Inhibition of vascular endothelial growth factor gene expression by T7-siRNAs in cultured human retinal pigment epithelial cells.
BACKGROUND
Retinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.


METHODS
Human primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence.


RESULTS
Three pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P < 0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).


CONCLUSIONS
T7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.
Li
Guang-yu
GY
Department of Ophthalmology, Second Hospital, Jilin University, Changchun 130041, China. liguangyu_box@yahoo.com.cn
Fan
Bin
B
Wu
Ya-zhen
YZ
Wang
Xin-rui
XR
Wang
Yao-hui
YH
Wu
Jia-xiang
JX
D001483:Base Sequence; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000628:therapy*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000494:pharmacology*; D014158:Transcription, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism*
D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases