[Establishment of RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter and its biological effects on prostate cancer cells].
OBJECTIVE
To construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter and investigate its targeted inhibition effects in androgen-responsive prostate cancer cells LNCaP.


METHODS
To clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the pGensil-1 plasmid containing U6 promoter and EGFP. U6 promoter was replaced by hPSA promoter. Then, the recombinant EGFP-hPSA-siCXCR4 fragment was sub-cloned into pLXSN, which was evaluated by restriction enzyme. The pLXSN-EGFP-hPSA-siCXCR4 was transfected into PA317 cells with Lipofectamine 2000. The virus obtained from transfected PA317 cells was transfected into PC-3m, LNCaP and MCF-7 cells, respectively. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western blot. The invasion ability of prostate carcinoma cells was detected by Transwell experiment.


RESULTS
The recombinant pLXSN-hPSA-siCXCR4 was successfully constructed. The expression of CXCR4 mRNA and protein in LNCaP cells was blocked by pLXSN-hPSA-siCXCR4. The expression inhibition rate was (81.53 +/- 10.22)% at mRNA level detected by semi-quantitive RT-PCR and (90.52 +/- 9.31)% at protein level detected by Western blot, respectively, in LNCaP cells at 48 h. The expression of CXCR4 mRNA and protein was effectively inhibited by sequence-specific hPSA-siCXCR4 in LNCaP cells, but not in PC-3m and MCF-7 cells. The results of Transwell experiment showed that the number of cells in down-pore of micro-membrane was 139.9 +/- 14. 2 in the treated group, significantly less in comparison with 348.4 +/- 36. 4 in the controlled group (P < 0.05). However, the number of PC-3m and MCF-7 cells in down-pore of micro-membrane was not significantly different among the control and treated groups (P > 0.05).


CONCLUSIONS
The downstream siRNA controlled by hPSA promoter in retrovirus system can be expressed selectively in androgen-responsive prostate carcinoma cells, showing an apparent targeting character. RNAi targeted to CXCR4 driven by hPSA promoter has a potential value in gene therapy of androgen-responsive prostate cancer.
Du
Yue-feng
YF
Department of Urology, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Xing
Yi-fei
YF
Zeng
Fu-qing
FQ
Lu
Peng
P
Liu
Xian-yin
XY
Xiao
Ya-jun
YJ
D000818:Animals; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D051379:Mice; D041681:NIH 3T3 Cells; D009361:Neoplasm Invasiveness; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017430:Prostate-Specific Antigen / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; D017430:Prostate-Specific Antigen