Short hairpin RNA silencing the expression of insulin-like growth factor binding protein 3 in corpus cavernosal smooth muscle cell in rat

Abstract

Objective To screen the most effective short hairpin RNA (shRNA) which down-regulate the insulin-like growth factor binding protein 3 (IGFBP-3) in corpus cavernosal smooth muscle cell (CCSMC) in vitro. Methods 4 pair IGFBP-3 small interfering RNA (siRNA), and plasmid constructs for U6 promoter plasmid vector (pGPU6/GFP/Neo-IGFBP-3-shRNA) and negative control vector were constructed. Corpus cavernosal smooth muscle cells (CCSMC) were primary cultured and divided into 6 groups: the CCSMCs-only, transfection pGPU6/GFP/Neo-IGFBP-3 (611) group, transfection pGPU6/GFP/Neo-IGFBP-3 (526) group, transfection pGPU6/GFP/Neo-IGFBP-3 (866) group, transfection pGPU6/GFP/Neo-IGFBP-3 (710) group and the negative control group (transfection with pGPU6/GFP/Neo-shNC). The silencing effection was determined by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. Results CCSMCs transfected with shRNA showed an obviously decrease of IGFBP-3 in mRNA and protein levels compared with CCSMCs transfected with negative control vector and the CCSMCs-only in transfection pGPU6/GFP/Neo-IGFBP-3 (611) group, pGPU6/GFP/Neo-IGFBP-3 (526) group and pGPU6/GFP/Neo-IGFBP-3 (866) group. The most inhibiting effective were observed in trasnfection pGPU6/GFP/Neo-shIGFBP3-526 group. The 72% and 79% inhibiting effection were determined in real-time PCR and Western blotting. Conclusion shRNA can obviously silence the expression of IGFBP-3 in CCSMCs. It may be an effective methods to improved erection in vivo. Key words: Erectile dysfunction; Insulin-like growth factor binding protein 3; Gene Therapy; siRNA Technology; Rats