Antisense oligonucleotide targeting c-fos mRNA limits retinal pigment epithelial cell proliferation: a key step in the progression of proliferative vitreoretinopathy.
The purpose of this work was to investigate the effect of c-fos antisense oligonucleotide (c-fos-AS-ON) on proliferative vitreoretinopathy (PVR). Cultures of human retinal pigment epithelial (hRPE) cells were established from adult human corneal donors. These cells were positively stained for cytokeratins. C-fos-AS-ON effect on serum-stimulated cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine (BrdU) into cellular DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were respectively performed to quantify the serum-stimulated c-fos gene mRNA and protein expression in hRPE cells. Eight rabbits (16 eyes) were divided into c-fos-AS-ON treatment group and control group. 2.5 x 10(5) cultured hRPE cells were injected into the vitreous cavity of eyes to establish a PVR model. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection and by pathological study on days 28 post-injection. The results showed that blocking the expression of c-fos by the addition of c-fos-AS-ON to the culture medium significantly inhibited the hRPE cells proliferation. This effect of c-fos-AS-ON was found to be sequence specific (the use of a sense or a mismatch sense oligonucleotide had no such an effect) and dose-dependent (0.375 microM was the lowest effective dose tested). Growth inhibition by c-fos-AS-ON remained for at least 72 h. By using RT-PCR and Western blotting, we found that the c-fos-AS-ON could specifically inhibit c-fos mRNA and protein synthesis in cultured hRPE cells. Though the eyes injected with c-fos-AS-ON also developed features of PVR, the severities of days 14, 21 and 28 post-injection were significantly lower than those in the control eyes (P<0.05). We conclude that c-fos-AS-ON can inhibit cultured hRPE cell proliferation, which mechanism may relate to blocking the expression of c-fos and can reduce the prevalence of experimental PVR. These findings establish a rationale for investigating the potential use of a c-fos-AS-ON as a novel therapeutical tool in the treatment of PVR.
Zhang
Li
L
Department of Ophthalmology, People's Hospital, Peking University, Beijing, China.
Li
Xiaoxin
X
Zhao
Mingwei
M
He
Peiying
P
Yu
Weidong
W
Dong
Jianqiang
J
Liu
Guodong
G
Li
Chunan
C
Shi
Xuan
X
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D018450:Disease Progression; D018390:Gene Targeting / Q000379:methods; D016762:Genes, fos / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008297:Male; D016376:Oligonucleotides, Antisense; D010857:Pigment Epithelium of Eye / Q000473:pathology*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection; D018630:Vitreoretinopathy, Proliferative / Q000473:pathology* / Q000517:prevention & control
D016376:Oligonucleotides, Antisense; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D007633:Keratins