TRIM24通过调控MRN复合物参与DNA双链断裂修复的机制研究

DNA double-strand breaks (DSBs) are among the most serious types of DNA damage and their signaling and repair is critical for all cells and organisms. The MRE11-RAD50-NBS1 (MRN) complex plays a central role in recognition of the damaged DNA and precisely orchestrated activation of repair signaling pathways. Whereas the precise mechanisms in regulation of MRN complex in response to DSBs remain obscure. Knockdown of tripartite motif-containing 24 (TRIM24) sensitizes human cancer cells to DSBs, which showed decreased survival rate and increased apoptosis upon γ-irradiation or etoposide treatment. In the absence of TRIM24, the activation of DSBs repair signaling initiated by MRN complex was dramatically diminished. In addition, the interactions between TRIM24 and all three components of MRN complex are inducible. These results suggest TRIM24 is a key regulator of MRN complex in response to DSBs. In order to confirm this hypothesis, we will use a variety of approaches of molecular biology and cell biology to investigate the role of TRIM24 in regulation of MRN complex, and the relationship between TRIM24 and radiosensitivity/chemosensitivity of cancer cells. This study will offer new insight into the molecular mechanisms of TRIM24 in DSBs repair, and could possibly lead to novel strategy for anti-cancer drug development base on targeting key factors in DNA damage response pathways.

DNA双链断裂（DSBs）是最为严重的DNA损伤类型，MRE11-RAD50-NBS1（MRN）复合物是介导DSBs修复的关键，然而我们对MRN复合物的调控机制仍知之甚少。我们在预实验中发现TRIM24敲减使肿瘤细胞对于DSBs变得过度敏感。MRN复合物所介导的DSBs修复信号通路在TRIM24缺失的细胞中不能被激活，且DSBs能够诱导TRIM24与MRE11、RAD50和NBS1之间产生相互作用。这些结果提示TRIM24可能通过调控MRN复合物而在DSBs修复过程中起到关键作用。为了证实以上推测，我们将结合多种分子生物学和细胞生物学手段揭示TRIM24调控MRN复合物的分子机制，并进一步探索TRIM24与肿瘤细胞放化疗敏感性之间的关系。本研究将加深我们对DNA损伤修复机制的理解，也将为以DNA损伤修复关键蛋白作为靶点的抗肿瘤药物设计提供新的线索和思路。

在此项目的研究过程中，我们发现缺失TRIM24会导致细胞对DSBs的修复能力减弱，尤其是通过同源重组途径修复DSBs的能力显著下降。TRIM24能够在DSBs产生后被迅速招募至DNA受损位点，通过其羧基端PHD-Bromo结构域直接结合MRE11，并促进MRN复合物的招募。更深入的研究表明，TRIM24在DSBs应答中发挥作用依赖于ATM的激酶活性，ATM对TRIM24第768位和第808位丝氨酸的磷酸化能够促进其在DSBs的招募以及与MRN复合物组成元件产生相互作用，进而介导MRN复合物的招募及下游修复信号通路的激活。更重要的是，我们通过生物信息学方法对TCGA-LIHC数据集进行了分析，发现TRIM24在肝癌中高表达，并且TRIM24表达水平越高则患者生存周期越短。化疗是肝癌的重要治疗方式之一，因此我们推测TRIM24可能是决定肝癌细胞对放化疗敏感与否的关键因素。随后的细胞实验和动物模型证实了我们的推测：TRIM24缺失会导致肝癌细胞中由MRN复合物介导的DSBs修复通路无法被有效激活，进而对Etoposide变得过度敏感；在皮下种植瘤模型中，普通肝癌细胞对Etoposide非常耐受，然而TRIM24敲除的肝癌细胞对Etoposide则十分敏感。综上所述，本项目的结果证明了TRIM24是一个新的DNA损伤应答关键蛋白，更提示了TRIM24是肿瘤治疗的潜在靶点。