Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial cells by RNA interference

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.

Keywords

RNA interference / p27Kip1 / plasmid / corneal endothelial cells

References

[1] JoyceN. C.. Cell cycle status in human corneal endothelium. Exp Eye Res, 2005, 81(6): 629-638

[2] LiJ., SunX. C., BonannoJ. A.. Role of NBC1 in apical and basolateral HCO₃⁻ permeabilities and transendothelial HCO₃⁻ fluxes in bovine corneal endothelium. Am J Physiol Cell Physiol, 2005, 288(3): 739-746

[3] JoyceN. C., ZhuC. C.. Human corneal endothelial cell proliferation: potential for use in regenerative medicine. Cornea, 2004, 23: 8-19

[4] JoyceN. C.. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res, 2003, 22(3): 359-361

[5] BoehmM., YoshimotoT., CrookM. F., et al.. A growth factor-dependent nuclear kinase phosphoylates p27Kip1 and regulates cell cycle progression. EMBO J, 2002, 21: 3390-3401

[6] LeeH. T., KayE. P.. Regulatory role of PI 3-kinase on expression of Cdk4 and p27, nuclear localization of Cdk4, and phosphorylation of p27 in corneal endothelial cells. Invest Ophthalmol Vis Sci, 2003, 44(4): 1521-1528

[7] WiederholtM., JentschT. J.. Cell culture of bovine corneal endothelial cells and its application to transport studies. Methods Enzymol, 1990, 192: 571-582

[8] JoyceN. C., SamuelE.. Expression of cell cycle-associated proteins in human and rabbit corneal endothelium in situ. Invest Ophthalmol Vis Sci, 1996, 37: 1566-1575