喉癌中PIN1基因过表达导致中心体扩增机制的研究

Our previous study suggested that laryngeal squamous cell cancer was closely related to centrosome amplification induced by abnormal expression of PIN1and CDK2 gene. In this project, NF-κB element on CDK2 promoter will be identified and the promoter-binding activity of NF-κb to CDK2 promoter will be detected. CDK2 induced by NF-κb which activated byPIN1 was analyzed to explore the centrosome amplification mechanism. Establishment of cell lines with different centrosome number (2-polar and multipolar spindle) by Subcloning Hep-2 cell line, as well as in HEK293 cell line (2N) to research PIN1 expression, centrosome amplification, cell division status, tumorigenicity in nude mice, and so on, which reveals the mechanism of centrosome amplification, chromosome spindle formation, error isolation, multipolar spindle cell division during human laryngeal carcinoma cells mitosis and Elucidates the regulating model of malignant transformation of cells. Microarray will be applied to screen specific miRNAs associating with centrosome amplification in subclone cell and discover the specific miRNAs. In vitro and in vivo experiments will be used to illustrate centrosome amplification mechanism regulated by miRNAs and provide a theoretical and experimental basis for targeting gene therapy for laryngeal carcinoma.

前期工作发现PIN1 、CDK2基因异常表达引起中心体扩增、染色体异倍体与喉癌发生密切相关。拟鉴定CDK2启动子区NF-κB反应元件，并检测NF-κB与CDK2启动子结合活性，探索PIN1通过激活NF-κB结合CDK2基因启动子持续表达引起中心体扩增的可能途径。亚克隆Hep-2细胞系建立含有不同中心体数目的亚克隆细胞系（2极和多极纺锤体），以及诱导HEK293细胞系（2N）成为2极纺锤体的异倍体细胞克隆，研究PIN1基因表达、中心体扩增、细胞分裂状态、裸鼠中致瘤能力等。从而揭示喉癌细胞有丝分裂过程中中心体扩增、纺锤体形成、染色体错误分离、多极纺锤体细胞分裂机制及细胞恶性转化的调节机制。利用Microarray筛查亚克隆细胞中心体扩增特异性miRNAs；发现与喉癌中心体扩增特异的miRNAs，通过体内、外实验阐明miRNAs参与调节中心体扩增机制。为喉癌靶基因治疗提供理论基础和实验依据。