Effects Of Livin Gene Expression Inhibition By SiRNA On Growth And Chemotherapeutic Sensitivity Of Human Bladder Cancer Cell EJ

Objective: Livin gene is the newest member of the inhibitor of apoptosis protein(IAP) family. It does not express in normal human bladder tissue, but in bladder cancer cells with high expression stimulates the proliferation of tumor cells, increase the rate of recurrence and it enables to increase the chemotherapeutic drug resistance of tumor cells. Therefore, it can be a therapeutic target and monitoring indicators of prognosis and recurrence of bladder cancer. This study we research the pathogenesis, provide a theoretical basis and experimental evidence to gene and chemotherapy of bladder cancer, by researching the effects of Livin gene expression inhibition by siRNA on growth and chemotherapeutic sensitivity of human bladder cancer cell EJ. Methods: 1. Watch the human bladder cancer cell EJ cells transfected through different conditions, to optimized conditions and efficiency rating of transfection. 2. Use Liposomal transfection techniques specific synthetic Livin siRNA into human bladder cancer EJ cells. 3. After Livin siRNA transfected in bladder cancer EJ cells 24 hours, use semi-quantitative RT-PCR detect the Livin mRNA expression of each group EJ cells. 4. Western Blot was used to detect the expression of Livin protein of each group EJ cells after transfection 48 hours. 5. Selection of THP acts on transfection and non-transfected cells, CCK-8 method to detect the proliferation inhibition rate of each group cells. 6. Apply flow cytometry to detect the apoptosis of each group cells. Results: 1. Negative control siRNA-FAM was transfected in human bladder cancer EJ cells. There was about 70% transfection efficiency with cell density at 3×105/well, and siRNA 100 pmol/well, Lipofectamine® 2000 reagent 5μl/well in six well play. At the same time in ninety-six well play, with cell density at 4×103/well, and siRNA 5pmol/well, Lipofectamine® 2000 reagent 0.25μl/well. 2. After Livin siRNA transfected in bladder cancer EJ cells 24 hours, the expression of Livin mRNA of the interference group is significantly reduce compared with the blank group and negative control group(p<0.05). 3. The expression of Livin protein slowed down significantly after transfected 48 hours(p<0.05). 4. The group of interference, chemotherapy and combined effect can all inhibit the proliferation of EJ cells with CCK-8. And the combined effect of group on cell proliferation inhibition rate was significantly higher than the group of interference effect alone and chemotherapy alone(p<0.05). 5. The results of flow cytometry showed that the group of interference, chemotherapy and combined effect can all induce apoptosis in bladder cancer EJ cells. And the combined effect group of the apoptosis rate was significantly higher than the group of chemotherapy alone and interference effects alone(p<0.05). Conclusions: 1. Livin gene plays an important role in promoting the development of bladder cancer. 2. Livin siRNA targeting specificity of the experimental design can effectively inhibit the expression of Livin gene in bladder cancer EJ cells. 3. By inhibiting the expression of Livin gene can inhibits the proliferation and induce apoptosis in human bladder cancer EJ cells. 4. The EJ cells transfected siRNA is sensitivity strengthen to chemotherapeutic drugs THP.