Duplexes of 21 nucleotide RNA specific for COX II mediates RNA interference in cultured bovine aortic coronary endothelial cells (BAECs).
The present study was conducted to utilize a double-stranded RNA (dsRNA) specific for cyclooxygenase (COX) II and demonstrate inhibition of the expression of COX II protein and its product PGE. A 21-dsRNA specific for COX II was introduced by lipofectamine into a primary cell culture of bovine aortic coronary endothelial cells (BAECs). BAECs basally express COX I but not COX II, and COX II expression is only apparent after stimulation with phorbol 12-myristate acetate (PMA). We first demonstrated that the lipofected fluorescent dsRNA-COX II is accumulated and localized within the cultured cells. We then demonstrated gene silencing of PMA-induced COX II protein expression by dsRNA-COX II using immuno-histochemistry. Western blot analysis and radioimmunoassay were used to quantitate the percent of inhibition. It was found that lipofected dsRNA-COX II reduced the percent of PMA-induced COX II enzyme by 36% and PGE production by 40%. There was no demonstrable effect of dsRNA-COX II or PMA on COX I expression.
D000818:Animals; D001011:Aorta; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000032:analysis / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007763:Lac Operon / Q000235:genetics; D008856:Microscopy, Fluorescence; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D013755:Tetradecanoylphorbol Acetate; D014162:Transfection
D007527:Isoenzymes; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate