RNA-interferentie op basis van onderzoek naar de functie van heat shock protein 27 tijdens cornea epitheliale wondheling
Summary
Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.
Abstract
Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.
Introduction
Corneale epitheliale cellen (LME) continu afgestoten in traanfilm, terwijl ze tegelijkertijd vervangen door cellen van de limbus en corneale epitheliale basale lagen. 1 verschillende extrinsieke stressoren kunnen de apoptose en afschilfering van CECs induceren. 2 De heat shock eiwitten (HSP) zijn sterk geconserveerd en kunnen worden onderverdeeld in twee families op basis van molecuulgrootte. 3 de grootste HSP-familie omvat HSP90, HSP70 en HSP60 en de kleine familie omvat HSP27. 4 de fosforylatie van HSP27 speelt een belangrijke rol in celoverleving spelen en is vereist voor celmigratie vanwege de rol van dit eiwit in actine remodellering. 5-7 Daarom hebben we geprobeerd om de mogelijke rol van HSP27 fosforylatie in CEC migratie en apoptose getest in een in vitro model van epitheliale wondgenezing.
RNA-interferentie (RNAi) met behulp van kleine of korte interfererende RNA (siRNA) heeft generated belangstelling voor zowel fundamenteel als toegepast biologie, omdat het mogelijk maakt de expressie van elk gen van belang worden overreden. 8 Hierin gebruikten we HSP27-specifiek siRNA om de bijdrage van HSP27 CEC wondgenezing en apoptose beoordelen. Traditionele werkwijzen voor RNAi gen knock-in cellen gebruiken synthetische RNA duplexen, waaronder twee niet-gemodificeerd 21-meer oligonucleotiden die kunnen worden samengesteld om siRNA's te maken. De RNAi siRNA dat we in deze studie is een eenvoudige en efficiënte methode om cellen te transfecteren, en dit reagens werkt met verschillende geïmmortaliseerde cellijnen. In deze studie tonen we de voor deze analyse methoden, met inbegrip van een scratch-geïnduceerde directionele wond assay, western blotting, siRNA transfectie assay, immunofluorescentietest, en flowcytometrie.
Protocol
Representative Results
Discussion
In this present study, we evaluated the potential role of HSP27 in corneal epithelial wounding using in vitro approaches. The critical steps involved siRNA transfection for HSP27 knock-down to observe the function of HSP27 in cells subjected to stress. Notably, a role for HSP27 was revealed by these experiments in epithelial cell migration and apoptosis during corneal epithelial wound healing. Unlike previous studies10 that used rat HSP27-specific siRNA to transfect vascular smooth muscle cells, we us…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Deze studie werd ondersteund door de Student Research Grant (13-14) van de Universiteit van Ulsan College of Medicine, Seoul, Korea en een subsidie (2014-464) van de Asan Institute for Life Sciences, Seoul, Korea.
Materials
| Biological safety cabinet | CHC LAB Co.Ltd, Daejeon, Republic of Korea | CHC-777A2-06 | Class II, Type A2 |
| Stealth RNAi™ siRNA | Thermo Fisher Scientific, Inc., Waltham, MA | RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA | |
| BEGMTM | Lonza, Inc., Walkersville, MD | CC-3171, CC4175 | Bronchial epithelium growth medium |
| Protease inhibitor | Sigma-Aldrich, Inc., St. Louis, MO | P8340 ,P7626 | 1 uM Pepstatin A, 1 uM Leupetin, 0.1 uM Aprotin |
| Bradford protein assay | Bio-Rad Laboratories, Hercules, CA | #500-0001 | Bradford protein assay |
| Nitrocellulose filters | Amersham, Little Chalfont, UK | RPN3032D | Western blotting membrane |
| Non-phosphorylated HSP27 | Abcam Inc., Cambridge, MA | ab12351 | 1:1000 dilution (Total HSP27) |
| Phosphorylated HSP27 (Ser85) | Abcam Inc., Cambridge, MA | ab5594 | 1: 1000 dilution HSP27 was phosphorylated at Ser85 |
| Lipofectamine® RNAiMAX reagent | Invitrogen, Carlsbad, CA | 13-778-075 | Transfection reagent |
| Phosphorylated Akt (Ser473) | Cell Signaling Technology, Danvers, MA | No. 4060 | 1: 1000 dilution Akt was phosphorylated at Ser473 (cell survival marker) |
| Non-phosphorylated Akt | Cell Signaling Technology, Danvers, MA | No. 4061 | 1:1000 dilution (Total Akt) |
| Bcl-2-associated X protein | Cell Signaling Technology, Danvers, MA | No. 4062 | 1: 1000 (anti-apoptotic protein marker) |
| GAPDH | Santa Cruz Biotechnology, Santa Cruz, CA | No. 4063 | 1:1000 loading control marker (house keeping gene) |
| Horseradish peroxidase-conjugated goat anti-rabbit antibodies | Thermo Fisher Scientific, Inc., Waltham, MA | NCI1460KR | 1:10000 dilution |
| OPTI-MEM | Invitrogen, Carlsbad, CA | 31985 | reduced serum medium for transfection |
| Image analysis software | Olympus, Inc., Tokyo, Japan | Image-Pro Plus 5.0 | |
| Skimed milk powder | Carl Roth GmbH + Co. KG, Karlstruhe, Germany | T145.2 | |
| Tris | Amresco LCC, Inc. Solon, OH | No-0497 | |
| Sodium Chloride | Amresco LCC, Inc. Solon, OH | No-0241 | |
| Six well culture plate | Thermo Fisher Scientific, Inc., Waltham, MA | 140675 | 35.00 mm diameter / well |
| 24-well culuture dish | Thermo Fisher Scientific, Inc., Waltham, MA | 142475 | |
| Orbital shaker | N-Bioteck, Inc., Seoul, South Korea | NB1015 | |
| Bovine serum albumin | Santa Cruz Biotechnology, Santa Cruz, CA | sc-2323 | |
| BDFACSCantoTM II | BD Biosciences, Franklin Lakes, NJ | Flow cytometry | |
| X-Ray Film | Kodak, Rochester, NY | Medical X-Ray Cassette with Green 400 Screen | |
| western blotting luminol reagent | Santa Cruz Biotechnology, Santa Cruz, CA | sc-2048 | |
| FITC Annexin V Apoptosis Detection Kit I | BD Biosciences, Franklin Lakes, NJ | 556547 |
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