去泛素化酶与双微体形成之间关系研究

Double minute chromosomes (DMs) is one of hallmarks of gene amplification and has a close association with progression and chemoresistance of tumors in eukaryotic cells. Ubiquitylation, as an important posttranslational modification, is also involved in generation of DMs. However, deubquitylating enzymes (DUBs) regulating ubiquitylated molecules in the process remain to be clarified. To elucidate the fuction of DUBs in generation of DMs, MYC/MYCN induced reporter assay is first used to screen DUBs expression library to find potential DUBs involved in DMs formation. Then several cellular and molecular biologic approachs will be chosen to validate exact DUBs played roles in DMs formation. Next DNA damage repairs related micro-array will be executed to find the signal pathway, in which validated DUBs reguate DMs formation. On the other hand, GST pull-down and proteomics methods will be used to identify the targets protein of interest DUBs. In the end, by mouse model the function of interest DUBs in treatment of clinical tumors will be evaluated. It will identify several DUBs involved in DMs formation, elucidate the association between deubiquitylating modification and generation of DMs, and allow new chemtherapy targets by this project.

双微体作为基因扩增的细胞遗传学标志物，常与肿瘤的恶性进展及耐药性的产生密切相关。泛素化修饰作为细胞内一种重要的翻译后修饰方式同样参与双微体的形成，但是这些重要蛋白的去泛素化调节还有待阐明。为明确去泛素化修饰在双微体形成过程中的作用，本研究利用双微体所携带的MYC/MYCN蛋白所激活的报告基因筛查人去泛素化酶表达文库，进一步通过相应细胞和分子生物学实验确认参与双微体形成的去泛素化酶；同时，利用DNA损伤修复相关micro-array对目的去泛素化酶参与双微体形成信号通路进行检测，并通过GST pull-down和蛋白质组学分析方法确认其作用靶点；最后，利用裸鼠成瘤模型在体内对目的去泛素化酶对双微体形成影响以及作为含双微体肿瘤治疗靶点进行评价。通过本研究将确认参与双微体形成的去泛素化酶，明确去泛素化修饰与双微体形成之间关系，同时还将为含双微体肿瘤治疗提供新的靶点。

双微体作为基因扩增的细胞遗传学标志物，常与肿瘤的恶性进展及耐药性的产生密切相关。泛素化修饰作为细胞内一种重要的翻译后修饰方式同样参与双微体的形成，但是这些重要蛋白的去泛素化调节还有待阐明。为明确去泛素化修饰在双微体形成过程中的作用，本研究利用双微体所携带的MYC/MYCN 蛋白所激活的报告基因筛查人去泛素化酶表达文库，进一步通过相应细胞和分子生物学实验确认参与双微体形成的去泛素化酶；同时，利用DNA 损伤修复相关micro-array 对目的去泛素化酶参与双微体形成信号通路进行检测，并通过GST pull-down 和蛋白质组学分析方法确认其作用靶点；最后，利用裸鼠成瘤模型在体内对目的去泛素化酶对双微体形成影响以及作为含双微体肿瘤治疗靶点进行评价。通过E-box报告基因筛查，我们确认USP13和USP14参与双微体的形成与维持。我们进一步确认USP14在神经母细胞瘤中通过调节MYCN/MYC稳定性发挥癌基因作用；临床肿瘤样本分析显示USP14高表达预示较差的病人预后；更为有意义的是，通过体外及体内实验证实抑制USP14表达不但抑制肿瘤细胞增殖，而且能够增强传统化疗药的细胞毒作用。这提示USP14可以在MYCN/MYC扩增肿瘤中作为新的药物作用靶点。