大肠癌中DNA复制蛋白对双微体染色质的复制、损伤和修复的影响及分子机制

Amplification of oncogenes and multidrug resistance genes, either in the form of homogeneously staining regions or in the form of double minute chromososmes, is an important factor which facilitates the malignant transformation of cells and contributes to their tumorigenicity. An important goal of colorectal cancer research is to decrease the number of double minute chromosomes from colorectal cancer cells, thereby decrease the improper amplification of oncogenes and multidrug resistance genes. Even though there are therapeutic advances in the past decade, some subclasses of colorectal cancer are still unresponsive to current therapies; therefore, research is needed for the development of novel therapies for the treatment of colorectal cancer. We propose to investigate the molecular mechanisms of how DNA replication proteins affect the formation and loss of double minute chromosomes in colorectal cancer cells. Using the RNAi technique, we first identify DNA replication proteins in which their knockdown is able to decrease the number of double minute chromosomes in colorectal cancer cell lines. We further confirm candidate proteins by observing the decrease in mRNA and protein expression of oncogenes carried on the double minute chromososmes, and the decrease in the tumorigenicity of the knockdown cell lines compared to parental cell lines. We will further investigate how replication stress caused by the knockdown of the candidate proteins affect the replication of double minutes chromatin, the damage on double minutes chromatin and checkpoint activation, and the repair of the damage. Compared to matched cell lines in which gene amplification occurs on homogeneously staining regions, our study will provide a general framework of the complex relationship between proteins involved in DNA replication and double minute chromosomes; it will also identify proteins which can be targeted and aid the development of future therapeutics for the treatment of double minutes-containing colorectal cancers.

细胞中染色体外双微体（DMs）携带扩增的癌基因及耐药基因是细胞发生恶性转化和成瘤的重要因素。大肠癌研究的一个重要目标是减少或去除细胞内的双微体，从而减少癌基因和耐药基因的扩增及其蛋白的功能。目前大肠癌的多种亚型对现有治疗不敏感，因此，大肠癌的治疗急需新生物靶点的研究。本研究以含有双微体的大肠癌细胞株为研究对象，探讨DNA复制蛋白对双微体形成及丢失的分子机制。运用RNA干扰技术，检测是否DNA复制蛋白的下调会减少大肠癌细胞中的双微体数目，进而检测DNA复制起始/过程低调控对双微体携带癌基因或耐药基因的表达水平，及对细胞成瘤能力的影响，确定候选基因。深入研究因细胞内复制压力对双微体染色质的复制、损伤、细胞周期检查点激活和损伤修复的影响及分子机制。与高扩增基因定位于染色体上均匀染色区的匹配细胞株相比照，我们的研究将提供DNA复制蛋白和双微体关系的总体框架，为将来治疗含双微体的大肠癌确定靶点基因。

细胞中染色体外双微体（DMs）携带扩增的癌基因及耐药基因是细胞发生恶性转化和成瘤的重要因素。大肠癌研究的一个重要目标是减少或去除细胞内的双微体，从而减少癌基因和耐药基因的扩增及其蛋白的功能。目前大肠癌的多种亚型对现有治疗不敏感，因此，大肠癌的治疗急需新生物靶点的研究。本研究以含有双微体的大肠癌细胞株为研究对象，探讨DNA复制蛋白对双微体形成及丢失的分子机制。研究发现DNA复制蛋白Cdc7大肠癌细胞中高表达，在大肠癌COLO320DM和COLO320HSR细胞中，通过运用Cdc7蛋白抑制剂PHA-767491，发现Cdc7抑制后，DNA复制起始/过程低调控使细胞中双微体数目减少，双微体和均质染色区上携带的癌基因扩增水平明显下降，说明Cdc7蛋白与大肠癌细胞中双微体和均质染色区形成相关。进而针对细胞周期检查点TP53蛋白，运用RNA干扰技术沉默后，双微体数目减少，携带癌基因扩增减少，DNA双链断裂得到修复。由此说明DNA复制蛋白Cdc7和细胞周期检查点蛋白TP53活性与肿瘤细胞双微体稳定维持和基因扩增密切相关。本研究发现为将来治疗含双微体的大肠癌确定靶点基因。.经过3年的努力，课题组完成了研究计划，研究发表文章4篇，包括SCI收录文章2篇，国家核心期刊文章2篇。同时，通过研究培养硕士研究生3名和博士研究生1名已经毕业，七年制学生2名在读。