Establishment of RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter and its biological effects on prostate cancer cells.
- Author:
Yue-feng DU
1
;
Yi-fei XING
;
Fu-qing ZENG
;
Peng LU
;
Xian-yin LIU
;
Ya-jun XIAO
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Male; Mice; NIH 3T3 Cells; Neoplasm Invasiveness; Plasmids; Promoter Regions, Genetic; Prostate-Specific Antigen; genetics; Prostatic Neoplasms; genetics; metabolism; pathology; RNA Interference; RNA, Messenger; metabolism; RNA, Small Interfering; genetics; Receptors, CXCR4; genetics; metabolism; Recombinant Proteins; genetics; metabolism; Retroviridae; genetics; Transfection
- From: Chinese Journal of Oncology 2007;29(7):489-494
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSTo 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.
RESULTSThe 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).
CONCLUSIONThe 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.
