| Part I Study of the asymmetric structure interfering RNA on ubiquitin specific peptidase22silencing in Bladder cancer EJ cellsObjective:To explore the effect of ubiqutin specific peptidase22asymmetric structure interfering RNA (USP22aiRNA) on efficacy and specific of USP22gene silencing in bladder EJ cells.Methods:Control siRNA and the target sequences of USP22small interfering RNAs (USP22siRNAs) were designed using RNAi algorithm available online. One sequence with the most silencing effect was chosen to design of three different length targeting sequences of USP22aiRNAs. EJ cells were transfected with control siRNA, USP22siRNAs or USP22aiRNAs for indicated times and concentrations, the USP22mRNA transcripts and proteins levels were determined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively, and the effects of the aiRNA-and siRNA-mediated silence of USP22gene expression were compared.Results:The USP22mRNA transcripts and proteins levels were significantly down-regulated by treated with USP22aiRNA in dose-and time-depended manners, as compared with tranditional USP22siRNA, the differences were significant (P<0.05). Moreover, the efficacy and specific of USP22aiRNA(15/21) are better than the traditional USP22siRNA.Conclusions:In contrast to USP22siRNA, USP22aiRNA(15/21) was shown to the same effect to down-regulated USP22gene expression levels. Furthermore, the aiRNA-mediated silence of USP22gene was durable, efficient and reduced nonspecific and off-target effects. The technology of aiRNA-mediated gene silencing may be a novel approach for the gene research. Part II Effect of USP22asymmetric structure interfering RNA against ubiquitin specific peptidase22on cell proliferation and apoptosis of bladder cancer EJ cellsObjective:To investigate the effect USP22asymmetric structure interfering RNA (USP22aiRNA) against ubiquitin specific peptidase22on the cell proliferation and apoptosis of bladder cancer EJ cells.Methods:EJ cells were transfected with USP22siRNA or USP22aiRNA(15/21), respectively. The cell viability was determined using MTT, EdU and plate clone formation assays. The distribution of cell cycle and the rate of cell apoptosis were assessed by flow cytometry.Results:In comparison with USP22siRNA, transfection with USP22aiRNA(15/21) was more potent in inhibiting the proliferation of EJ cells in vitro. Moreover, transfection with USP22aiRNA(15/21) induced higher frequency G1phase cell cycle arrest of EJ cells. However, both USP22siRNA and USP22aiRNA(15/21) did not trigger EJ cell apoptosis.Conclusions:USP22aiRNA(15/21) is more potent in inhibiting human bladder cancer EJ cells proliferation, which may provide a novel base for the design of targeting therapies for bladder tumor. Part Ⅲ Effect of USP22-mediated p53-Mdm2signaling pathway on the proliferation of bladder cancer EJ cellsAims:To investigate the effect of USP22-mediated p53-Mdm2(Mouse double-minute2) pathway on the proliferation of bladder cancer EJ cells.Methods:After transfected with USP22siRNA and USP22aiRNA(15/21) for48h, the mRNA transcripts and proteins levels of p53, p21and cyclin E were determined using qRT-PCR and Western blot, respectively. When EJ cells were co-transfected with p53siRNA and USP22siRNA or USP22aiRNA(15/21) for48h, the cell viability was measured by MTT assays and the distribution of cell cycle was assessed using FCM assays. After transfected with pcDNA-Mdm2, the mRNA transcripts and proteins levels of p53and USP22were detected using qRT-PCR and Western blot, respectively.Results:After silencing the expression of USP22by USP22siRNA and USP22aiRNA(15/21), the mRNA transcripts and proteins levels of p53and p21were up-regulated significantly. Conversely, the expression levels of cyclin E mRNA transcripts and proteins were down-regulated. In comparison with control group, the differences were significant (P<0.05). When EJ cells were transfected with USP22siRNA(15/21), together with p53siRNA, the cell viability and distribution of cell cycle were not significantly different, as compared with control group (P>0.05). However, after transfected with pcDNA-Mdm2, USP22siRNA or USP22aiRNA(15/21)-meidated the up-regulation of p53was blocked.Conclusions:Mdm2-p53signaling pathway is important in the USP22-mediated the regulation of proliferation and cell cycle in bladder cancer EJ cells. USP22-Mdm2-p53signaling pathway may provide a novel method for gene therapy of bladder cancer. Part IV Research of Gambogic acid on TNF-a-induced migration and invasion of human prostate cancer PC3cellsAim:To explore the mechanism underlying the Gambogic acid (GA) inhibits the TNF-a-induced invasion of human prostate cancer PC3cells.Methods:Tumor necrosis factor-α (TNF-α) was used as an invasive inducer to induce migration and invasion of PC3cells, then cells were treated with different concentrations of GA. The inhibitory effects of GA on TNF-a-induced migration and invasion were examined by migration and invasion assays, respectively. The phosphorylation of Akt and GSK-3β were measured by Western blot. The nuclear factor-kappaB (NF-kB) transcriptional activity was tested by luciferase reporter gene assay, and the NF-kB DNA translocation was analyzed by immunofluorescence assay and Western blot. In addition, the DNA binding activity of p65was detected using chromatin immunoprecipitation (ChIP) assays. Moreover, after silencing Snail gene by specific siRNA, the expression levels of invasiveness-associated genes were measured using qRT-PCR and Western-blot, respectively.Results:GA (0.5μM) significantly inhibits TNF-a-induced invasion of PC3cell. The TNF-a-mediated activation of PI3K/Akt and NF-kB was blocked by GA. Moreover, the regulation of Snail is likely involved in the anti-invasion effect of GA. After treatment with GA, the expression levels of Snail were significantly decreased in the TNF-a-stimulated PC3cells.Conclusion:GA inhibits TNF-a-induced PC3cell migration and invasion through inhibition of the NF-kB and PI3K/Akt signaling pathways, which may provide a novel method for therapy of human prostate cancer.
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