Investigação baseado no RNA de interferência da função da proteína de choque térmico 27 durante a cicatrização de feridas da córnea epitelial
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
Células epiteliais da córnea (PEC) são eliminados continuamente em filme lacrimal, enquanto eles são simultaneamente substituído por células do limbo e camadas basais do epitélio da córnea. 1 Vários factores de stress extrínsecos pode induzir a apoptose e descamação do PEC. 2 As proteínas de choque térmico (HSPs) são altamente conservados e podem ser divididos em duas famílias de acordo com o tamanho molecular. 3 a maior família HSP inclui HSP90, HSP70 e HSP60, e a família menor inclui HSP27. 4 a fosforilação de HSP27 é conhecida por desempenhar um papel importante na sobrevivência celular e é necessária para a migração de células devido ao papel desta proteína na remodelação da actina. 07/05 Portanto, uma tentativa para testar o potencial papel da fosforilação em HSP27 migração CEC e a apoptose em um modelo in vitro de reparação epitelial.
RNA de interferência (RNAi) utilizando quer ARN interferentes pequenos ou curtos (siARN) tem GEinteresse nerated tanto em biologia básica e aplicada, uma vez que potencialmente permite a expressão de qualquer gene de interesse a ser knocked-down. 8 Aqui, usamos siRNA específico do HSP27 para avaliar a contribuição de HSP27 para a cicatrização de feridas CEC e apoptose. ARNi métodos tradicionais para o gene knock-down em células utilizam duplexes de ARN sintéticos, incluindo dois oligonucleótidos 21-meros não modificados que podem ser montadas para criar ARNsi. O RNAi siRNA que usamos no presente estudo é um método simples e altamente eficiente para transfectar células, e este reagente trabalha com várias linhas de células imortalizadas. No presente estudo, demonstramos os métodos utilizados para esta análise, incluindo um ensaio de induzida por zero ferida direcional, western blot, ensaio de transfecção siRNA, imunofluorescência e citometria de fluxo.
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
Este estudo foi apoiado pela Research Grant Student (13-14) da Universidade de Ulsan College of Medicine, Seoul, Coreia do Sul e uma subvenção (2014-464) do Instituto Asan para as Ciências da Vida, Seoul, Coreia.
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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