IJCEM Copyright © 2008-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711
Int J Clin Exp Med 2013;6(2):126-132

Original Article
Prostate-targeted mTOR-shRNA inhibit prostate cancer cell growth in human tumor

Yue-Feng Du, Qing-Zhi Long, Ying Shi, Xiao-Gang Liu, Xu-Dong Li, Jin Zeng, Yong-Guang Gong, Xin-Yang Wang, Da-Lin He

Xi’an Jiaotong University, First Affiliated Hospital of Medical School, Department of Urology, Yanta West Road 67#, Xi’an, Shanxi,
710061, People’s Republic of China; Huazhong University of Science & Technology, Tongji Medical College, Union Hospital,
Department of Urology, Jiefang Avenue 1277#, Wuhan, Hubei, 430022, People’s Republic of China; Xi’an Jiaotong University, The Key
Laboratory of Biomedical In-formation Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Shaanxi,
People’s Republic of China

Received October 16, 2012; Accepted November 25, 2012; Epub January 26, 2013; Published February 6, 2013

Abstract: Objective: To construct a recombinant lentivirus vector driven by the PSMA promoter carrying mTOR-shRNA, and to obtain the
effect on the mTOR gene silencing in human prostate cancer xenografts. Methods: The complimentary oligos of small interference RNA
(siRNA) with hairpin structures targeting the mTOR gene and a negative control were synthesized, then ligated with pLV-PSMA-
promoter vector and sequenced. The recombinant vectors were then transfected with viral packaging mix into 293T cells, viral
supernatant was harvested to determine the titer. Prostate cancer cells infected by virus were harvested and the expression of mTOR
(LV-PSMA-shmTOR), target proteins and cell growth were detected by reverse transcription-PCR (RT-PCR), Western blot and MTT
separately. In established tumors derived from human prostate cancer cells, concentrated LV-PSMA-shmTOR lentivirus was injected
intravenously in the tail vein of C4-2b tumor bearing female severe combined immunodeficient (SCID) mice. Tumor volume and
immunohistochemistry was assessed. Results: Sequencing data showed that the constructed plasmids contained the correct
sequences of mTOR siRNA transcript templates. A vector producing cell line 293T was established, and the titer for transfection was
obtained. RT-PCR, Western blot and MTT analyses demonstrated that mTOR shRNA expression construct could suppress the
expression of mTOR and inhibit the prostate cancer cell growth, specially. The tumor growth was suppressed in nude mouse.
Conclusion: A PSMA driven lentivirus mediated siRNA targeting mTOR gene was successfully constructed, which decreased the
expression of mTOR and induced the prostate cancer cell growth in vitro and in vivo. It has set up a research platform for the gene
therapy of tumors which take mTOR as the target in the prostate cancer field. (IJCEM1210006).

Keywords: mTOR, PSMA, prostatic carcinoma, apoptosis

Address correspondence to: Da-lin He, Yanta West Road 277#, Xi’an, Shanxi, 710061, People’s Republic of China. Fax: +86 29
85323945; Tel: +86 29 85323940; E-mail: dlhe2010@163.com