Enquête basée sur l'interférence ARN de la fonction de la protéine de choc thermique 27 pendant la cicatrisation cornéenne épithéliale Wound
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
Les cellules épithéliales de la cornée (CECS) tombent en continu dans le film lacrymal, alors qu'ils sont simultanément remplacées par des cellules du limbe et des couches basales épithéliales de la cornée. 1 Divers facteurs de stress extrinsèques peuvent induire l'apoptose et la desquamation de CECs. 2 Les protéines de choc thermique (HSP) sont hautement conservée et peut être divisé en deux familles selon la taille moléculaire. 3 la plus grande famille HSP comprend HSP90, HSP70 et HSP60, et la plus petite famille comprend HSP27. 4 la phosphorylation de HSP27 est connue pour jouer un rôle important dans la survie des cellules et est nécessaire pour la migration cellulaire en raison du rôle de cette protéine dans l' actine remodelage. 5-7 par conséquent, nous avons tenté de tester le rôle potentiel de HSP27 phosphorylation dans la migration de la CCE et de l' apoptose dans un modèle in vitro de cicatrisation épithéliale.
L'interférence ARN (ARNi) en utilisant soit des petites ou courtes ARN interférents (siRNA) a gel' intérêt à la fois la biologie également exonéré fondamentale et appliquée, car elle permet potentiellement l'expression d'un gène d'intérêt à être renversé vers le bas. 8 Ici, nous avons utilisé siRNA spécifiques HSP27 pour évaluer la contribution des HSP27 à la guérison de la plaie de la CCE et de l' apoptose. Les méthodes traditionnelles d'ARNi pour le gene knock-down dans les cellules utilisent des duplexes d'ARN synthétiques, comprenant deux oligonucléotides 21-mères non modifiés qui peuvent être assemblés pour créer des ARNsi. L'ARNi siRNA que nous avons utilisé dans cette étude est une méthode simple et très efficace pour transfecter des cellules, et ce réactif travaille avec diverses lignées cellulaires immortalisées. Dans la présente étude, nous démontrons les méthodes utilisées pour cette analyse, y compris un essai induit scratch-plaie directionnelle, western blot, siRNA essai de transfection, immunofluorescence et cytométrie en flux.
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
Cette étude a été soutenue par la subvention de recherche pour étudiants (13-14) de l'Université d'Ulsan College of Medicine, Seoul, Corée et une subvention (2014-464) de l'Institut Asan for Life Sciences, Séoul, Corée.
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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