Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial cells by RNA interference.
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.
Huang
Yukan
Y
Department of Ophthalmology, Union hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. huangyk1975@163.com
Zhang
Mingchang
M
Wang
Yong
Y
Fan
Keshun
K
Zhang
Guanghong
G
Zhou
Yanli
Y
D000818:Animals; D002417:Cattle; D049109:Cell Proliferation; D003315:Cornea / Q000166:cytology; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; D050760:Cyclin-Dependent Kinase Inhibitor p27; C022616:thiazolyl blue