赖氨酸甲基转移酶SET8调控对肿瘤细胞重离子辐射敏感性的影响及其机制研究

SET8 is a sole lysine methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20me1) reported so far in higher eukaryotes. H4K20me1 is closely related to the regulation of DNA replication, chromosome condensation and the activation of DNA replication checkpoints. Deficiency of SET8 results in massive DNA damage, cell cycle arrest and induction of apoptosis. High LET heavy ion radiation has advantages such as high relative biological effectiveness (RBE), low efficiency of cell damage repair, higher penetration into tissue as well as more precisely and intensely targeted to the tumor cells compared to the traditional low LET ionizing radiation like X-ray and γ-ray, and thus seems to be a potentially more effective radiation therapy. In the proposed project, we are going to knock down SET8 by siRNA transfection in cancer cell and then the cells are irradiated by the high LET carbon ions beam. The cellular survival, cell cycle and apoptosis, etc., will be investigated to assess if there is an additive or synergic effect between SET8 downregulation and heavy ion radiotherapy for cancer therapy. The related signaling pathway mechanisms will be investigated by high through sequence, western blot and DSB repair protein foci confocal in situ to assess the role of SET8 signal pathway in the repair of the clustered DSBs induced by heavy ions.

SET8蛋白是迄今发现唯一能够特异性单甲基化组蛋白H4K20的赖氨酸甲基转移酶，其催化的H4K20单甲基化修饰在DNA复制、染色体固缩及细胞周期检验点激活中发挥重要调控作用。SET8缺失将导致DNA损伤，细胞周期阻滞，甚至发生细胞凋亡。高LET重离子辐射具有相对生物学效应高、组织穿透力强、能量能更准确集中地传送到特定靶点等特点，已成为当前癌症临床放疗中很有应用前景的一项技术。本项目研究拟通过SET8-siRNA敲低技术处理肿瘤细胞，联合重离子辐射处理，检测细胞存活、细胞周期阻滞、细胞凋亡等生物学终点，分析辐射增敏效果，探讨将SET8生物学调控功能与重离子辐照放疗优势进行结合用于临床治疗的可能性；采用高通量测序、western blot分析、DSB位点相关修复蛋白共聚焦成像等方法，检测分析相关信号传导机理，研判SET8相关信号通路在重离子诱导的DNA团簇损伤修复中的作用。

SET8蛋白是迄今发现唯一能够特异性单甲基化组蛋白H4K20的赖氨酸甲基转移酶，其催化的H4K20单甲基化修饰在DNA复制、染色体固缩及细胞周期检验点激活中发挥重要调控作用。SET8缺失将导致DNA损伤，细胞周期阻滞，甚至发生细胞凋亡。SET8异常表达对辐射效应的影响及机理研究，目前还未有报道。我们的研究显示：细胞内SET8响应辐射应答，低LET的X射线与高LET的碳离子辐射均可诱导SET8表达水平在辐照后4至48 h发生下调。SET8低表达增加了肿瘤细胞辐射敏感性；SET8高表达，肿瘤细胞的辐射抗性增加。. 差异基因表达谱分析显示，RNF8在SET8敲低前后表达差异明显，RNF8作为E3泛素连接酶广泛参与DNA修复过程，并影响DNA损伤位点处53BP1的召募；此外，与细胞周期和凋亡相关基因BARD1、PAK6和CDK10等在SET8敲低前后表达差异亦明显。SET8敲低后细胞损伤修复关键基因表达下降；SET8过表达后细胞损伤修复关键基因表达升高。为进一步探究SET8通过影响DNA损伤修复基因表达和功能来调控肿瘤细胞辐射敏感性的内在机理，我们分别对H4K20me1和H4K20me0进行特异性Pull-down实验，并通过蛋白质谱检测和数据库GO分析，发现A549细胞中多个DNA损伤修复基因能够与H4K20单甲基化的组蛋白H4结合。我们下一步的工作将聚焦于这些基因与H4K20单甲基化相互作用对DNA损伤修复及辐射增敏影响的机制研究。. 我们的研究结果表明，SET8是一个放疗的潜在辅助增敏靶点；本项目的研究促进了有关组蛋白修饰在辐射损伤生物效应上的作用理解。