The Study Of Short-hairpin RNA Targeting Prostate Cancer Nucleostemin Driven By PSMA Promoter And Enhancer In Animal Models

Objective:Through the made of subcutaneous tumor-bearing nude mice model of prostate cancer and the comparison of treatment results, to further study the Cellular specificity of shRNA transcription driven by PSMAe/p to silence NS gene expression and the effect of gene silencing on the NS; to further explore the gene transfer capability of Tf-PEG-PEI in vivo.Method:Modify the Non-viral vector polyethylenimine (PEI) into the Tf-PEI-PEG; bacterial transformation, amplification, extraction and purification of plasmid were made on the two plasmids pPSMAe/p-UPRT, PSMAe/p- shNS-poly (A), we have the restriction enzyme digestion and sequencing to ensure that the sequence is correct;Culturing the PC-3 cell that do not express PSMA and the LNCap cell that express PSMA, LNCap cells and PC-3 cells were inoculated into nude mice to establish xenograft tumor model. Xenograft tumors were injected with recombinant plasmid, investigating the volume of tumors during therapy and measuring the final weight of tumors, Immunohistochemistry was used to detect the expression of NS in each group. Western blot was used to detect the expression of NS in each group. To further demonstrate the cellular specificity of shRNA transcription targeting gene driven by PSMAe/p and the high gene transfer ability of Tf-PEG-PEI. Provid experimental foundation and theoretical basis for the treatment of prostate cance.Result:In the nude mice LNCap cells xenograft tumor model, the tumor volume was calculated and the tumor growth curves was drawn. In the course of treatment, the tumor volume of the interference group which used the PEI or Tf-PEG-PEI as the carrier reduced significantly, there was statistical significance between the interference group and the other three groups in the tumor volume (P<0.05), especially in the first cycle of the treatment the tumor grow more slowly. The larger the tumor, the worse the treatment. Significance was found in the tumor volume between the PEI group and Tf-PEG-PEI group (P<0.05). There is no significant difference in the tumor volume among the other three groups (P>0.05). In the nude mice PC3 cells xenograft model, no difference was detected among the five groups in the tumor volume (P>0.05). After treatment, the nude mice were killed and the tumor tissue was taken out and weighted. In the nude mice LNCap cells xenograft tumor model, the weight of tumor tissue of the interference group which used the PEI or Tf-PEG-PEI as the carrier was significantly lighter, there was statistical significance between the interference group and the other three groups in the tumor volume (P<0.05). There was statistical significance in the weight of tumor tissue between PEI group and Tf-PEG-PEI group (P<0.05). The other three groups had no significant difference in the weight of tumor tissue (P>0.05). In the nude mice PC3 cells xenograft model, there was no difference in the weight of tumor tissue among the five groups (P>0.05). In the nude mice LNCap cells xenograft tumor model, the tumor tissue underwent NS immunohistochemical staining, the interference group which used the PEI or Tf-PEG-PEI as the carrier compared with the other three groups, the NS staining positive rate significantly decreased (P<0.01), and the degree of nucleus and cytoplasm staining was weaker. PEI group and Tf-PEG-PEI group had significant difference in the NS staining positive rate (P<0.05). There was no difference among the rest groups in the NS staining positive rate (P>0.05). NS gene expression was detected by western blot, the interference group which used the PEI or Tf-PEG-PEI as the carrier compared with the other three groups, NS expression was decreased significantly, there was statistical significance between the interference group and the other three groups (P<0.05). There was difference in the NS expression between the PEI group and Tf-PEG-PEI group (P<0.05). There was no difference among the rest groups in the NS expression (P>0.05). In the nude mice PC3 cells xenograft model, no difference was detected among the five groups in the NS expression (P>0.05).Conclusion:Tf-PEG-PEI has a high capacity of targeted gene transfer ability, it can transfecte vitro gene into the prostate cancer tissue from Prostate cancer tumor model in nude mice efficiently, and modified by PEG greatly reduced its toxicity, is a safe, efficient RNA interference delivery systems. PSMAe/p can drive shRNA' expression specialy in LNCap cells which express PSMA, but can not in the PC-3 cells. The shRNA transcription targeting NS gene driven by PSMAe/p has cellular specificity.Utilizing cell specific promoter is a effective strategy for targeting gene therapy in prostate cancer.