Duplexes of 21 nucleotide RNA specific for COX II mediates RNA interference in cultured bovine aortic coronary endothelial cells (BAECs)

Prostaglandins Other Lipid Mediat. 2003 Jul;71(3-4):119-29. doi: 10.1016/s1098-8823(03)00043-1.

Abstract

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.

MeSH terms

  • Animals
  • Aorta
  • Blotting, Western
  • Cattle
  • Cells, Cultured
  • Cyclooxygenase 1
  • Cyclooxygenase 2
  • Dinoprostone / analysis
  • Dinoprostone / metabolism
  • Endothelium, Vascular / enzymology*
  • Gene Expression Regulation, Enzymologic / drug effects
  • Isoenzymes / biosynthesis
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Lac Operon / genetics
  • Microscopy, Fluorescence
  • Prostaglandin-Endoperoxide Synthases / biosynthesis
  • Prostaglandin-Endoperoxide Synthases / genetics
  • Prostaglandin-Endoperoxide Synthases / metabolism*
  • RNA Interference*
  • RNA, Double-Stranded / metabolism*
  • RNA, Messenger / metabolism
  • RNA, Small Interfering
  • Tetradecanoylphorbol Acetate
  • Transfection

Substances

  • Isoenzymes
  • RNA, Double-Stranded
  • RNA, Messenger
  • RNA, Small Interfering
  • Cyclooxygenase 1
  • Cyclooxygenase 2
  • Prostaglandin-Endoperoxide Synthases
  • Dinoprostone
  • Tetradecanoylphorbol Acetate