[Simultaneous inhibition of both hTERT and androgen receptor gene expression in LNCaP cells using single shRNA vector].
OBJECTIVE
To explore the feasibility of inhibition of hTERT and androgen receptor (AR) gene expression simultaneously in LNCaP cells by single shRNA vector.


METHODS
Templates DNA of both hTERT and AR siRNA were inserted into Pgenesil vector to construct a new vector Pgenesil-hTERT-AR-shRNA by RNAi-DNA vector technology. Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA vectors were transfected into prostate cancer LNCaP cells respectively. The levels of AR mRNA, apoptosis and proliferation of each cell group were determined by FQ-PCR, Annexin V method and MTT.


RESULTS
The level of hTERT mRNA of control group cells and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.51 +/- 0.08) x 10(8), (7.32 +/- 0.43) x 10(7), (2.94 +/- 0.15) x 10(6), (4.45 +/- 0.25) x 10(7) and (3.17 +/- 0.18) x 10(6) (copies/ml) respectively. The level of AR mRNA of control cell groups and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.92 +/- 0.11) x 10(5), (6.47 +/- 0.32) x 10(5), (3.70 +/- 0.24) x 10(4), (1.22 +/- 0.06) x 10(4) and (7.21 +/- 0.41) x 10(3) (copies/ml) respectively. These data indicate that the expression of hTERT or AR gene could be significantly inhibited by Pgenesil-hTERT-shRNA or Pgenesil-AR-shRNA while Pgenesil-hTERT-AR-shRNA could simultaneously inhibit both hTERT and AR gene expression. The apoptosis rate and the inhibition rate of cell growth of Pgenesil-hTERT-AR-shRNA group were significantly higher than those of Pgenesil-hTERT-shRNA group or Pgenesil-AR-shRNA group (P < 0.05).


CONCLUSIONS
It is feasible to inhibit both hTERT and AR gene expression simultaneously by single shRNA vector. It will be a new research strategy of gene therapy for prostate cancer.
Department of Urology, Sir Run RUN Shaw Hospital, Zhejiang University Medical College, Hangzhou, China. ligonghui1970@hotmail.com
Gong-hui
GH
Li
Sheng
S
Cheng
You-feng
YF
Zhou
Zhao-dian
ZD
Chen
D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D010957:Plasmids; D034741:RNA, Small Interfering; D011944:Receptors, Androgen / Q000378:metabolism*; D019098:Telomerase / Q000378:metabolism*; D014162:Transfection
D034741:RNA, Small Interfering; D011944:Receptors, Androgen; C509186:TERT protein, human; D019098:Telomerase