RNA-Interferenz-basierte Untersuchung der Funktion von Heat Shock Protein 27 während Hornhautepithels Wundheilung
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
Hornhautepithelzellen (LME) werden kontinuierlich in Tränenfilm vergossen, während sie von Zellen aus dem Limbus und Hornhautepithels basalen Schichten gleichzeitig ersetzt werden. 1 Verschiedene extrinsische Stressoren können die Apoptose und Schuppung der LME induzieren. 2 Die Hitzeschockproteine (HSP) hoch konserviert und können in zwei Familien nach Molekülgrße unterteilen. 3 die größte Familie HSP umfasst HSP90, HSP70 und HSP60, und die kleinere Familie umfasst HSP27. 4 ist die Phosphorylierung von HSP27 ist bekannt , eine wichtige Rolle in Zellüberleben zu spielen und ist für die Zellmigration erforderlich wegen der Rolle dieses Proteins in die Aktin – Umbildung. 5-7 Daher wir die mögliche Rolle von HSP27 – Phosphorylierung in CEC Migration und Apoptose in einem in vitro – Modell von epithelialen Wundheilung zu testen versucht.
RNA-Interferenz (RNAi) entweder kleiner oder short interfering RNAs (siRNA) mit GEnerated Interesse sowohl in der Grundlagen- und angewandten Biologie, da sie möglicherweise die Expression jedes Gens von Interesse erlaubt zerlegten werden. 8 Hier haben wir HSP27-spezifische siRNA , den Beitrag von HSP27 CEC Wundheilung und Apoptose zu bewerten. Traditionelle RNAi Methoden für die Gen-knock-down in Zellen verwenden synthetische RNA-Duplexe, darunter zwei unmodifizierten 21-mer Oligonukleotide, die zusammengesetzt werden können siRNAs zu erstellen. Die RNAi siRNA, die wir in dieser vorliegenden Untersuchung verwendet wird, ist eine einfache und hocheffiziente Methode, um Zellen zu transfizieren, und dieses Reagens arbeitet mit verschiedenen immortalisierten Zelllinien. In der vorliegenden Studie zeigen wir die Methoden für diese Analyse verwendet, einschließlich eines Kratzers induzierten Richtungs Wunden-Assay, Western Blot, siRNA-Transfektion Assay, Immunfluoreszenztest und Durchflusszytometrie.
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
Diese Studie wurde von der Studenten Research Grant (13-14) der University of Ulsan College of Medicine, Seoul, Korea und einen Zuschuss (2014-464) aus dem Asan-Institut für Life Sciences, Seoul, Korea unterstützt.
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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