| Livin is the latest found IAP family member, containing one BIR do-main and one RING domain, which can potently inhibit both the intrinsic and the extrinsic cell apoptosis. It has been reported that Livin was highly expressed in many type of cancers including melanoma, lung cancer, colon cancer et al, but not expressed in their normal counterparts, and the positive expression of Livin was often correlated with the poor prognosis and resis-tance to chemotherapy. In our previous study, we have found that Livin was highly expressed in bladder cancer tissues, but how dose Livin regulate bladder cancer proliferation; apoptosis and chemosensitivity need further study.Objective:(1) Examining the expression of Livin in bladder cancer cell lines and knockdown its expression by RNAi to observe the effect on bladder cancer cell proliferation, apoptosis and chemosensitivity.(2) To determine whether could Livin, XIAP and Survivin synergistically regulate proliferation, apoptosis and chemosensitivity in bladder cancer. (3) Inves-tigating the optimal parameter of ultrasound destruction of microbubble mediated siRNA transfection to explore a possible gene delivery strategy for bladder cancer gene therapy.Methods:RT-PCR was used to exam the mRNA expression of the two Livin isoforms in bladder cancer cell lines including EJ, T24,5637, and BIU-87. Immunocytochemistry and Western blot assays were performed to determine the Livin protein expression in bladder cancer cell lines. Trypan blue staining and colony formation assay was performed to analyze the ef-fect of Livin knockdown on T24 cell proliferation. TUNEL assay was per-formed to analyze the effect of Livin knockdown on T24 cell sensitivity to proapoptotic stimuli. Combined knockdown of Livin, XIAP, and Survivin was achieved by siRNAs. MTT assay and colony formation assay were performed to analyze the inhibitory effect of combined IAPs knockdown on T24 cell growth. Annexin-V—PI double staining was performed to analyze the effect of combined IAPs knockdown on T24 cell apoptosis. FITC la-beled siRNA was transfected into T24 cells by ultrasound destruction of microbubble. Transfection efficiencies were observed by fluorescence mi-croscope and flow cytometry and the MTT assay was performed to observe the effect on cell viability.Results:Livin gene was highly expressed in the four kinds of bladder cancer cell lines. However, only the Livin-αisoform expressed in bladder cancer cell lines, but not Livin-β, both at the mRNA and the protein level. Silencing Livin expression could significantly inhibit T24 cell proliferation, promote sensitivity to proapoptotic stimuli. Combined knockdown of all three genes (Livin, XIAP and Survivin) synergistically inhibited prolifera-tion and transformation ability, and promoted cell apoptotic sensitivity to chemotherapy in the high grade bladder cancer T24 cells. Furthermore, combined knockdown of Livin, XIAP and Survivin markedly increased the abundance of active Caspase-3, active Caspase-7 and active Caspase-9, as well as increased the abundance of the cytosolic Smac. Ultrasound destruc-tion of microbubble has an obvious effect of promoting gene transfection. However, the efficiency of microbbuble mediated transfection of siRNA to T24 cell is about 30% which is still lower than the traditional liposome mediated gene transfection.Conclusion:Knockdown of Livin expression could significantly in-hibit T24 cell growth ability, promote sensitivity to proapoptotic stimuli. The combined knockdown of the three IAP genes (Livin, XIAP, and Sur-vivin) could greatly increase the spontaneous cell apoptosis and have a synergistic effect on regulation of cell growth and apoptosis. Microbubble mediated siRNA transfection might be a potent gene delivery technique in cancer gene therapy. |
