XRCC4基因对胶质母细胞瘤放疗敏感性的影响及作用机制

Radiotherapy is one of the leading approaches for glioblastoma multiforme (GBM) treatment, however, the intrinsic radioresistance of tumor cells limits the clinical efficacy of radiation therapy. Therefore, discovering a new appoach to improve the radiosensitivity of GBM is essential to maximally sensitize tumor cells to radiation-induced killing. Our previous study demonstrated that XRCC4, DNA double strands repair gene, was overexpressed in GBM and XRCC4 knockdown by artificial miRNA interference could efficiently sensitize the human GBM cells to ionizing radiation (IR)-induced killing by 2 to 3 times. However, its mechanisms are still unknown.In the present study, we will investigate the effects of the XRCC4 knockdown on the radiosensitization of GBM cells using clonogenic surviving assay and xenograft tumor nude mice model in vitro and in vivo. We will also detect the cell cycle and apoptosis by Flow Cytometry Analysis (FCAS) and Annexin V/PI, DNA double strand breaks by immunofluorescence staining of γH2AX and 53BP1, DNA damage and repair by comet assay, Reactive Oxygen Species (ROS) by DCFH-DA fluorescent probes and chromosomal aberrations changes by chromosome analysis. Furthermore, the protein level of genes involved in cell cycle, apoptosis and DNA repair singaling pathway were evaluated by Western blot and immunohistochemistry in vitro and in vivo.The aim of this project is to determine the mechanism of XRCC4-mediated radiosensitization effects of GBM cells in molecular, cellular, and the overall levels, which will provide new ideas and avenues for the radiothrapeutic of GBM as well as other types of cancers.

胶质母细胞瘤（GBM）内在的抗辐射特性极大地限制了放射治疗的临床疗效，如何提高GBM对放射治疗的敏感性一直是国内外研究的热点。我们前期发现：采用人工miRNA 干扰技术抑制DNA修复基因XRCC4后使胶质瘤的放射敏感性提高了2-3倍。然而，其作用机制尚不清楚。本课题拟通过体外体内实验相结合，采用细胞克隆形成和裸鼠荷瘤模型验证XRCC4对GBM放射敏感性的影响；拟采用流式细胞仪和Annexin V/PI检测细胞周期分布与凋亡率、γH2AX和53BP1荧光染色检测DNA双链断裂、彗星实验检测DNA损伤修复、DCFH-DA探针检测细胞活性氧ROS水平、染色体分析检测染色体畸变；采用Western blot和免疫组织化学染色检测细胞周期、凋亡和DNA修复通路蛋白的表达变化，从分子、细胞和整体水平探讨XRCC4基因影响GBM放射治疗敏感性的作用机制，为GBM及其他类型肿瘤的放射增敏治疗提供指导意义。

胶质母细胞瘤（GBM）内在的抗辐射特性极大地限制了放射治疗的临床疗效，如何提高GBM对放射治疗的敏感性一直是国内外研究的热点。本课题研究证实，amiR-XRCC4可在体外和体内有效抑制XRCC4基因的表达，且使胶质母细胞瘤的放疗杀伤作用增加了2-3倍。抑制XRCC4不改变胶质母细胞瘤的细胞周期和细胞凋亡水平，但明显增加细胞活性氧ROS水平，并显著增加γ-H2AX和53BP1的表达，从分子、细胞和整体水平明确了XRCC4基因影响GBM放射治疗敏感性的作用机制，为GBM及其他类型肿瘤的放射增敏治疗提供了重要指导意义。