MIIP基因缺失诱导染色体不稳定性及其在结直肠癌发生发展中的作用研究

Chromosome instability (CIN) is important in the development and progression of colorectal cancer (CRC). However, the mechanism of CIN remains unclear. A hypothesis of this project is raised based on our preliminary bioinformatics analyses and in vitro results. MIIP haploinsufficiency leads to reduced binding between MIIP and Cdc20, increases the activity of APC/C, promotes the degradation of cyclin B1, securin and Topo IIα, which induce CIN and promote CRC development and progression. In this project, an orthotopic CRC mouse model, a MIIP knockout mouse model and tumor tissues from CRC patients will be used to verify the role of MIIP as a tumor suppressor gene in CRC. The binding of MIIP with Cdc20 will be further characterized using site-directed mutagenesis and coimmunoprecipitation assay. The effect of MIIP deletion on degradation of cyclin B1, securin and Topo Iiα and their activity will be tested together with the change in cell cycle and mitosis. With these data, we will illustrate the mechanism through which MIIP controlls CIN which will be further demonstrated in CRC animol models and patient samples using array-Comparative Genomic Hybridization and immunohistochemistry staining assays.

染色体不稳定性（CIN）与结直肠癌的发生发展密切相关，但CIN的调节机制未明。本研究在前期生物信息学分析和体外实验的基础上，提出MIIP基因缺失通过降低与Cdc20的结合而增强APC/C的泛素连接酶活性，促进cyclin B1、securin和Topo IIα的降解,进而引起有丝分裂时机和姐妹染色单体分离的异常，从而诱导CIN，最终促进结直肠癌发生发展的假说。拟通过裸鼠原位移植瘤模型、MIIP基因敲除小鼠模型和临床病例进一步验证MIIP缺失促进结直肠癌的发生发展；利用位点突变和免疫共沉淀技术证实MIIP与Cdc20结合，并检测MIIP缺失对cyclin B1、securin和Topo IIα降解、活性的调控及其对细胞周期和染色体分裂的影响，阐述MIIP缺失调控CIN的机制；通过微阵列-比较基因组杂交和免疫组化等技术在动物模型和临床病例中进一步验证MIIP缺失促进CIN及其调控机制。

本项目应用锌指核酸酶技术在结直肠癌（CRC）细胞中构建了MIIP单拷贝缺失的稳定克隆，并证实MIIP单拷贝缺失导致克隆形成增加、细胞迁移和侵袭能力增强；siRNA实验在两个CRC细胞系中证实MIIP降表达也导致克隆形成增加、细胞迁移和侵袭能力增强。动物实验进一步证实，MIIP单拷贝缺失/降表达促进了CRC的进展。在526例人CRC组织标本中进一步证实，MIIP单拷贝缺失/降表达与结直肠腺瘤-腺癌序列转化密切相关，并与CRC淋巴结、远处转移密切相关。对TCGA CRC数据库的分析显示，MIIP单拷贝缺失与MIIP低表达、淋巴结转移、远处转移密切相关，并与染色体不稳定性（CIN）呈正相关。核型分析和光谱核型分析证实MIIP单拷贝缺失能够诱导CIN。机制研究证实了MIIP与Cdc20 的结合位点，MIIP竞争性抑制APC与Cdc20 结合，抑制下游securin 和cyclin B1的降解，进而调控染色体分裂及细胞周期；并且，MIIP单拷贝缺失降低了拓扑异构酶II的DNA酶切活性，导致不正确的染色体分裂，进而增强CIN。通过本项目，我们首次报道MIIP在CRC中是一个新的潜在的肿瘤抑制基因，为阐述CRC的发生发展机制提供了一个新的方向和实验依据，为治疗CRC提示了新的潜在的靶点；并且，本项目首次报道MIIP是一个CIN抑制基因，为阐述CIN的分子机制提供新的思路，为从CIN干预角度治疗肿瘤提供新的靶点和实验基础；另外，目前多种拓扑异构酶抑制剂用于CRC和其它肿瘤的治疗，本项目的体外实验已经证实MIIP能够影响和反映拓扑异构酶的活性，进一步分析MIIP状态与使用拓扑异构酶抑制剂的患者的疗效、生存之间的关系，将有助于临床选择适合拓扑异构酶抑制剂治疗的患者进行精准治疗。