Study Of Short-Hairpin RNA Targeting Nucleostemin Driven By PSMA Promoter And Enhancer In Prostate Cancer Therapy

Partâ… Construction and verification of shRNA recombinant plasmid targeting interference EGFPObjective Utilizing three different termination signals to construct three eukaryotic expression recombinant plasmids targeting interference EGFP(enhanced green fluorescent protein)which are driven and regulated by prostate specific membrane antigen promoter and enhancer(PSMAe/p).Methods The three designed interference sequences targeting EGFP using poly(A)which is the termination signal of plasmid pSilencer^TM4.1-CMV neo,minipoly(A)(AAUAAA hexamer), poly(U)(TTTTTT)as termination signals respectively were cloned into the pPSMAe/p vector,which was ligated after Salâ… and BamHâ… digestion.Poly(A)and minipoly(A)termination signals belong to RNA polymeraseâ…¡promoter,whereas poly(U)belongs to RNA polymeraseâ…¢promoter.The recombinant plasmids were verified by PCR of the bacterium liquids,restriction enzyme digestion and gene sequencing.Results The artificial DNA sequences were successfully inserted into the pPSMAe/p carrier respectively.The enzyme digesting spectra and sequencing results of the recombinant plasmids were correct.Conclusion The eukaryotic expression recombinant plasmids utilizing different termination signals targeting EGFP:pPSMAe/p-shEGFP-poly(A),pPSMAe/p-shEGFP-minipoly(A),pPSMAe/p-shEGFP-poly(U) were successfully constructed.Partâ…¡Study of Cell specificity of shRNA targeting EGFP driven by PSMA enhancer and promoterObjective To study the effects and cell specificity of prostate specific membrane antigen promoter and enhancer(PSMAe/p)in driving short hairpin RNA (shRNA)transcription and explore which terminator signal is more effective. in human prostate cancer cell lines LNCaP and PC-3.The designed shRNA sequence targeting NS with poly(A)termination signal was cloned into the pPSMAe/p -shEGFP-poly(A)vector,which was ligated after Sal I and BamH I digestion. Recombinant plasmid pPSMAe/p-shNS-poly(A)was transfected into human prostate cell lines LNCaP and PC-3 with Lipofectamine^TM2000.The mRNA and protein levels of NS were detected by semi-quantitative RT-PCR and Western blot.The changes of cell morphology,cell cycle,proliferation ability and apoptosis were also studied after down-regulating the NS gene level.LNCaP 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.Results Both LNCaP cells and PC-3 cells express NS protein.We can determine the successful construction of recombinant plasmid pPSMAe/p-shNS-poly(A)by enzyme digestion and sequence analysis.The expression level of NS gene in LNCaP cells was downregulated,cells became larger and showed more pseudopodia,having a tendency to differentiate,the detection of cell cycle showed a decrease of S stage and an increase of G1 stage,cell proliferation ability in vitro and tumorigenesis ability in nude mice were discounted,the tumor volume and final weight were also decreased after knocking down NS gene.The downregulation of NS gene expression level wasn't conspicuous in PC-3 cells,cell cycle and cell proliferation ability didn't change obviously.Conclusion 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.NS may act as an important G1/S regulator to regulate the malignant proliferation of LNCaP cells.NS gene may serve as an ideal therapeutic target for prostate cancer.