Knockdown Of PDE5 Expression By Adenovirus-mediated Transfer Of Short Hairpin RNAs In Smooth Muscle Cells Of Rat Corpus Cavernosum

Objective To downregulate the expression of PDE5 gene by adenovirus-mediated specific short hairpin RNAs in smooth muscle cells of rat corpus cavernosum and to investigate the feasibility of gene therapy for erectile dysfunction (ED).Methods A recombinant adenovirus, rAd-rPDE5-shRNA, targeting two sites of the PDE5 gene of Rat was constructed. The rAd-rPDE5-shRNA was transfected into the smooth muscle cells of rat corpus cavernosum. The efficiency of transfection was evaluated by cell counting of EGFP expressed cells under fluorescence microscopy and the expression of PDE5 gene was analyzed by RT-PCR and Western blot at 48h post-transfection.Results rAd-rPDE5-shRNA was identified by both endonuclease digestion and PCR of extracted recombinant adenovirus genome. 48h after the transfection of rAd-rPDE5-shRNA at a MOI of 30pfu per cell, expression of PDE5 in the smooth muscle cells of rat corpus cavernosum was inhibited by (80.78±2.30)﹪at mRNA level and (67.39±3.33)﹪at protein level.Conclusion The shRNAs delivered by the recombinant adenovirus can stably and effectively inhibit the expression of PDE5 in smooth muscle cells of rat corpus cavernosum with a high transfection efficiency; two siRNAs that were expressed by one recombinant adenovirus can significantly suppress the expression of target gene. It provides a research basis for ED gene therapy by RNA interference.