Expression Of Smac Gene Under The Control Of Uroplakin Ib Promoter And Its Promoting-Apoptosis Effect On Target Bladder Cancer Cells

Part 1Detection of the expression of second mitochondria derived activator of caspase in transitional cell cancer of bladderObjective To detect the expression of second mitochondria-derived activator of caspase (Smac) in transitional cell cancer of bladder (TCC) and discuss its significance. Methods Smac was detected in 15 specimens of normal bladder epithelium and 72 specimens of TCC by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry at the level of gene and protein, respectively. Results Both the differences of Smac protein and mRNA expressions between normal mucous membrane of bladder and gradeâ… TCC had no statistical significance(P>0.05). The expressions of Smac protein and mRNA in TCC decreased gradually and significantly with the increase of grade of TCC (P<0.01 and P<0.05, respectively). In invasive TCC, the expressions of Smac protein and mRNA were higher than those of in superficial TCC (P<0.01). Conclusions Normal bladder epithelium has high expression of Smac while TCC has low expression of Smac. The expression of Smac is closely related to the grade and stage of TCC.Part 2The study of tissue specificity of Chinese Uroplakin Ib and cloning and identification of its promoterObjectives To study the tissue specificity of Chinese uroplakin Ib and clone and identify its promoter. Methods The expression of uroplakin Ib of 32 specimens of bladder cancer, 16 of normal mucous membrane of bladder , 15 of normal mucous membrane of small intestine, 16 of normal mucous membrane of esophagus, 19 of normal parenchyma of kidney, 20 of normal parenchyma of liver, 8 of normal skin and 8 of normal cardiac muscle were detected with RT-PCR. Genomic DNA was extracted from normal mucous membrane of bladder of human. Uroplakin Ib promoter was cloned and identified with electrophoresis on agarose and sequencing. Results The expression of uroplakin Ib was detected from all specimens of normal mucous membrane of bladder, 15 of 16 of grade I bladder cancer(93.8%), 7 of 9 of gradeâ…¡bladder cancer(77.8%), 4 of 7 of gradeâ…¢bladder cancer(57.1%). There were no expression of uroplakin Ib in the specimens of normal mucous membrane of esophagus and small intestine, parenchyma of kidney and liver, skin and cardiac muscle. The Chinese uroplakin Ib promoter (234bp) was cloned successfully. Conclusions Chinese uroplakin Ib has high urothelium specificity. The uroplakin Ib promoter, a short promoter, is fit for the research of target-directional gene therapy of bladder cancer well. We have cloned Chinese uroplakin Ib promoter successfully,which lay the foundation of subsequent research. Part 3Construction of eukaryotic expression vector carrying Smac gene under control of human Uroplakinâ… b promoterObjective To construct of eukaryotic expression vector carrying Smac gene under control of human Uroplakinâ… b promoter. Methods Internal CMV and T7 promoters were deleted from eukaryotic expression vector pcDNA3.1-Smac and displaced by human Uroplakinâ… b promoter. New plasmid was sent for sequence analysis. The plasmid DNA was digested with two enzymses at two sites, and compared with standard markers in gel electrophoresis. The plasmid was sent for sequence analysis also. Results The eukaryotic expression vector pcDNA3.1-UpIb promoter-Smac carrying Smac gene under control of human Uroplakinâ… b promoter was constructed successfully. Conclusion The new vector is of great significance for bladder cancer-targeted gene therapy.Part 4The bladder cancer-specificity and activity of UpIb promoter's promoting the expression of Smac geneObjective To study the bladder cancer-specificity and activity of UpIb promoter's promoting the expression of Smac gene. Method Smac mRNA and protein were detected in BIU87 cell line and many other non-bladder cancer cell lines before and after transfection with pcDNA3.1-UpIb promoter-Smac including UpIb promoter and Smac gene by RT-PCR and immunohistochemical method respectively. Results The expression of Smac mRNA in BIU87 cell line increased about 2.1 times after transfection . The Smac protein-positive cell ratio of BIU-87 cell line was about 25.6% before transfection and it increased to about 70.5% after transfection. There was no significant difference about the expression of Smac mRNA and protein in other non-bladder cancer cell lines before and after transfection. Conclusions UpIb promoter could promote the expression of Smac gene with bladder cancer-specificity and considerable activity, which laid the foundation of target gene therapy for bladder cancer.Part 5Transfection of bladder cancer cells with pcDNA3.1-UpIb promoter-Smac and the effectiveness of promoting apoptosisObjectives To transfect bladder cancer cell line with eukaryotic expression plasmid pcDNA3.1-UpIb promoter-Smac carrying UpIb promoter and Smac gene and study the effectiveness of promoting apoptosis. Methods The bladder cancer cell line BIU-87 was transfected with plasmid by liposome. After 24 hours, the expression of Smac was detected by RT-PCR. Low-dose mitocycin C induced the apoptosis of BIU-87 cells. Then the apoptosis was detected by upside-down microscope, Wrighs-Gimesa, DNA agarose gel electrophoresis technique, TUNEL fluorescence-labelled technique and flow cytometry. Results In all groups exposed to mitocycin C, typically morphological features of apoptosis could be detected by upside-down microscope and Wrighs-Gimesa and DNA ladder could be detected by DNA agarose gel electrophoresis. It was detected that the apoptosis rate of BIU-87 cells transfected with pcDNA3.1-UpIb promoter-Smac was significantly higher than that of cells without transfection by TUNEL fluorescence-labelled technique and flow cytometry. Conclusions The transfection of BIU-87 cell with pcDNA3.1-UpIb promoter-Smac could increase the expression of Smac and promote the apoptosis of BIU-87 cells induced by mitocycin C effectively. It provided a new target gene therapy method for bladder cancer that the plasmid and apoptosis-induced drug were applied cooperatively.Part 6The expression of pcDNA3.1-UpIb promoter-Smac and its effect on apoptosis of bladder cancer cells after intratumoral injectionObjective To investigate the expression and effect on apoptosis of tumor cells of intratumoral injection of the mixture of liposome and bladder cancer-special expressing plasmid carrying Smac gene (pcDNA3.1-UpIb promoter-Smac). Methods The mixtures of liposome and pcDNA3.1-UpIb promoter-Smac were injected directly into the human bladder cancer xenografts in nude mice. Then, apoptosis of cancer cells was induced by injection of Mitomycin C into tumors. The expression of Smac mRNA was measured by RT-PCR. The expression of Smac protein was detected by immunohistochemical method. The apoptosis of cancer cells was observed by HE stain, DNA Ladder analysis and TUNEL-Fluorescence method. Results After intratumoral injection of the mixture of liposome and pcDNA3.1-UpIb promoter-Smac, the expression of Smac gene in tumor increased and the ratio of apoptosis induced by Mitomycin C rose. Conclusions The direct injection of the mixture of liposome and pcDNA3.1-UpIb promoter-Smac into human bladder cancer xenograft in nude mice could make Smac gene express in tumor effectively and promote apoptosis of cancer cells notably, which laid the foundation for the clinical application of pcDNA3.1-UpIb promoter-Smac and such gene therapy mode.