Experimental Study
Short hairpin RNA silencing the expression of insulin-like growth factor binding protein 3 in corpus cavernosal smooth muscle cell in rat
Pu Xiaoyong, Zhou Xiangxue, Xiao Hengjun, Lin Chuqi, Peng Weihua, Bi Xuecheng, Li Dong, Liu Jiumin
Published 2017-08-08
Cite as Chin J Exp Surg, 2017,34(08): 1367-1370. DOI: 10.3760/cma.j.issn.1001-9030.2017.08.033
Abstract
ObjectiveTo 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.
Methods4 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.
ResultsCCSMCs 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.
ConclusionshRNA 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
Contributor Information
Pu Xiaoyong
Department of Urology, Guangdong General Hospital, Guangdong Academy of Meidical Sciences, Guangzhou 510080, China
Zhou Xiangxue
Department of Neurology, the East Apartment of the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510700, China
Xiao Hengjun
Department of Urology, the Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510600, China
Lin Chuqi
Department of Urology, Guangdong General Hospital, Guangdong Academy of Meidical Sciences, Guangzhou 510080, China
Peng Weihua
Department of Urology, Guangdong General Hospital, Guangdong Academy of Meidical Sciences, Guangzhou 510080, China
Bi Xuecheng
Department of Urology, Guangdong General Hospital, Guangdong Academy of Meidical Sciences, Guangzhou 510080, China
Li Dong
Department of Urology, Guangdong General Hospital, Guangdong Academy of Meidical Sciences, Guangzhou 510080, China
Liu Jiumin
Department of Urology, Guangdong General Hospital, Guangdong Academy of Meidical Sciences, Guangzhou 510080, China
