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.
Written by:
Du YF, Long QZ, Shi Y, Liu XG, Li XD, Zeng J, Gong YG, Wang XY, He DL. Are you the author?
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.
Reference: Int J Clin Exp Med. 2013;6(2):126-32.
PubMed Abstract
PMID: 23386916
UroToday.com Investigative Urology Section
