Research Of The Effect Of UPRT/5-FU Suicide Gene System On Prostate Cancer Improved By The Targeted Regulation With Prostate-specific Membrane Antigen Promoter And Enhancer

Objective In this study, it is aim to construct the recombinant expression plasmid pPSMA promoter/enhancer -UPRT which UPRT gene is regulated by PSMA promoter/enhancer.To explore the specially targeted killing role of the pPSMA promoter/enhancer -UPRT/5-FU suicide gene system to prostate cancer.Methods CD PSMA promoter and enhancer DNA sequences are amplified from LNCaP cells by use of polymerase chain reaction (PCR). PSMA promoter and enhancer DNA sequences are cloned into the multiple clone site (MCS) of plasmid pEGFP-1 to construct recombinant plasmid pPSMA promoter /enhancer -EGFP. UPRT gene is amplified from E. coli JM109 by PCR. UPRT gene is cloned into the recombinant expression plasmid pPSMA promoter /enhancer -EGFP to replace the EGFP sequence digested by two restriction enzymes, therefore the expression UPRT is induced by its up-stream prostate-specific membrane antigen promoter and enhancer. (2) It is observed by immunohistochemical assay for PSMA expression in prostate cancer cell linesand non-prostate cancer cell lines. pPSMA promoter/enhancer -EGFP is transfected into several cell lines, including prostate cancer cell lines and non-prostate cancer cell lines by liposome transfection, and the targeted-regulating expression of PSMA promoter/enhancer in the different cells is observed by green fluorescence. pPSMA promoter/enhancer -EGFP and pPSMA promoter/enhancer -UPRT is respectively transfected into LNCaP cell line, to screen the cell line that can express stably with antibiotic G418. ③ pPSMA promoter/enhancer -UPRT is transfected into LNCaP cells, cells survival rate is measured by MTT after acted by the treatment of UPRT/5-FU suicide gene system; cells apoptosis rate and change of cell cycle are measured by FCM. The amount of 5-FU is measured by HPLC to testify the function of UPRT ④ Various percentage of LNCaP cells that include transfected and non-transfected are mixed to observe the bystander effect of suicide gene system UPRT/5-FU.Results ① The recombinant expression plasmid pPSMA promoter/enhancer -EGFP and the recombinant expression plasmid pPSMA promoter/enhancer -UPRT are successfully constructed. Every recombinant plasmid's sequences has been verified without deletion and mutation.②Only prostate cancer androgen-dependent LNCaP cell line express PSMA positive. After the transfection of pPSMA promoter/enhancer-EGFP into various cell lines, there is green fluorescence only in the LNCaP cell line, which indicated that targeted-regulating of PSMA enhancer/promoter is specificity for prostate cancer.③ Cells survival rate measured in vitro by MTT in the group of UPRT/5-FU is lower than the group of 5-FU. IC50 of 5-FU group is 5.5 times than that of UPRT/5-FU group. The cytotoxic effects on LNCaP cells isstronger in UPRT/5-FU group.④ Analyzed by FCM, apoptosis rate of UPRT/5-FU group is 40%~52%, 5-FU group is 26%~28%, the normal cells group is 5%, it is obvious that UPRT/5-FU suicide gene system has high apoptosis rate, When UPRT gene expresses, 5-FU can make toxic metabolites quickly and directly to kill the tumor cells. With the concentration of 5-FU increasing, apoptosis rate is dose dependent.5-FU can influence the cell cycle turning from G1 to S phase.⑤ Extracellular 5-FU concentration measured by HPLC: two groups are decreasing with time going. But after 2h, the 5-FU' concentration of UPRT/5-FU group is lower than that of 5-FU group.It is shown indirectly that 5-FU can produce toxic metabolites quickly under action of UPRT.⑥ With the transfected LNCaP cells becoming more and more , cells survival rate go down gradually. It doesn't show obvious bystander effect after using UPRT/5-FU suicide gene system.Conclusion In this study, We succeed to construct the recombinant expression plasmid pPSMA promoter/enhancer -UPRT by molecular biology skills. Targeted gene -UPRT gene is regulated specifically and expressed by prostate - specific membrane antigen promoter and enhancer in prostate cancer androgen-dependent LNCaP cell line.To confirm UPRT/5-FU suicide gene system by several methods, UPRT can make 5-FU toxic metabolites quickly and directly, so UPRT/5-FU suicide gene system is stronger to kill the LNCaP cells and induce tumor cells apoptosis than 5-FU.