生物节律基因CLOCK与大肠癌预后的相关性及分子机制研究

The preventive and therapeutic situation on colorectal cancer in our country is becoming increasingly serious. However, the serum tumor marker for the diagnosis of colorectal cancer is extremely limited. Thus, it is an urgent need to explore more novel tumor markers. Increasing evidences have indicated that CLOCK, a family member of the circadian genes, can control the expression of many so-called clock-controlled genes (CCGs), especially including c-Myc, P21, P53. The CLOCK gene may contribute to the development and progression of many kinds of diseases including cancer, but little work has been focus on its role in colorectal cancer. Two SNPs (rs3749474 and rs1801260) located in the CLOCK gene are significantly associated with colorectal caner overall survival in our earlier studies and the related article has been published in 《Cancer》 in 2012. Based on that, this project will further analysis the association of expression level of CLOCK gene and SNP genotype with prognosis of colorectal cancer patients. We will use siRNA technology to interfere the expression level of the CLOCK gene in colorectal cancer cells, and then its effect on biological activity of tumor cells will be analyzed and the related fold changes of its target gene such as c-Myc, P21, P53 and etc will also be detected. The whole-genome expression microarray and pathway analysis will be conducted to identify the network formed by CLOCK-influenced genes. In the end of this study, an in-depth discussion of the molecular mechanism of CLOCK gene invovled in the progression of colorectal cancer will be conducted to display the experimental evidence and theoretical support for further research on new tumor marker in colorectal cancer.

我国大肠癌防治形势日益严峻，临床用于评估大肠癌预后的血清学指标又极为匮乏，亟待寻找新型分子标志物。目前大量研究已证实生物节律基因CLOCK可通过调控c-Myc、P21、P53等钟控基因的表达，在多种疾病包括肿瘤的发生及进展中发挥重要作用，但在大肠癌领域的研究较少。2012年项目申请者在《Cancer》杂志上首次报道了CLOCK基因rs3749474及rs1801260位点与大肠癌患者预后显著相关。本项目拟在此基础上进一步探讨CLOCK基因蛋白表达水平与SNP基因型及大肠癌患者预后之间的相关性，利用siRNA干涉技术下调大肠癌细胞中CLOCK基因的表达，检测细胞生物学功能改变及c-Myc、P21、P53等钟控基因的表达变化，并通过全基因组表达芯片构建CLOCK基因的分子调控网络，深入探讨CLOCK基因影响大肠癌患者预后的分子作用机制，为大肠癌新型分子标志物的研究提供实验证据及理论支持。

我国大肠癌防治形势日益严峻，临床用于评估大肠癌预后的血清学指标又极为匮乏，亟待寻找新型分子标志物。目前大量研究已证实生物节律基因CLOCK可通过调控C-Myc、P21、P53等钟控基因的表达，在多种疾病包括肿瘤的发生及进展中发挥重要作用，但在大肠癌领域的研究较少。2012年项目申请者在Cancer杂志上首次报道了CLOCK基因rs3749474及rs1801260位点与大肠癌患者预后显著相关。在此基础上本项目完成了550例大肠癌样本CLOCK免疫组化检测及SNP基因型检测，通过相关性分析发现携带rs3749474及rs1801260突变基因型患者的CLOCK表达水平较野生型有下降趋势，在TNM分级较高、分化较差及死亡的大肠癌患者中CLOCK表达水平较高，且较高的CLOCK表达水平与患者较差的预后显著相关。此外我们利用siRNA干涉技术下调大肠癌细胞中CLOCK基因的表达，发现干涉后大肠癌细胞的活力水平、增殖能力、粘附能力和侵袭能力均有所减弱，但细胞凋亡水平增加。干涉CLOCK基因后P21、P53蛋白表达水平升高，C-Myc、Cyclin D1及WEE1表达水平降低。进一步研究将干涉组及对照组大肠癌细胞株通过全基因组表达芯片对基因表达谱进行分析，筛选获得24个差异表达基因，并发现PRKDC、UBC等差异表达基因在AKT、ERK、PI3K和FGFR等重要信号通路中出现富集。本项目的开展深入探讨了CLOCK基因影响大肠癌患者预后的分子作用机制，为大肠癌新型分子标志物的研究提供实验证据及理论支持。