Primary Studies On The Expression Of EGFP-specific SiRNA Driven By PSMA Enhancer And Promoter In Prostate Cancer

Objective:To construct the recombinant eukaryotic expression plasmid pPSMAenhancer/ promoter-siEGFP-polyA in which EGFP-shRNA is driven by prostate-specific membrane antigen enhancer and promoter, and, explore its specific driving role in prostate cancer cell lines.Methods:1. The recombinant plasmid pPSMAe/p-siEGFP-polyA was constructed by cloning the artificial DNA fragment of EGFP-specific shRNA and polyadenylation termination signals into the multiple clone site of plasmid pEGFPe/p which contained PSMA enhancer and promoter DNA sequences.2. The recombinant plasmid pPSMAe/p-siEGFP-polyA and plasmid pEGFP-C1 were transfected into several cell lines, including prostate cancer cell lines (LNCaP, PC-3, DU145) and non-prostate cancer cell line (MCF-7), by liposome-mediated transfection. The expression of EGFP-shRNA under the targeted regulate of PSMA enhancer/promoter in the different cells was observed by green fluorescence. The expression of EGFP mRNA in the different cells after transfection was investigated with RT-PCR. The semi-quantitative assay of EGFP in the different cells after transfection was made with Western blotting.Results:1. The recombinant eukaryotic expression plasmid pPSMAe/p-siEGFP-polyA was successfully constructed, and its correctness was confirmed by sequence analysis.2. After the transfection of the recombinant plasmid pPSMAe/p-siEGFP-polyA and the plasmid pEGFP-C1 into various cell lines, the ratio of green fluorescence positive cells decreased significantly (P＜0.001) only in the LNCaP experimental group, which expresses PSMA specially. While in the cell lines which don't express PSMA ,such as PC-3, DU145 and MCF-7, there was no significant difference (P＞0.05) in the ratio of green fluorescence positive cells with the control group compared.3. From RT-PCR, the expression level of EGFP mRNA decreased significantly (P＜0.001) in the LNCaP experimental group, compared with the control group. To the cell lines of PC-3,DU145 and MCF-7, there was no statistical difference (P＞0.05) between the experimental group and the control one.4. The western blotting demonstrated: the protein level of EGFP reduced significantly (P＜0.001) in the LNCaP experimental group contrasted with the control group. And no statistical difference (P＞0.05) was found between the experimental group and the control one in the cell lines of PC-3,DU145 and MCF-7.Conclusion:The recombinant expression plasmid pPSMAe/p-siEGFP-polyA in which EGFP-shRNA is driven by prostate-specific membrane antigen enhancer and promoter was constructed successfully. And the study has confirmed that pPSMAe/p-siEGFP-polyA can interfere with the expression of EGFP gene in prostate cancer cell line which expresses PSMA specifically. From the present study, it has been demonstrated that the PSMAenhancer/promoter play a specially targeted driving role to shRNA in prostate cancer.