Silencing of PSMA by RNAi influences biological behavior of prostate cancer cell line LNCaP |
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| Authors: | CAO Kai-yuan ZHANG Tian XU Lin YUAN Guang-qing TIAN Xiao-dong HUANG Xiao-rong QIU Shao-peng |
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| Institution: | 1.Research Center for Clinical Laboratory Standard, 2Department of Immunology, School of Preclinical Medicine, 3Basic Medical Experimental Teaching Center, Zhongshan Medical College, 4Department of Urinary Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China. E-mail: urology@vip.163.com |
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| Abstract: | AIM: To study the blocking effect of shRNA on the expression of PSMA gene in LNCaP cell line by using shRNA eukaryotic expression vector. METHODS: Three pairs of DNA templates coding shRNA, synthesized against PSMA and cloned into the vector pSilencer 2.1-U6-neo, which was named pSilencer 2.1-U6-neo-shRNA, were identified by restriction endonuclease digestion analysis and DNA sequencing. LNCaP cells were then transfected with these three pSilencer 2.1-U6-neo-shRNAs and the negative control pSilencer 2.1-U6-neo-NC. After G418 selection, the cells were selected and the interfering effect was detected by RT-PCR and Western blotting. The biological behaviours of the transfected LNCaP cells were also tested. RESULTS: Restriction endonuclease digestion analysis and DNA sequencing results all showed that the 3 target segments were cloned into pSilencer 2.1-U6- neo vector respectively. After transfected into LNCaP cells, the inhibitory ratio of PSMA mRNA was 33.15%, 9.26% and 41.97% respectively, and that of PSMA protein was 26.26%, 6.47%, 40.69% respectively. The p-shRNA3 was chosen to test the cell growth and its invasive power in vitro. The results showed that after interfering, the invasiveness of LNCaP cells were enhanced. CONCLUSION: The vector-based shRNA on PSMA gene effectively knocks down the PSMA gene expression. The successful construction of PSMA shRNA makes it possible for further study of the interaction between PSMA and prostate cancer. |
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