basado en la interferencia de ARN Investigación de la Función de la proteína de choque térmico 27 durante la curación de la herida corneal 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
Las células epiteliales corneales (CEC) se desprenden continuamente en la película lagrimal, mientras que se sustituyen de forma simultánea por las células desde el limbo y capas basales epiteliales corneales. 1 Varios factores estresantes extrínsecos pueden inducir la apoptosis y la descamación de CECs. 2 Las proteínas de choque térmico (HSPs) están muy conservadas y se puede dividir en dos familias según su tamaño molecular. 3 la familia HSP más grande incluye HSP90, HSP70, y Hsp60, y la familia más pequeña incluye HSP27. 4 la fosforilación de HSP27 se sabe que juega un papel importante en la supervivencia celular y es necesario para la migración celular a causa del papel de esta proteína en la remodelación de la actina. 5-7 por lo tanto, hemos intentado probar el posible papel de la fosforilación de HSP27 en la migración de CEC y la apoptosis en un modelo in vitro de la curación de heridas epiteliales.
ARN de interferencia (RNAi), utilizando tanto las pequeñas o cortas ARN de interferencia (siRNA) tiene generated interés tanto en la biología básica y aplicada, ya que potencialmente permite la expresión de cualquier gen de interés para ser derribado. 8 En esto, hemos utilizado siRNA-HSP27 específico para evaluar la contribución de HSP27 para la cicatrización de heridas CCA y la apoptosis. Los métodos tradicionales de ARNi para genes knock-down en células utilizan dúplex de ARN sintéticos, incluyendo dos oligonucleótidos 21-mer no modificados que se pueden ensamblar para crear siRNAs. La RNAi siRNA que hemos utilizado en el presente estudio es un método simple y altamente eficaz para transfectar células, y este reactivo trabaja con diversas líneas de células inmortalizadas. En el presente estudio, se demuestra los métodos utilizados para este análisis, incluyendo un ensayo de cero inducida por la herida direccional, el Western Blot, ensayo de transfección siRNA, ensayo de inmunofluorescencia y citometría de flujo.
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 estudio fue apoyado por la Beca de Investigación para Estudiantes (13-14) de la Universidad de Ulsan College of Medicine, Seúl, Corea y una subvención (2014-464) del Instituto Asan de Ciencias de la Vida, Seúl, Corea.
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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