泛素特异肽酶22基因通过Myc调控人膀胱癌EJ细胞增殖的机制研究

Silencing ubiquitin specific peptidase 22 inhibits the cell proliferation of human bladder cancer EJ cells via down-regulated Myc

目的 研究非对称小干扰RNA（asymmetric structure of interfering RNA,aiRNA）沉默泛素特异肽酶22（ubiquitin specficpeptidase 22,USP22）基因对膀胱癌EJ细胞增殖的影响及其相关机制.方法 2010年8月至2012年3月,我们设计并合成USP22 aiRNA及阴性对照aiRNA（NC-aiRNA） USP22 aiRNA序列结构包括长度分别为15 bp和21 bp的正义链和反义链,将此aiRNA命名为USP22 aiRNA（15/21）,利用脂质体转染膀胱癌EJ细胞.实验分3组：对照组、NC-aiRNA组和USP22 aiRNA（15/21）组转染后48 h,通过逆转录-聚合酶链反应（RT-PCR）和Western印迹法分别检测USP22、Myc、cyclin D1和cyclin E1基因mRNA和蛋白表达水平,噻唑蓝（MTT）法检测EJ细胞的增殖活性,荧光素酶报告基因检测系统检测Myc启动子活性 利用染色质免疫共沉淀法（chromatin immunoprecipitation,ChIP）分析沉默USP22基因后转录因子Sp1与Myc基因启动子的结合情况结果 与对照组相比,转染USP22 aiRNA（15/21） 48 h后EJ细胞中的USP22、Myc、cyclin D1和cyclin E1的mRNA和蛋白表达水平分别下调了（87.4±5.2）％和（91.2±7.3）％、（69.2±4.3）％和（61.0±6.6）％、（78.5±4.1）和（67.4± 5.5）％、（81.0±5.5）％和（78.3±4.0）％（P＜0.05）.MTT结果显示,USP22aiRNA（15/21）组EJ细胞的增殖活性明显受到抑制,在24、48、72和96h4个时间点的细胞生长活性分别为（85.4±5.7）％、（71.3±8.4）％、（52.5±6.7）％和（45.8±6.4）％（P＜0.05）.荧光素酶报告基因检测结果显示,USP22 aiRNA（15/21）组Myc启动子的转录活性下调（65.5±4.2）％（P＜0.05）.ChIP结果进一步显示,沉默USP22抑制了转录因子Sp1与Myc启动子的结合,USP22 aiRNA（15/21）组Sp1与Myc启动子的结合能力相比对照组下调了（48.0±3.2）％（P＜0.05）.结论 USP22 aiRNA（15/21）可能通过抑制Myc基因启动子与转录因子Sp1的结合,下调Myc的表达,从而抑制膀胱癌EJ细胞的增殖.

Objective To investigate the role of silencing USP22 gene by aiRNA on the cell proliferation in human bladder cancer EJ cells.Methods USP22 aiRNA and NC-aiRNA were designed and synthesized.The structure of USP22 aiRNA had 15 bp sense sequence and 21 bp antisense sequence and was named USP22 aiRNA （15/21）.The EJ cells were transfected with USP22 aiRNA （15/21） or NC-aiRNA for 48 h.The mRNA and protein levels of USP22,Myc,cyclin D1 and cyclin E1 were measured by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot,respectively.The proliferation rate of EJ cells was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-Diphenyl tetrazolium bromide （MTT） assay.The transcriptional activity of Myc gene was determined by luciferase reporter gene assay.The ability of Sp1 binding to Myc promoter was tested by a ChIP assay.Results Compared with control group,the mRNA and protein levels of USP22,Myc,cyclin D1 and cyclin E1 in USP22 aiRNA （15/21） group were reduced by （87.4±5.2）％ and （91.2±7.3）％,（69.2±4.3）％ and （61.0±6.6）％,（78.5±4.1）％ and （67.4±5.5） ％,（81.0±5.5）％ and （78.3 4.0）％ （P＜0.05）.MTT assay demonstrated that the cell viability at 24,48,72,96 h in the USP22 aiRNA （15/21） group was （85.4±5.7）％,（71.3±8.4）％,（52.5±6.7）％ and （45.8±6.4） ％ （P＜0.05） and luc,iferase reporter gene assay showed that the transcriptional activity of Myc in USP22 aiRNA （15/21） group was decreased by （65.5±4.2） ％ （P＜0.05）.Moreover,ChIP assay confirmed that the ability of Sp1 binding to the Myc promoter in USP22 aiRNA （15/21） group was inhibited by （48.0±3.2） ％ in comparison with control group （P＜0.05）.Conclusions These results suggested that silencing USP22 inhibited the EJ cells proliferation by blocking Sp1 binding to the Myc promoter.