Establishment of RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter and its biological effects on prostate cancer cells / 中华肿瘤杂志
Chinese Journal of Oncology
; (12): 489-494, 2007.
Article
在 Zh
| WPRIM
| ID: wpr-298567
Responsible library:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>
全文:
1
索引:
WPRIM
主要主题:
Pathology
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Plasmids
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Prostatic Neoplasms
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Retroviridae
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Recombinant Proteins
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RNA, Messenger
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Transfection
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Gene Expression Regulation, Neoplastic
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Promoter Regions, Genetic
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Prostate-Specific Antigen
限制:
Animals
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Humans
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Male
语言:
Zh
期刊:
Chinese Journal of Oncology
年:
2007
类型:
Article