Inhibition of proliferation of rabbit lens epithelial cells by S-phase kinase-interacting protein 2 targeting small interfering RNA.
OBJECTIVE
Improper proliferation of lens epithelial cells is causally related to posterior capsule opacification. In the present study, we investigated whether small interfering RNA (siRNA)-mediated gene silencing of S-phase kinase-interacting protein 2 (Skp2) can be employed to inhibit rabbit lens epithelial cell (rLEC) proliferation by increasing the p27(kip1) level.


METHODS
A plasmid containing Skp2 siRNA was used to decrease the high constitutive level of Skp2 protein in rLECs, which can lead to consequent degradation of p27(kip1). Protein expression of Skp2 and p27(kip1) was detected by immunocytochemistry and western blot. Cell viability was measured using the 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 against proliferating cell nuclear antigen.


RESULTS
Immunocytochemistry and western blot showed a decreased level of Skp2 and increased level of p27(kip1) in cells transfected with pSkp2 siRNA but not in vehicle transfection and uninfected cells. MTT assay showed that cell viability significantly declined in rLECs transfected with Skp2 siRNA. Skp2 siRNA transfected cells showed significantly less 59-bromodeoxyuridine- and proliferating cell nuclear antigen-positive staining compared with control cells.


CONCLUSIONS
Skp2 siRNA inhibits cell proliferation and decreases cell viability of rLECs in vitro by suppression of p27(kip1) downregulation. Our findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel gene therapy for posterior capsule opacification induced by LEC abnormal proliferation.
Department of Ophthalmology, First Clinic College of Harbin Medical University, Harbin, China.
Ying
Y
Su
Feng
F
Wang
Qinghui
Q
Yan
Yan
Y
Teng
Hao
H
Cui
D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D015536:Down-Regulation; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D020868:Gene Silencing; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011817:Rabbits; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D013194:Staining and Labeling; D014162:Transfection; D015854:Up-Regulation
D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering; D044786:S-Phase Kinase-Associated Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D001973:Bromodeoxyuridine