Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial cells by RNA interference.
- Author:
Yukan, HUANG
;
Mingchang, ZHANG
;
Yong, WANG
;
Keshun, FAN
;
Guanghong, ZHANG
;
Yanli, ZHOU
- Publication Type:Journal Article
- MeSH:
Cell Proliferation;
Cornea/cytology;
Cyclin-Dependent Kinase Inhibitor p27/*metabolism;
Endothelial Cells/*cytology;
Endothelial Cells/metabolism;
Gene Expression Regulation;
Models, Biological;
Plasmids/metabolism;
RNA Interference;
RNA, Messenger/metabolism;
RNA, Small Interfering/metabolism;
Reverse Transcriptase Polymerase Chain Reaction;
Tetrazolium Salts/pharmacology;
Thiazoles/pharmacology;
Transfection
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2008;28(2):211-5
- CountryChina
- Language:English
-
Abstract:
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.
