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Genetics

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Published: September 27th, 2016

DOI:

10.3791/54280

1Department of Ophthalmology, Saevit Eye Hospital, 2Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center
* These authors contributed equally

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.

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.

Corneal epithelial cells (CECs) are continuously shed into tear film, while they are simultaneously replaced by cells from the limbus and corneal epithelial basal layers.1 Various extrinsic stressors can induce the apoptosis and desquamation of CECs.2 The heat shock proteins (HSPs) are highly conserved and can be divided into two families according to molecular size.3 The largest HSP family includes HSP90, HSP70, and HSP60, and the smaller family includes HSP27.4 The phosphorylation of HSP27 is known to play an important role in cell survival and is required for cell migration because of the role of this protein in actin rem....

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1. Cell Line

  1. Culture 106 telomerase-immortalized human corneal epithelial cells (HCECs) in a 6-well plate (density: 1039.9 cell/mm2) in a 37 °C incubator with a 5% CO2 atmosphere using bronchial epithelium growth medium (BEGM) until they reach 95% confluence.

2. Western Blot Analysis after Creating Epithelial Scratch Wounds

  1. Streak a sterile 200 µl pipette tip across the surface of a well of confluent cultured HCECs four .......

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The expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding compared with unwounded HCECs13. Western blot analysis revealed that the expression of phosphorylated HSP27 and phosphorylated Akt were both significantly reduced, whereas the expression of Bax was significantly increased in HSP27-specific siRNA-transfected HCECs (Figure 1A-E). The phosphorylated HSP27 expression was reduced by 30% and 40% in 10 nM and 50 .......

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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.......

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This study was supported by the Student Research Grant (13-14) of University of Ulsan College of Medicine, Seoul, Korea and a grant (2014-464) from the Asan Institute for Life Sciences, Seoul, Korea.

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Name Company Catalog Number Comments
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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