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.
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

Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kipl-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endo- thelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combi- nation was used as negative control (pGenesil-HK). The recombination of four plamids was con- firmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kipl was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kipl mRNA and p27Kipl 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 pro- liferation rates of the pGenesil-P3 group, the pGenesii-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kipl on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesiI-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kipl could down-regulate the expression of p27Kipl effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.