DNA修复蛋白RAD51和MSH2的合成致死作用研究

Synthetic lethality is an important direction in cancer-drug development. Two genes are said to be in a synthetic lethal relationship if a mutation in either gene alone is not lethal but mutations in both cause the death of the cell. Such a gene can then be the target for developing an anticancer drug, and these drugs could represent a non-toxic, tumor-selective treatment for cancers. Two papers published together in 2005 first showed how synthetic lethality could be applied to cells that are deficient in a DNA-repair pathway. Mutations in the DNA mismatch repair protein MSH2 are common in human tumor cells. So it will be really meaningful for the treatment of MSH2 deficiency tumor if we can find the paired gene of synthetic lethal. Reference has been reported that methotrexate is selectively lethal to tumour cells with defects in the DNA mismatch repair gene MSH2, we found in our pre-experimental study that methotrexate can affect the expression of homology recombination repair protein RAD51, and RAD51 overexpression can reverse the lethal effects of methotrexate. So, whether RAD51 and MSH2 is a lethal pair of defective DNA repair synthesis genes to improve the targeting of MSH2 deficiency oncology treatment is of important significance. This project, using in vitro and vivo experiments, will study the synthetic lethal interactions between RAD51 and MSH2 as well as the effect of RAD51 on the adaptation of homology recombination repair pathway after MSH2 mutation or deficiency. Through this study, we hope to provide new insights into the treatment of MSH2 deficiency oncology, and facilitate the effective use of RAD51 inhibitors.

"合成致死"机制是抗癌药物研发的方向之一，寻找DNA修复缺陷的合成致死配对基因对提高临床肿瘤治疗的靶向性有重要意义。文献报道氨甲喋呤对DNA错配修复基因MSH2缺陷肿瘤具有特异的杀伤作用。本项目前期工作发现，氨甲喋呤可抑制肿瘤细胞同源重组修复蛋白RAD51的表达,且RAD51的高表达可以挽救氨甲喋呤对MSH2缺陷肿瘤细胞的杀伤作用。因此，RAD51和MSH2是否为合成致死的配对基因，是本课题研究的焦点。本项目拟通过观察抑制RAD51对MSH2缺陷肿瘤的杀伤作用来阐明RAD51和MSH2间的合成致死关系，并探讨RAD51在MSH2缺陷引起的同源重组修复功能改变中的代偿作用。通过本研究为MSH2缺陷肿瘤的治疗提供新思路，为临床合理有效应用RAD51抑制剂提供帮助。

MSH2功能缺陷在很多肿瘤中被发现，尤其在结直肠肿瘤中多见。结直肠癌最常用的是以5-FU为基础药物的化疗方案，但对于有MSH2缺陷的结直肠癌患者，该方案的治疗效果则较差。因此，现急需优化现有的MSH2缺陷肿瘤的治疗方法。合成致死是癌症药物研发的重要方向，该方向研究的药物能够在不损伤正常组织的同时，靶向杀伤肿瘤细胞。而本项目前期发现氨甲喋呤可抑制肿瘤细胞同源重组修复蛋白RAD51的表达,且RAD51的高表达可以挽救氨甲喋呤对MSH2缺陷肿瘤细胞的杀伤作用，而文献报道氨甲喋呤对DNA错配修复基因MSH2缺陷肿瘤具有特异的杀伤作用。 .因此，本项目通过观察抑制RAD51对MSH2缺陷肿瘤的杀伤作用来阐明RAD51和MSH2间的合成致死关系，并探讨这种合成致死作用可能的机制。结果表明1)　结肠癌细胞系MSH2表达水平与其对RAD51抑制剂RI－1的敏感性呈负相关。2)　 抑制RAD51对结肠癌细胞的杀伤作用受细胞中MSH2表达水平的影响，二者存在合成致死作用。3)RAD51高表达对RAD51/MSH2合成致死作用有挽救效应。4)MSH2与RAD51的表达水平间无明显相关性。5）MSH2缺陷肿瘤细胞的DNA修复能力减弱，但不改变其HR修复能力。6）MSH2以不依赖于RAD51的方式参与DSB修复.本研究证实RAD51和MSH2是合成致死的配对基因，因此采用RAD51抑制剂可靶向杀伤MSH2缺陷或低表达的肿瘤细胞，有较好的应用前景。近年连续报道多种可下调RAD51表达的药物如emodin,gefitinib和DIDS等，本研究结果也可为指导本类药物的合理有效应用提供参考。