각막 상피 창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사
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
그들은 동시에 윤부 각막 상피 기저 층에서 세포로 대체하는 동안 각막 상피 세포 (CECs)이 지속적으로, 눈물 막으로 흘려된다. (1) 다양한 외부 스트레스가 CECs의 세포 사멸 및 박리를 유도 할 수 있습니다. 2 열 충격 단백질들 (HSPs) 고도로 보존되어 있으며, 분자의 크기에 따라 두 개 패밀리로 분할 될 수있다. (3) 최대 HSP 계열은 HSP90, HSP70, 및 HSP60 및 작은 가족 HSP27 포함을 포함한다. 4 HSP27의 인산화는 세포 생존에 중요한 역할을하는 것으로 알려져 때문에 굴지의 리모델링이 단백질의 역할의 세포 이동이 필요합니다. 5-7 따라서, 우리는 상피 상처 치유의 체외 모델에서 CEC 마이그레이션 및 세포 자멸사에서 HSP27 인산화의 잠재적 인 역할을 테스트하려고했습니다.
RNA 간섭 (RNAi의) 작거나 짧은 간섭 RNA를 하나를 사용하여 (siRNA의)은 GE있다잠재적으로 허용하는 두 기초 및 응용 생물학 nerated 관심이 관심의 유전자의 발현이 노크 다운 될 수 있습니다. (8) 여기서, 우리는 CEC 상처 치유와 세포 자멸사에 대한 HSP27의 기여를 평가하기 위해 HSP27 특정 siRNA를 사용했다. 세포에서 유전자 노크 다운에 대한 기존의 RNAi 방법은 siRNA를을 만드는 조립 될 수있는 두 개의 수정되지 않은 21 메르 올리고 뉴클레오티드를 포함하는 합성 RNA 이중 가닥을 사용합니다. 우리가 본 연구에 사용 된 siRNA의 RNAi의 세포를 형질하는 간단하고 효율적인 방법이며,이 시약은 다양한 불멸화 세포주와 함께 작동한다. 이 연구에서는, 우리는 스크래치에 의한 방향 상처 분석, 웨스턴 블로 팅, siRNA를 형질 분석, 면역 형광 분석을 포함하여이 분석에 사용되는 방법을 설명하고, 유동 세포 계측법.
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
이 연구는 의학, 서울, 울산 대학교 의과 대학 학생 연구 그랜트 (13 ~ 14)과 아산 생명 과학 연구소, 서울, 한국에서 보조금 (2014-464)에 의해 지원되었다.
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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