BTG2招募CCR4-NOT复合体在电离辐射后肝癌细胞凋亡/修复通路选择中的作用机理研究

The B-cell translocation gene-2 (BTG2) is initially as one of the ionizing radiation responsive genes and induced by p53,which regulate cell cycle progression and DNA damage repair.however,it is not clear the mechanism of BTG2 makes the choice of the pathway of apoptosis or DNA repair in cells following ionizing radation.The CCR4-NOT complex(CNOT) is an important chaperone-molecular for BTG2,which binds to BTG2 to regulate the expression of a variety of functional protein.We previously data showed the survival fraction in HepG2 cells with overexpression of the BTG2 were significantly lower than that of un-transfected HepG2 cells after ionizing radation.The expression of BTG2 and the CNOT comlex in HepG2 cells was increased after ionizing radation,and the combination of the two proteins was also increased.BTG2 binds to CNOT to regulate cell cycle progression. Accordingly, we propose that the combination of BTG2 and CNOT may be an important mechanism of pathway choice of apoptosis or DNA damage repair. In this study, we evaluated the interaction between these two proteins to make the choice of the pathway of apotosis or DNA repair in different liver cancer cells with different p53 expression,and the interaction between the two proteins was demonstrated by gene knockout,pull-down,CHIP and ROS activation and so on. In conclusion, these findings suggest a plausible mechanism by which BTG2 protects cells against oxidative stress through the engagement of CNOT complex to decide the fate of cancer cells, and provide a theoretical and experimental basis to explain the reason for sensitizing liver cancer cells to radiotherapy.

BTG2是p53诱导的早期反应型辐射敏感基因，具有促凋亡和DNA损伤修复双功能，然目前电离辐射后BTG2参与细胞凋亡/修复通路作用的选择机制尚不明确。CCR4-NOT复合体(CNOT)是介导BTG2功能的重要伴侣分子，其与BTG2结合调控多种功能蛋白的表达。我们前期研究发现电离辐射后转染BTG2的HepG2细胞死亡率显著高于未转染组；电离辐射诱导BTG2与CNOT8表达增高，结合增加；BTG2与CNOT相互结合参与细胞周期的调控。据此我们提出BTG2与CNOT结合可能是细胞凋亡/DNA损伤修复通路选择的重要机制。本课题以不同p53表达状态的肝癌细胞为平台，辅以基因敲除、pull-down、CHIP和ROS激活等方法，研究电离辐射后BTG2与CNOT结合及其在细胞凋亡/修复通路选择中的作用，揭示BTG2在电离辐射早期决定肿瘤细胞命运的分子机制，为诠释肝癌放疗敏感性问题提供理论依据和实验基础。

BTG2 是 p53 诱导的早期反应型辐射敏感基因，具有促凋亡和 DNA 损伤修复双功能，然目前电离辐射后 BTG2 参与细胞凋亡/修复通路作用的选择机制尚不明确。CCR4-NOT复合体(CNOT)是介导 BTG2 功能的重要伴侣分子，其与 BTG2 结合调控多种功能蛋白的表达。本课题前期研究发现BTG2的表达水平影响电离辐射后肝癌细胞的存活，而电离辐射诱导 肝癌细胞中BTG2 与 CNOT 表达增高， 结合增加； BTG2 与 CNOT 相互结合参与细胞周期的调控。本课题在前期研究基础上，拟以不同 p53表达状态的肝癌细胞和成瘤裸鼠为平台，辅以基因敲除、WB、 CHIP 和 ROS 激活等方法，研究电离辐射后BTG2 与 CNOT 结合及其在细胞凋亡/修复通路选择中的作用。本研究结果初步证实：BTG2与CNOT在肝癌细胞中表达具有相关性；BTG2与CNOT可以作用于肝癌细胞的生存、迁移和凋亡；进一步通过裸鼠动物模型，在体观察BTG2增强肝癌细胞移植瘤放疗敏感性的作用。总之，本课题的研究是对前期工作的进一步拓展和延伸，把目前认为对肿瘤放疗敏感性有重要作用的基因BTG2和CCR4复合体联系起来，不仅有助于进一步阐明BTG2基因放疗增敏的机制，同时也有助于探索肝癌放疗抵抗中相关信号通路的选择模式，为解决今后对不同基因状态的临床肝癌患者实施个体化的基因治疗提供理论和实验基础。