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 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 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 pro-liferation 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 in-creased 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.