Study Of Short-Hairpin RNA Targeting Prostate Cancer Antigen-1 Driven By PSMA Promoter/Enhancer In Prostate Cancer Cells

Part I Construction of shRNA recombinant plasmid targeting interference prostate cancer antigen-1Objective To construct eukaryotic expression restructuring plasmid PSMAe/p-shPCA-1 which is regulated by prostate-specific membrane antigen enhancer/promoter.Methods To synthetic sequences of DNA which is corresponding template that is people prostate cancer antigen-1 gene shRNA.Then, The artificial DNA sequences are directionally cloned to pSIREN-RetroQ-ZsGreen which contains BamH I and EcoR I. After that, the artificial DNA sequences are transferred to PSMA enhancer/promoter which is Contains BamH EcoR I double enzyme cutting site. The recombinant plasmids are detected by PCR, restriction enzyme digestion, agarose electrophoresis, and gene sequencing identification.Results The artificial DNA sequences are inserted successfully into the PSMAe/p carrier. Results of double enzyme cut map of restructuring plasmid carrier and targeted sequential accord with expected results.Conclusion We construct successfully eukaryotic plasmid expressing the carrier PSMAe/p-shPCA-1 which is drived by prostate-specific membrane antigen enhancer/promoter. Partâ…¡Study of Cell of shRNA targeting PCA-1 driven by PSMA enhancer/promoteObjective To observe different prostate cancer cells after cell transfection that prostate cancer antigen-1 gene was inhibited, and to explore relationship of PCA-1 genes and prostate cancer progression, malign hyperplasia, transfer infiltration capacity.Methods First by immunohistochemical method, to detect prostate cancer antigen-1 expression in prostate cancer cell lines LNCaP and PC-3; Recombinant plasmid PSMAe/p-shPCA-1 is transfected into human prostate cell lines LNCaP and PC-3 through LipofectamineTM 2000. After that, by cell scratches experiment to observe change of cells migration ability, by MTT method to detect cell proliferation in vitro and by Transwell experiment to detect cells infiltrating ability after interference; Using FCM to change of PCA-1 in different cells after interference.Results LNCaP and PC-3 have PCA-1 protein expression; LNCaP cell migration significantly decreased, cell proliferation ability obviously down-regulated in vitro and infiltrating ability weakens.However, cell migration, proliferation ability and infiltrating ability of PC-3 change obviously.Conclusion The shRNA transcription targeting PCA-1 gene driven by PSMAe/p has cellular specificity. PCA-1 gene plays a very important role in LNCaP cell proliferation, transfer, infiltration of malignant.PCA-1gene may serve as an ideal therapeutic target for prostate cancer.