[Short hairpin RNA targeting vascular endothelial growth factor effectively inhibits expression of vascular endothelial growth factor in human retinal pigment epithelium].
OBJECTIVE
To design shRNA targeted to human vascular endothelial growth factor (VEGF) and to evaluate the effect of VEGF. shRNA on expression of VEGF in human retinal pigment epithelium (RPE) cells in vitro.


METHODS
Human RPE cells were isolated with enzyme-assisted microdissection. The cells were identified by immunohistochemical method with antibody to cytokeratin and S-100. Plasma DNA was identified via restriction enzyme EcoRI and SamI. shRNAs (P1, P2) specific for human VEGF were designed. DNA expression vector is pSilencer 4.1-CMV of Ambion company. P3 is negative control nonspecific shRNA. There are 5 groups. Group 1: VEGF in cultured human RPE exposed to 100 micromol/L CoCl2 30 h; Group 2: VEGF in cultured human RPE in normal culture medium; Group 3, 4, 5: VEGF in cultured human RPE exposed to 100 micromol/L CoCl2 30 h after P1, P2, P3 transfection, respectively. VEGF level in conditioned media was measured by Western blot.


RESULTS
The cells in culture could be stained with both cytokeratin and S-100 antibodies. The length of two fragment was 3.3 kb and 1.6 kb, respectively, which indicated that the extraction and purification were successful. The expression of VEGF in RPE was increased significantly (P < 0. 001) in group 1 as compared with group 2. Hypoxia-induced upregulation of human VEGF is halted by siRNA application in vitro (P < 0. 001 and P < 0. 001 in group 3 and 4 compared with group 1, respectively). shRNAs targeted hVEGF effectively and specifically inhibited hypoxia-induced VEGF levels in human RPE. The level of VEGF was reduced 65.9% and 52.4% in groups 3 and 4, respectively. There was no difference between group 5 and 1 (P = 0. 147). There was no difference of beta-actin production in RPE cells among groups.


CONCLUSIONS
Delivery of shRNA can be used in vitro to target specific RNAs of VEGF and to reduce the level of the specific protein product (VEGF) in the targeted cells (human RPE). This work established the basis for the using of RNA interference in studies of retinal biology and for the treatment of a variety of retinal angiogenic diseases, especially the choroidal neovascularization.
Cai
Chun-mei
CM
Beijing Institute of Ophthalmology, Beijing Tongren Ophthalmologic Center, Capital University of Medical Sciences, Beijing 100730, China. chunmeicai66@yahoo.com.cn
Sun
Bao-chen
BC
Liu
Xu-yang
XY
D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics*
D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A