Inhibition of cell proliferation of Tenon's capsule fibroblast by S-phase kinase-interacting protein 2 targeting SiRNA through increasing p27 protein level.
OBJECTIVE
Although antiproliferative drugs have been used to prevent scarring after filtration surgery in patients with glaucoma, there are complications associated with their use. In the present study, the authors investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be used to increase p27(kip1) level and inhibit cell proliferation in rabbit Tenon's capsule fibroblast (rTF).


METHODS
A plasmid containing Skp2 siRNA was used to decrease the high constitutive level of Skp2 protein in rTF, which can lead to consequent degradation of p27(kip1). Cell proliferation was assayed by immunocytochemistry using antibodies against 59-bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). Skp2 siRNA was delivered to a trabeculectomy animal model to study the effect on rTF proliferation in vivo.


RESULTS
Immunocytochemistry and Western blot analysis showed a decreased level of Skp2 and an increased level of p27(kip1) in cells transfected with pSkp2 siRNA but not in vehicle transfection and uninfected cells in vitro and in vivo. MTT assay showed that cell viability significantly declined in rTF transfected with Skp2 siRNA. Skp2 siRNA-transfected cells showed significantly less BrdU- and PCNA-positive staining than control cells in vitro and in vivo. Infiltration bleb was detected in the Skp2 siRNA group 14 days after trabeculectomy.


CONCLUSIONS
Skp2 siRNA inhibited cell proliferation and decreased cell viability of rTF in vivo and in vitro. These findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel gene therapy to treat scarring after glaucoma surgery by the suppression of p27(kip1) downregulation.
Wang
Feng
F
Department of Ophthalmology, First Clinical College, Harbin Medical University, Harbin, China.
Qi
Lin Xin
LX
Su
Ying
Y
Yan
Qing Hui
QH
Teng
Yan
Y
D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D003239:Connective Tissue Cells / Q000378:metabolism / Q000473:pathology; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D020868:Gene Silencing / Q000187:drug effects; D007150:Immunohistochemistry; D008297:Male; D008856:Microscopy, Fluorescence; D010957:Plasmids; 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; D014130:Trabeculectomy; D014162:Transfection; D014746:Vimentin / Q000378:metabolism
D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering; D044786:S-Phase Kinase-Associated Proteins; D014746:Vimentin; D050760:Cyclin-Dependent Kinase Inhibitor p27