Study Of Cellular Specificity Of Short-hairpin RNA Transcription Driven By PSMAe/p And Gene Transfer Ability Of Tf-PEG-PEI

ObjectiveBased on selection of NS as "target gene",use RNA interference techniques, by creating a non-viral vector system to transfer the interfere plasmid effectively and targetly into the prostate cancer cell in vivo and vitro, so interfere sequence can express specitly in the prostate cancer cell, silencing the specific cancer gene, which provided experimental and theoretical basis for the treatment of prostate cancer.MethodsBy constructing targeting gene transfect non-viral vector Tf-PEG-PEI,using PEI and Tf-PEG-PEI to transfer plasmid pEGFP-C1 and recombinant plasmid targeting interference EGFP which are driven and regulated by prostate specific membrane antigen promoter and enhancer(PSMAe/p) into human prostate cancer cell lines LNCaP which highly express PSMA, then the suppression effect of EGFP was assayed by fluorescence microscope.The mRNA and protein levels of EGFP were detected by real time RT-PCR and Western blot in each group cells.Then use cells which does not express PSMA as comparation,including human prostate cancer cell line PC-3,human bladder cancer cell line T24 and human embryo kidney 293 cell(HEK293).So to demonstrate the cellular specificity of shRNA transcription targeting gene driven by PSMAe/p and the high gene transfer ability of Tf-PEG-PEI. Using shRNA sequence targeting NS to replace the shRNA sequence targeting EGFP of recombinant plasmid pPSMAe/p-shEGFP-poly(A) to construct the new recombinant plasmid,then transfect it into human prostate cancer cell lines LNCaP and PC-3 by Tf-PEG-PEI to detect the changes of cell morphology, cell cycle, proliferation ability,and the expression of NS gene and protein. 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. ResultsFluorescence microscopy showed that:after interference plasmid and pEGFP-C1 plasmid were cotransfected into LNCaP cells, the interference group's expression of fluorescence decreased which use PEI and Tf-PEG-PEI as a carrier respectively, There were significant differences compared with the control group (P<0.01).The expression of EGFP mRNA and protein in interference group compared with the control group were decreased, the difference was statistically significant (P<0.01). After interference plasmid and pEGFP-C1 plasmid were cotransfected into PC-3,T24 and HEK293 cells, there were no significant difference in fluorescence expression between each groups (P>0.05).EGFP mRNA and protein levels were no significantly difference. Compare the groups that transfer the same plasmid using PEI or Tf-PEG-PEI as carrier, there were no significant difference (P> 0.05)on fluorescence, EGFP mRNA expression and EGFP protein expression, but Tf-PEG-PEI group is better in mprphology, survival, slightly higher transfection efficiency. Both prostate cancer cell lines LNCaP and PC-3 express the NS protein. After the interference plasmid pPSMAe/p-shNS-poly (A) was transfected into LNCaP cells, the level of NS gene expression 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 was discounted 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.ConclusionsTf-PEG-PEI has a high capacity of targeted gene transfer ability, it can transfecte vitro gene into cultured cells 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, T24 cells and HEK293 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. 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.