| Part I Expression of Survivin in bladder cancer cell linesObjective: To detect the expression of Survivin gene and protein in several bladder cell lines, preparing for the next part of experiments.Methods: Three bladder cancer cell lines T24, SCaBER and 5637, were cultured in vitro. The expression of Survivin mRNA was assessed by RT-PCR, and its protein expression was determined by Western-blot. The results were compared with that of 5637 cell line to determine which cell line can be used in the next part of experiments. Tnat is, 5637 cell line was the positive control.Results: ①RT-PCR: The Survivin/β-actin ratio was used to represent the expression of Survivin mRNA. The ratio of T24, SCaBER and 5637 cell lines were 0.747±0.274,0.599±0.129 and 0.699±0.184, respectively. All the results had no significant difference, P>0.05.Compared with 5637 cell line, T24 and SCaBER also had high expression of Survivin mRNA. ②Western-blot: The Survivin/β-actin ratio was used to represent the expression of Survivin protein. The ratio of T24, SCaBER and 5637 cell lines were 0.799±0.121,0.429±0.053 and 0.769±0.051 ,respectively.The results between T24 and 5637 had no significant difference, P>0.05.The one between SCaBER and 5637 had significant difference, P<0.05.It means that the expression of Survivin protein of T24 was as high as that of 5637 and the protein expression of SCaBER was about 55.8% compared with that of 5637 cell.Conclusions: Compared with 5637 cell line, T24 cell line had also high expression of Survivin mRNA and protein; SCaBER cell line had high expression of Survivin mRNA but lower protein expression. The results could hint some possible biological behaviors of the two cell lines.They both can be used in the next part of the experiments.Part II SiRNA inhibits expression of Survivin in bladder cancer cell lines and construction of Survivin-siRNA plasmid vectorObjective: To pick out the siRNA which can most effectively inhibit expressionof Survivin in T24 and SCaBER bladder cancer cell lines and then construct its plasmid vector.Methods: 6 siRNA were chemical synthesized: 4 of them were used to inhibit Survivin expression of T24 and SCaBER cell lines, the other two were non-silencing siRNA and FITC-siRNA. They were all transfected into T24 and SCaBER cell lines, respectively. Survivin mRNA were detected 48h after transfection by RT-PCR and Survivin protein were done by Western-blot 72h later. The most effective siRNA can be picked out by this means and then construct the Survivin-siRNA plasmid vector. The vector was transfected in the same way to know the transfection rate and if it was poisonous to the cell.Results: ①The transfection rate of siRNA were high enough in both T24 and SCaBER cell lines and it was even higher in T24.②Results of RT-PCR:The NO.1 siRNA was the most effective siRNA in T24 cell line and the inhibiting rate was over 60%.The NO.3 siRNA was the most effective siRNA in SCaBER cell line but the inhibiting rate was less than 40%. All the results had significant difference, P< 0.05.③Results of Western-blot: The NO.1 siRNA was the most effective siRNA in T24 cell line and the inhibiting rate was about 60%. All the results had significant difference, P<0.05 .None of the siRNA could inhibit Surviving protein effectively in SCaBER cell line. All the results had no significant difference, P>0.05.④In another Western-blot experiment, Survivin protein were detected continuously lday,2days,3days,4days,5days,6days and 7days after transfection. The most effective inhibition "time window" was 48-96h after transfection. The inhibiting rate was even higher than 80%. All the results had significant difference, P<0.05.⑤The NO.1 siRNA gene sequence was used to construct Survivin-siRNA plasmid vector pGC silencer? U6/Neo/GFP/RNAi and the negative control plasmid pGC silencer? U6/Neo/GFP/NON RNAi was constructed at the same time. They could be transfercted into T24 cell line. The transferction rates were high enough and the negative control vector was atoxic to the cell.Conclusions: ①The NO.1 siRNA can inhibit Survivin expression most effectively in T24 cell line. ②None of the siRNAs can inhibit Survivin expression effectively in SCaBER cell line. ③The plasmid vectors pGC silencer? U6/Neo/GFP/RNAi and the negative control plasmid vectors pGC silencer? U6/Neo/GFP/NON RNAi could be transfercted into T24 cell line and the negative control vector was atoxic to the cell.Part III Inhibition of Survivin to suppress T24 cell line proliferation and invasion using Survivin-siRNA techniqueObjective: To observe the influence of siRNA on growth, viability, apoptosis, cell cycle and invasion ability of T24 cell line and then to explore the mechanism. Methods: Experiments included interference group(Survivin-siRNA added), negative control group(non-slience siRNA added) , blank control group( no siRNA added) and FITC group(FITC-siRNA added). Cell viability were determined by MTT assay. Apoptosis and cell cycle was assessed by nuclear staining with hoechst 33258 and flow cytometry. The migration and invasion ability of T24 cell were detected by erasion trace test and transwell chemoinvasion assay.Results: ?Compared with control groups, the cell viability of interference group depressed 72h after siRNA transfected and was 40～50% lower 6 days later. It confirmed that the siRNA could inhibit Survivin gene effectively. All the results had significant difference, P<0.05. ①Nuclear staining with hoechst 33258 showed cell apoptosis in interference group while control groups showed seldom. But the apoptosis rate was only about 30%. Flow cytometry confirmed apoptosis in interference group but no G2/M arrest could be found. All the results had significant difference, P<0.05.③Compared with control groups, the lineations were much wider in interference group than those of control groups. And the counts of cell penetrating through membrane of Transwell chamber were much less than those of control groups. All the results had significant difference, P<0.05. Conclusions: ①Survivin-siRNA can inhibit T24 cell viability and proliferation obviously. ②Survivin-siRNA can induce T24 cell apoptosis. ③Survivin-siRNA can inhibit the migration and invasion ability of T24 cell line.
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