Inhibitory effect of PTIª²1(PCª²3) on PCª²3 cell line by using shortª²hairpin siRNA expression vector pEGFP/U6 in vitro

    SONG Bin1, WANG He1, ZHAO Jing2, QIN Weiª²Jun1, YANG Anª²Gang2

    1Department of Urology Surgery, Xijing Hospital, 2Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct the shortª²hairpin RNA (shRNA) eukaryotic expression vector specific to prostate carcinoma tumorª²inducing gene 1 (PCª²3) [PTIª²1 (PCª²3)], which is overexpressed in human prostate carcinoma PCª²3 cell line, and to observe the silencing effect of the established vector on PTIª²1 (PCª²3). METHODS: The shRNA specific to PTIª²1 (PCª²3) was synthesized and inserted into pEGFP/U6 by gene recombination technology to construct the expression vector, pEGFP/U6ª²mPs, which was then transfected into the cultured PCª²3 cells. The cell total RNA was extracted to perform RTª²PCR and the whole cell protein was extracted and detected by Western blot with rabbitª²antiª²human PTIª²1 (PCª²3) polyclonal antibody. RESULTS: pEGFP/U6ª²mPs expression vector was successfully constructed. Lipofectamine2000 mediated gene transfection of pEGFP/U6ª²mPs led to cellª²death at 48h after transfection. The downª²regulations of both the PTIª²1 (PCª²3) mRNA level and the PTIª²1 (PCª²3) protein level were also observed. CONCLUSION: The pEGFP/U6ª²mPs expression vector can inhibit the expression of PTIª²1 (PCª²3) gene in human prostate carcinoma PCª²3 cell line, which may downª²regulate the level of PTIª²1 protein expression. "Translational infidelity", the role of PTIª²1 (PCª²3) protein, is probably degraded, which may be responsible for the death of the cancer cells. The PTIª²1 (PCª²3) gene is further confirmed as a dominantª²acting oncogene. The specificity of the RNA interference may lead to a novel approach of clinical gene therapy in treating prostate carcinoma. ......