Construction And In Vitro Evaluation Of Tissue-specific Prostate-specific Antigen Promoter/Enhancer-regulated Vectors

Objective: To amplify prostate specific antigen (PSA) promoter/enhancer fragmentswith the PCR method, and construct three specific plasmids of PSAP1-Luc, PSAP2-Luc,and PSAE-Luc. To evaluate the expression feature of vectors harboring PSApromoter/enhancer: screening the highest expression efficiency among the three plasmidsand evaluating whether the PSA promoter/enhancer possess some tissue-specific activity inreporter gene analysis.Methods: According to the PSA5’-flank region sequences (GenBank AccessionNumber U37672.1), three pairs of primers to amplify different lengths of thepromoter/enhancer were designed. PSA enhancer (PSAE) and promoter (PSAP) fragmentswere amplified using the PCR method. The designed PCR products were flanked by therestriction enzyme sites of Sac I and Bgl II that were intentionally added onto the end ofthe two PSA promoters (PSAP1and PSAP2) and the PSA enhancer (PSAE). Two PSApromoters, one PSA enhancer and pTAL-Luc vector, were used to construct three newvectors of PSAP1-Luc, PSAP2-Luc, and PSAE-Luc. These recombinant plasmids wereco-transfected with a β-galactosidase plasmid into three cell lines of PC-3, Hela, andMCF-7, respectively. All transfections were performed at least three times in triplicates.β-Galactosidase levels were measured by Multi-Mode Microplate Readers andluminescence was measured with MicrolumatPlus. The L/G values were calculated bydividing the activities of luciferase by that of β-galactosidase. The expression levels ofβ-galactosidase luciferase were used as a control.Results: Three PCR products were successfully amplified with the three designedprimers. The recombinant plasmids PSAP1-Luc, PSAP2-Luc, and PSAE-Luc weresuccessfully constructed. Prostate cancer cell PC-3expressed a higher luciferase activitythan that of cervical-cancer cell Hela and breast cancer cell MCF-7after being transfectedwith one of the three plasmids (P＜0.05). Among the three plasmids in the three types of cells studied, the highest luciferase level was observed with the PSAP2-Luc transfection (P＜0.01).Conclusion: The present study has demonstrated that all three PSA-related vectorscan possess some tissue-specific activity in the prostate originated cancer cell line of PC-3as compared with other cell lines originated from the other tissues. Among the threepromoters/enhancer evaluated, the PSA promoter2containing620bp showed the highestpromoter activity without compromising tissue-specificity, which may provide theexperimental basis for multi-modal imaging monitoring for the evaluation of the possibilityof treating prostate cancer by PSA promoter-driving TALENs targeting androgen receptorgene.