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Ji-Ye Wei, Jon Chatterton, Carl Romano, Kristina Rhoades; High Throughput siRNA Screen to Identify Targets of RPE Cells Protection from 7-Ketocholesterol Induced Oxidative Damage. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1790.
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© ARVO (1962-2015); The Authors (2016-present)
The use of short interfering RNA (siRNA) for knockdown of gene expression has become a powerful tool in pharmaceutical industry for new drug target identification. In the present study, we used Dharmacon’s siRNA “druggable” library to explore this strategy to identify new targets for protection of human RPE cells treated with 7-keto cholesterol (7kCh) in a high throughput, cell-based assay. We were looking for genes that when silenced protect cells from 7kCh toxicity. Hits would have higher cell viability compared to non-targeting control.
ARPE-19 cells were seeded in 96-well plates at 4,000 cells per well, as optimized for the screen. Reverse transfection was executed in this experiment with select transfection conditions resulting in 85% knockdown and 80% cell viability or higher. siRNA at 50nM mixed with Dharmafect4 at 0.2ug/well were incubated at room temperature for 30 minutes. Twenty-eight hours after transfection, media is replaced with either vehicle or treatment media containing 13uM or 15uM of 7kCh. Then 48 hours after transfection, media is replaced with either vehicle or treatment media containing 22uM or 25uM of 7KCh. 7-ketocholesterol (5-CHOLESTEN-3b-OL-7-ONE) was resuspended in 37 °C 45% 2-hydroxylpropyl-b-cyclodextrin at a concentration of 10 mM. 7kCh working dilutions of 1 mM were subsequently made in DMEM:/F12 containing 0.1 % FBS and NaBicarb. As negative controls vehicle alone suspended 2-HP-b-cyclodextrin were included in each experiment. CellTiter Fluor cell viability assessment assay (Promega) was used here, an indicator of metabolically active cells. The plates are then incubated for 2 hours and fluorescence was measured in a microplate ELISA reader with the following parameters: excitation = 380 nM / emission =505 nM.
181 siRNAs were identified as protective (at least 1.2 fold increased survival) in the higher concentrations of 7kCh. Notably, knockdown of several members of caspase pathways, G-protein coupled receptor families and basement membrane modifying enzymes demonstrated consistent increased survival at 25uM of 7kCh. These 181 siRNAs also caused significant protection in the lower concentration of 7kCh.
The high throughput cell-based siRNA gene knockdown screening assays is a useful method to identify novel targets for human RPE cell protection from 7-KCh induced oxidative stress damage in vitro.
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