1. ¹Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan, ROC
2. ²Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, Tainan, Taiwan, ROC
3. ³Institute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, Taiwan, ROC
4. ⁴Department of Pediatrics, Chi Mei Foundation Medical Center, Tainan, Taiwan, ROC
5. ⁵Department of Obstetrics and Gynecology, Chi Mei Foundation Medical Center, Tainan, Taiwan, ROC

Correspondence: Dr WT Chang, Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, No 1, University Road, Tainan 701, Taiwan, ROC. E-mail: wtchang@mail.ncku.edu.tw

⁶These authors contributed equally to this work.

Received 2 May 2008; Revised 23 October 2008; Accepted 26 November 2006; Published online 23 January 2009.

Abstract

Coexpression of multiple shRNAs can simultaneously inhibit multiple genes or target multiple sites on a single gene. These approaches can be used for dissecting complex signaling pathways and even be applied to targeting multiple genes in cancer therapy. Here we established a simple and efficient multiple shRNAs expression system based on pSUPER, the most popular expression vector in mammalian cells. A series of head-to-tail tandem array multiple shRNAs expression vectors were constructed containing different combinations of six shRNA expression cassettes targeting genes involved in cell proliferation and survival pathways: Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFB. In HeLa and HEK293 cells, the multiple shRNAs expression constructs could efficiently and simultaneously induce inhibition of all six genes. We further evaluated the inhibition effects of the multiple shRNAs expression vectors on the human prostate cancer cell line PC3, which contains different cell variants with distinct oncogenic signaling alterations. The results revealed that the multiple shRNAs expression system could inhibit all six genes and was much more efficient in inducing apoptosis in the PC3 cells. Our results suggest that the multitarget shRNAs expression system could be an effective strategy in cancer therapy and be applied to any other DNA vector-based shRNA expression system.