Inhibition of angiotensin-converting enzyme expression in rat vascular endothelial cells by RNA interference

Objective To selectively knockdown the expression of angiotensin-converting enzyme(ACE) in rat vascular endothelial cells(ECs) by RNA interference.Methods Recombinant plasmids expressing ACE gene-specific shRNA(pACE-shRNA) were constructed.Primary cultures of rat aortic endothelial cells were transfected with pACE-shRNA with different ratios of transfection reagent.Transfection efficiency was determined by fluorescence microscopy and cell viability was calculated by MTT assay.Rat aortic endothelial cells were then transfected with the optimal condition.Total RNA and total protein were extracted from endothelial cells before transfection and 24,48,72 hours after transfection.ACE mRNA and protein expression were analyzed by semi-quantitative RT-PCR and Western blot.Results The optimal ratio of pACE-shRNA to transfection reagent was 1.0 μg to 4 μl.With this transfection condition,transfection efficiency was(81.2±6.2)% and cell viability was(90.2±4.1)%.Expression of ACE mRNA and protein in endothelial cells were significantly reduced 48 hours after pACE-shRNA transfection,and was nearly undetectable after 72 hours.Conclusions RNA interference can knockdown ACE expression in cultured Ecs,which maybe provide a new gene therapy tool for cardiovascular diseases.