Indagine basata RNA interferenza della funzione di shock termico proteine 27 durante la guarigione della ferita corneale epiteliale
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
Corneali cellule epiteliali (PEC) sono continuamente capannone in film lacrimale, mentre sono allo stesso tempo sostituite da cellule del limbus e corneali strati basali epiteliali. 1 vari fattori di stress estrinseci possono indurre l'apoptosi e desquamazione della CEC. 2 Le proteine da shock termico (HSP) sono altamente conservati e possono essere suddivisi in due famiglie secondo dimensione molecolare. 3 la più grande famiglia HSP comprende HSP90, HSP70 e HSP60, e la famiglia più piccola comprende HSP27. 4 la fosforilazione di HSP27 è noto a svolgere un ruolo importante nella sopravvivenza cellulare ed è necessario per la migrazione delle cellule a causa del ruolo di questa proteina in actina rimodellamento. 5-7 Pertanto, abbiamo cercato di verificare il ruolo potenziale di HSP27 fosforilazione nella migrazione CEC e l'apoptosi in un modello in vitro di cellule epiteliali guarigione della ferita.
RNA interference (RNAi) utilizzando sia piccoli o brevi RNA interferenti (siRNA) ha geinteressi nerated sia in biologia di base e applicata, in quanto consente potenzialmente l'espressione di un gene di interesse da abbattuto. 8 In questo, abbiamo usato specifici siRNA-HSP27 per valutare il contributo di HSP27 a CEC guarigione della ferita e l'apoptosi. i metodi tradizionali per RNAi gene knock-down in cellule utilizzano duplex RNA sintetici, tra cui due non modificati oligonucleotidi 21-mer che possono essere assemblati per creare siRNA. La RNAi siRNA che abbiamo usato in questo studio è un metodo semplice e molto efficace trasfezione le cellule, e questo reagente funziona con varie linee cellulari immortalizzate. In questo studio, abbiamo dimostrato i metodi utilizzati per questa analisi, tra cui un saggio di zero-indotta ferita direzionale, western blotting, saggio di transfezione siRNA, test di immunofluorescenza e citometria a flusso.
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
Questo studio è stato sostenuto dalla Student Research Grant (13-14) dell'Università di Ulsan College of Medicine, Seoul, Corea e una borsa di studio (2014-464) presso l'Istituto di Scienze della Vita Asan, Seoul, 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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