Inhibition of PCNA antisense oligonucleotides mediated by liposome on mRNA expression and proliferation of h-RPE cells.
The proliferating cell nuclear antigen (PCNA) gene expression was blocked and retinal pigment epithelium (RPE) proliferation was inhibited by using antisense oligonucleotides (AS-ODN) mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy (PVR). RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN (0.07, 0.28 and 1.12 micro mol/L and sense oligonucleotides (S-ODN with the same concentrations as AS-ODN) mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by 3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression of PCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density (AOD) was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN (0.28 and 1.12 micro mol/L) markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant (P < 0.05 and P < 0.01, repectively) as compared with blank control group. AOD was well correlated with CPM (r = 0.975). It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.
Chen
Jianbin
J
Department of Ophthalmology, Tongji Hospital, Tongji Medial College, Huazhong University of Science and Technology, Wuhan 430030, China.
Xiang
Nan
N
Xu
Lili
L
Zeng
Shuiqing
S
D049109:Cell Proliferation; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D008081:Liposomes; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D008081:Liposomes; D016376:Oligonucleotides, Antisense; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger