन कारक,,, द्वारा परिभाषित, EMT की प्रक्रिया के दौरान ट्रांसक्रिप्शनल विनियमन के महत्व का वर्णन किया है उपकला आकारिकी और एक धुरी के आकार mesenchymal phenotype 3-8 से लाभ की तरह पत्थर. RNAs के जीनोम चौड़ा विश्लेषण के माध्यम से हाल के अध्ययनों जिसका splicing पैटर्न उपकला या mesenchymal phenotypes 9,10 या तो साथ जुड़े रहे हैं जीनों का एक समूह है कि वहां मौजूद पता चला. हमारी प्रयोगशाला से कार्य कार्यात्मक वैकल्पिक splicing और EMT जुड़े. कोशिका की सतह आसंजन अणु CD44 का अध्ययन करके, हम CD44 altern कि प्रदर्शनative splicing कसकर EMT के दौरान नियंत्रित किया जाता है, और अधिक महत्वपूर्ण बात, कारणतः स्विचन कि CD44 ब्याह isoform 11 EMT के लिए योगदान देता है.
वैकल्पिक रूप से 12-14 spliced रहे मानव बहु एक्सॉन जीनों के 95% अप करने के लिए के रूप में वैकल्पिक splicing, जीन विनियमन की एक व्यापक और संरक्षित मॉडल का प्रतिनिधित्व करता है. एक जीन से कई प्रोटीन उत्पादों पैदा करके, वैकल्पिक splicing मानव जीनोम को जटिलता का एक और परत जोड़ने, प्रोटीन विविधता के लिए एक आवश्यक तंत्र का गठन किया. जैसे, वैकल्पिक splicing के अनियंत्रण संभावित मानव रोगों के कारण गहरा जैविक प्रभाव को जन्म दे सकता है. दरअसल, रोगों में न्यायपालिका वैकल्पिक splicing spliceosome मशीनरी एन्कोडिंग जीन में उत्परिवर्तन आमतौर myelodysplastic सिंड्रोम 26-28 में पाया जाता है कि हाल के निष्कर्षों सहित, एक दशक से अधिक 15-25 के लिए प्रलेखित किया गया है. इसलिए, वैकल्पिक रूप से spliced मैं का पता लगाने के लिए विश्वसनीय तरीकों को विकसितsoforms EMT सहित विविध जैविक प्रक्रियाओं के अध्ययन में काफी महत्व की है.
यहाँ हम एक inducible EMT मॉडल का उपयोग वैकल्पिक splicing में परिवर्तन का पता लगाने के लिए एक प्रोटोकॉल प्रदान करते हैं. ब्याह isoforms पीसीआर प्राइमरों डिजाइन और पता लगाने के लिए तरीकों EMT दौरान वैकल्पिक splicing के अध्ययन के लिए, लेकिन यह भी अन्य जैविक प्रक्रियाओं में वैकल्पिक splicing के अध्ययन के लिए न केवल उपयुक्त हैं. EMT दौरान वैकल्पिक splicing जांच बेहतर इस प्रकार के कैंसर मेटास्टेसिस के इलाज के लिए प्रभावी रणनीति के विकास की सुविधा, EMT और ट्यूमर मेटास्टेसिस के तंत्र को समझने के क्रम में आवश्यक है.
Protocol
Representative Results
Discussion
यहाँ वर्णित प्रक्रिया एक inducible EMT मॉडल में वैकल्पिक splicing का पता लगाने में सक्षम बनाता है. ब्याह isoform अभिव्यक्ति के इस तरह, गतिशील परिवर्तन EMT के समय पाठ्यक्रम में कब्जा कर लिया जा सकता है. संयुक्त राष्ट्र से सं…
Declarações
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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