Study of short-hairpin RNA targeting EGFP driven specificly by PSMA enhancer/promoter

Objective:To study the effects of prostate specific membrane antigen promoter and enhancer(PSMAe/p) in driving short hairpin RNA(shRNA) transcription and explore which terminator is most effective.Methods:The designed interference sequence targeting EGFP using poly(A),minipoly(A),poly(U) termination signals respectively were cloned into the PSMAe/p vector,which was ligated after SalⅠand BamHⅠdigestion.Than the recombinant plasmids and pEGFP-C1 plasmid were co-transfected into PC-3 and LNCaP cells using Lipofectamine2000.The suppression effect of EGFP was assayed by fluorescence microscope and flow cytometry.mRNA and protein levels of EGFP were detected by RT-PCR and Western blot in each group cells.Results:The recombinant plasmids were constructed successfully.The expression of EGFP was decreased in the LNCaP cells which were co-transfected pEGFP-C1 and each interfering plasmids.In comparison with that of control group,there were significant difference(P0.01).The levels of EGFP in pPSMAe/p-shEGFP-poly(A) group decreased more obviously than that of pPSMAe/p-shEGFP-minipoly(A) group and pPSMAe/p-shEGFP-poly(U) group.In the PC-3 cells,the levels of EGFP had no difference(P0.05).Conclusion:pPSMAe/p-shEGFP expression vectors having different termination signals targeting EGFP are successfully constructed.PSMAe/p can drive shRNA transcription specificly.Poly(A) signal is most effective in three termination signals.