Objective： To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib （UpIb） promoter. Methods： For the directionality of Smac expression in the transitional cell carcinoma of bladder, internal CMV and T7 promoter sequences in eukaryotic expression vector pcDNA3.1-Smac were replaced with UpIb promoter to construct a new plasmid. The plasmid DNA was identified by gel electrophoresis after being double digested at respective sites, and then the sequence was analyzed. The expression of Smac mRNA and protein in BIU87 cell line were detected after the transfection by using the newly constructed vector. Results： The Smac gene-carrying and UpIb promoter-regulated eukaryotic expression vector pcDNA3-UpIb-promoter-Smac was successfully constructed. The expression of Smac mRNA was approximately increased by 2.1 times and the expression of Smac protein was increased in about 71% BIU87 cells. Conclusion： The new vector can be effectively expressed in bladder cancer cells and be of great significance for bladder cancer-targeted gene therapy.
Objective： To detect the expression of second mitochondria-derived activator of caspase （Smac） in bladder cancer and discuss its clinical significance. Methods： Smac was detected in 15 specimens of normal bladder epithelium and 72 specimens of bladder cancer by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry at the level of gene and protein, respectively. Results： The differences of both Smac protein and mRNA expressions between normal mucous membrane of bladder and grade i bladder cancer had no statistical significance （ P 〉 0.05 ）. The expressions of Smac protein and its mRNA in bladder cancer decreased gradually with the advance of bladder cancer （ P 〈 0.01 and P 〈 0.05, respectively）. In invasive bladder cancer, the expressions of Smac protein and its mRNA were higher than those in superficial bladder cancer （ P 〈 0.01）. Conclusions： Normal bladder epithelium has high expression of Smac while bladder cancer has low expression of Smac. The expression of Smac is closely related to the grade and stage of bladder cancer. Detection of Smac expression helps to judge the grade and stage of bladder cancer and Smac gene might become a valid target for gene therapy of bladder cancer.
Guiyi Liao Fuqing Zeng Xianghui Yue Liang Wang Fangmin Cheng
The expression of survivin, a member of inhibitor of apoptosis （IAP） family, was examined in bladder transitional cell cancer （BTCC） tissue and adjacent normal tissues to examine its clinical implication in the development of BTCC. Thirty specimens of bladder cancer were detected for the expression of survivin by using immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction （RT-QPCR） in BTCC tissue and adjacent normal tissues. Our results showed that the positive rate of survivin immunostaining specimen were 0 and 60% （18/30） in the adjacent normal tissues, bladder cancer, respectively. The - △ △CT value of survivin in bladder cancer tissue was 10.2829 （9.0034-11.5624） times that in the adjacent normal tissues. The expressions of survivin were correlated with the pathological grades of tumor and clinical stages. It is concluded that there was only weak expression of survivin mRNA in the adjacent normal tissues, but the expression of survivin mRNA in bladder cancer tissue was much higher than that in the adjacent normal tissues and the expression of survivin was correlated with pathological grades and clinical stages of tumor.
