组蛋白H3K9甲基化抑制肿瘤干细胞自我复制的基因调节研究

Selfrenew mechanism is the crucial problem in the stme cell research. Recently, histon modulation acquired attention of most scientists in this field. According to our previous study, inhibition of G9a,the H3K9 methyltransferase,can promote the selfrenew of glioma cancer stem cells, as well as increase the expression of cancer stem cell marker CD133 and Sox2.Therefore we hypothesize that H3K9 methylation may inhibit the cancer stem cell selfrenew via a G9a-H3K9-Sox2 pathway.For this purpose, we planned the following subjects:to further confirm the inhibitory effect of H3K9 methylation on the selfrenew of glioma cancer stem cells;then to study the inhibitory mechanisms of H3K9 methylation on Sox2 and CD133;to investigate the downstream mechanisms of Sox2 on the selfrenew of cancer stem cells; to test if there are any feedback regulaion of Sox2 on the G9a，whether Sox2 can directly binds to the promoter of G9a and whether they have any protein-protein interactions etc.In general, we hope to find evidences for the hypothesis that H3K9 methylation inhibit the selfrenew of cancer stem cells via the G9a-H3K9-Sox2 loop. This study would provide new insights to the cancer stem cell research.

自我复制的调节机制是干细胞研究的核心问题，组蛋白修饰对肿瘤干细胞自我复制的调节是近来关注的热点。我们的前期研究发现，抑制组蛋白H3K9甲基化和G9a（H3K9甲基转移酶）可以促进胶质瘤干细胞的自我复制，并促进CD133/Sox2等干细胞标志分子的表达。我们推测，H3K9甲基化是抑制肿瘤干细胞自我复制的重要机制，通过G9a-H3K9-Sox2通路起作用。为此我们计划：进一步明确H3K9甲基化抑制肿瘤干细胞自我复制的现象和可能的机制；确定H3K9甲基化对CD133/Sox2表达的调节机制；研究Sox2是否对G9a和H3K9有反馈性的调控、是否与G9a有蛋白-蛋白相互作用或直接的转录水平的调控；研究H3K9甲基化与肿瘤干细胞耐药性以及与胶质瘤肿瘤复发间的关系。通过以上实验，阐明H3K9甲基化调节肿瘤干细胞自我复制的机制，为研究肿瘤干细胞的表观遗传学调控提供新的思路和证据。

本项目围绕组蛋白H3K9二甲基化（H3K9me2）修饰对肿瘤干细胞的自我复制的作用展开研究，主要有以下几方面的重要发现：（1）H3K9me2修饰在成球培养的胶质瘤干细胞中呈低表达；（2）组蛋白甲基转移酶G9a对结束肿瘤干细胞的自我复制并走向分化起关键作用，抑制其活性可以维持肿瘤干细胞的自我复制，促进干细胞特性相关基因的表达；（3）G9a和H3K9me2对一系列刺激敏感，例如，癫痫放电、氧化应激、射线、但对TNF等炎症因子不敏感；（4）ChIP-Seq筛选研究发现了一系列与自我复制和分化相关的基因，都受到G9a和H3K9me2调控；（5）ChIP实验证明Sox2 的启动子区和增强子区都受到H3K9me2修饰，并参与自我复制的调控；（6）G9a和H3K9me2对基因的抑制需要另外一个转录因子对其进行招募，它调节肿瘤干细胞自我复制的实验并非每次都完全能重复，可能与它所修饰的下游靶基因的不确定性有关，这促使我们寻找与其合作的另外一个转录因子。