RNA interference-produced autoregulation of inducible nitric oxide synthase expression

Abstract

Vector-mediated delivery of short-hairpin RNA (shRNA) to regulate gene expression holds a great therapeutic promise. We hypothesize that gene expression can be autoregulated with RNA interference. We used inducible nitric oxide synthase (iNOS) as a gene model to test this hypothesis. Lipopolysaccharide dose-dependently increased iNOS in rat aortic smooth muscle cells and the nitrite production from these cells. These increases were attenuated in cells transfected with plasmids containing code for iNOS shRNA whose expression was controlled by an iNOS promoter. The production of shRNA was lipopolysaccharide dose-dependent. The lipopolysaccharide-induced iNOS expression in rat C6 glioma cells also was attenuated by transfection with plasmids containing the iNOS shRNA code. These results provide proof-of-concept evidence for using RNA interference technique to achieve autoregulation of gene expression.

Fig. 1. Effectiveness of the silencing effects of iNOS shRNAs and the induction of iNOS shRNA production

A: A diagram showing the basic structure of the plasmids used in the study. B: Human 293FT cells were co-transfected with the plasmid pCMV-iNOS and various pCMV-shRNA plasmids indicated in the figure. RNA was prepared at 24 h after the transfection. Results are means ± S.D. (n = 3). ^ P < 0.05 compared with pCMV-N group. C: RASMCs were transfected with various plasmids indicated in the figure. Twenty four hours later, they were incubated with various concentrations of lipopolysaccharide for 12 h and then harvested for shRNA4 quantification. Results are means ± S.D. (n = 3). * P < 0.05 compared with piNOS-shRNA4 at 0 µg/ml lipopolysaccharide.

Fig. 2. Does-response of lipopolysaccharide to induce iNOS expression and nitrite production

A: RASMCs were transfected with various plasmids indicated in the figure. Twenty four hours later, they were incubated with various concentrations of lipopolysaccharide for 12 h and then harvested for iNOS mRNA quantification. Results were normalized by the level of iNOS mRNA in cells transfected with piNOS-N at 0 µg/ml lipopolysaccharide. Reduction of iNOS mRNA abundance in cells transfected with piNOS-shRNA3 or piNOS-shRNA4 was calculated by subtracting mRNA levels in the cells transfected with piNOS-N from the mRNA levels in the cells transfected with those plasmids containing iNOS shRNA codes. Results are means ± S.D. (n = 3). * P < 0.05 compared with piNOS-N at 0 µg/ml lipopolysaccharide. B: The culture medium from the cells used in panel A was collected for nitrite measurement. Reduction of nitrite concentrations in cells transfected with piNOS-shRNA3 or piNOS-shRNA4 was calculated by subtracting nitrite concentrations of the cells transfected with piNOS-N from the concentration of the cells transfected with those plasmids containing iNOS shRNA codes. Results are means ± S.D. (n = 6). * P < 0.05 compared with piNOS-N at 0 µg/ml lipopolysaccharide.

Fig. 3. Silencing effects of iNOS shRNAs in C6 cells

A and B: Rat C6 cells were transfected with the plasmid piNOS-N that contained a code for green fluorescent protein. Photos were taken 24 h later. Panel A is a phase contrast picture to show all cells. Panel B is a fluorescent image to show cells transfected by the vector. C: C6 cells were transfected with or without plasmids indicated in the figure and were sorted based on their expression of green fluorescent protein. Cells that were transfected successfully by the corresponding plasmids were exposed to 1 µg/ml lipopolysaccharide for 3 h before they were harvested for real-time PCR for quantification of iNOS and actin mRNA. The iNOS results were normalized by actin data from the same sample. Results are means ± SD (n = 6). * P < 0.05 compared with iNOS-N group.