Inhibition ofproliferation of rabbit lens epithelial cells by S-phase kinase-interacting protein 2 targeting small interfering RNA

摘要

Purpose: Improper proliferation of lensepithelial cells is causally related to posterior capsuleopacification. In the present study, we investigated whether smallinterfering RNA (siRNA)-mediated gene silencing of S-phasekinase-interacting protein 2 (Skp2) can be employed to inhibit rabbitlens epithelial cell (rLEC) proliferation by increasing the p27kip1level. Methods: A plasmid containing Skp2siRNA was used to decrease the high constitutive level of Skp2 proteinin rLECs, which can lead to consequent degradation of p27kip1.Proteinexpression of Skp2 and p27kip1 was detected byimmunocytochemistry and western blot. Cell viability was measured usingthe tetrazolium reduction(3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide [MTT])assay. Cell proliferation was assayed by cell counts,immunocytochemistry, and western blot by using antibodies againstproliferating cell nuclear antigen. Results: Immunocytochemistry and westernblot showed a decreased level of Skp2 and increased level of p27kip1in cells transfected with pSkp2 siRNA but not in vehicletransfection and uninfected cells. MTT assay showed that cell viabilitysignificantly declined in rLECs transfected with Skp2 siRNA. Skp2siRNA transfected cells showed significantly less 59-bromodeoxyuridine-and proliferating cell nuclear antigen-positive staining compared withcontrol cells. Conclusions: Skp2 siRNA inhibitscell proliferation and decreases cell viability of rLECs in vitro bysuppression of p27kip1 downregulation. Our findings suggestthat siRNA-mediated gene silencing of Skp2 can be a novel genetherapy for posterior capsule opacification induced by LEC abnormalproliferation.