Effect of p27Kip1 Inhibition on Proliferation of Bovine Corneal Endothelial Cells by RNA Interference

• Published in: Journal of Huazhong University of Science and Technology. Medical sciences Vol. 28; no. 2; pp. 211 - 215
• Main Author: 黄渝侃 张明昌 王勇 范可顺 张光红 周莉
• Format: Journal Article
• Language: English
• Published: Heidelberg Huazhong University of Science and Technology 01.04.2008
• Subjects: Animals; Cattle; Cell Proliferation; Cornea - cytology; Cyclin-Dependent Kinase Inhibitor p27 - metabolism; Endothelial Cells - cytology; Endothelial Cells - metabolism; Gene Expression Regulation; Medicine; Medicine & Public Health; Models, Biological; Plasmids - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger - metabolism; RNA, Small Interfering - metabolism; Tetrazolium Salts - pharmacology; Thiazoles - pharmacology; Transfection; 内皮细胞; 核糖核酸; plasmid; p27Kip1; corneal endothelial cells; RNA interference
• ISSN: 1672-0733; 1993-1352
• DOI: 10.1007/s11596-008-0225-3

Three plasmids （pGenesil-P1, pGenesil-P2, pGenesil-P3） with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells （bCEC） were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control （pGenesil-HK）. The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group （non-transfected group）, pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry （FCM）. Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference （RNAi） may be an effective means to promote the proliferation of CEC.