摘要目的:应用大样本临床测序数据联合体外细胞筛选放疗抵抗相关基因，探讨放疗诱导含锌指和BTB结构域蛋白质7A(ZBTB7A)过表达在胶质母细胞瘤(GBM)放疗抵抗中的作用。方法:纳入中国脑胶质瘤基因组图谱计划数据库中966例脑胶质瘤标本的测序和临床数据筛选及验证GBM放疗抵抗候选基因：(1)纳入226例原发GBM(pGBM)与134例手术＋放疗后复发的GBM(rGBM)非配对肿瘤标本，以及15例首次诊断为pGBM、手术＋放疗后复发再次行手术切除的配对肿瘤标本的测序数据筛选GBM放疗抵抗候选基因，并验证ZBTB7A是否为放疗抵抗相关基因；(2)比较异柠檬酸脱氢酶(IDH)野生型与突变型胶质瘤标本ZBTB7A mRNA表达量的差异(数据集1样本量分别为286、356例，数据集2分别为149、175例)；(3)比较ZBTB7A高表达组(148例)与低表达组(148例)GBM患者生存期的差异。纳入2019年4月至2022年12月于首都医科大学附属北京天坛医院手术并经病理学诊断的pGBM与rGBM肿瘤标本(各12例)，采用免疫组织化学染色法检测两组肿瘤组织中ZBTB7A蛋白表达水平的差异。采用高能X线照射人脑GBM细胞株U87、LN229和U251(单次13 Gy，照射6.5 min)，4、8 h后采用定量PCR方法检测细胞ZBTB7A mRNA的表达量；照射U87和LN229细胞(单次5 Gy，照射2.5 min)，1、2、4、8、12 h后采用蛋白质免疫印迹法检测ZBTB7A蛋白及DNA损伤标志物H2AX蛋白的表达量。采用小干扰RNA慢病毒(shRNA)感染法敲低U87细胞ZBTB7A的表达后，采用高能X线照射(单次照射剂量为5 Gy，照射2.5 min)，1 h后行彗星实验检测细胞的DNA损伤情况；采用平板克隆实验检测细胞的增殖情况。结果:非配对和配对肿瘤标本测序数据交叉筛选显示，rGBM组ZBTB7A mRNA的表达量均高于pGBM组(均 P<0.05)。IDH野生型ZBTB7A mRNA表达量高于IDH突变型；ZBTB7A高表达GBM患者的生存期短于低表达者；免疫组织化学染色证实ZBTB7A蛋白在rGBM肿瘤标本中的表达量高于pGBM，上述数据差异均有统计学意义(均 P<0.05)。与未照射组比较，照射组细胞的ZBTB7A mRNA和蛋白的表达量均升高(均 P<0.05)；H2AX蛋白表达量在照射1 h后开始增加，4 h后逐渐降低。X线照射后，慧星实验结果显示，与阴性对照组比较，ZBTB7A敲低组的DNA损伤显著增加；平板克隆实验结果显示，ZBTB7A敲低组细胞增殖活性显著降低(均 P<0.05)。 结论:ZBTB7A在rGBM中高表达，表达量高与患者的生存期短有关；放疗可诱导GBM细胞ZBTB7A过表达；ZBTB7A可能参与GBM的放疗抵抗。

abstractsObjective:To screen candidate genes associated with radiation resistance by large-sample RNA-sequence and cell experiments; to explore the role of Zinc Finger And BTB Domain Containing Protein 7A (ZBTB7A) in radioresistance of glioblastoma (GBM).Methods:A total of 996 gliomas samples with clinical information and RNA sequencing data were extracted from Chinese Glioma Genome Atlas (CGGA): (1) We compared the data of RNA-seq between 226 primary GBM and 134 recurrent GBM, and 15 paired primary and recurrent GBM to validate the relationship between ZBTB7A and radiation resistance; (2) We compared the ZBTB7A mRNA expression between IDH mutant gliomas and IDH wild-type gliomas; (3) Survival analysis was used to explore the association of ZBTB7A mRNA expression with patients survival (148 low-expression samples vs. 148 high-expression samples ). Patients with primary GBM and recurrent GBM who underwent surgery at Beijing Tiantan Hospital Affiliated to Capital Medical University from April 2019 to December 2022 and were pathologically diagnosed (12 cases each) were included. The protein expression of ZBTB7A was detected in those samples by immunohistochemistry. The GBM cell lines (U87, U251 and LN229) received radiation by high-energy X-ray(single exposure: 13 Gy, 6.5 min). The mRNA expression of ZBTB7A were detected by quantitative PCR in 4 h and 8 h after high-energy X-ray exposure. The protein expression of ZBTB7A in U87 and LN229 cell lines were detected by Western blot at 1 h, 2 h, 4 h, 8 h and 12 h after high-energy X-ray exposure (single exposure: 5 Gy, 2.5 min). U87 cells were infected by ZBTB7A shRNA lentivirus and then received radiation by high-energy X-ray (single exposure: 5 Gy, 2.5 min). The DNA damage were detected by comet assay at 1 h after high-energy X-ray exposure. Colony formation assay confirmed the association of ZBTB7A with radiation resistance in U87 cells after high-energy X-ray exposure.Results:The crossover analysis based on the RNA sequencing data of unpaired and paired samples showed that ZBTB7A was significant up-regulated in recurrent GBM (both P<0.05). The mRNA expression of ZBTB7A was higher in IDH wild-type gliomas than IDH mutant gliomas ( P<0.05). Patients with high expression of ZBTB7A had a shorter overall survival ( P<0.05) and progression-free survival ( P<0.05) than patients with low expression. Immunohistochemistry showed that the protein of ZBTB7A was higher in recurrent GBM than primary GBM ( P<0.05). Comparing with U87 and LN229 cells without high-energy X-ray exposure, the mRNA and protein expressions were significantly increased in cells with high-energy X-ray exposure. The protein of H2AX was also increased at 1 h after high-energy X-ray exposure, but it began to decrease at 4 h after high-energy X-ray exposure. ZBTB7A was knocked down in U87 cells by shRNA lentivirus. The DNA damage was significantly increased in cell with high-energy X-ray exposure compared with control cells ( P<0.05). The proliferative activity of U87 cells with high-energy X-ray exposure was also lower than control cells ( P<0.05). Conclusions:The mRNA and protein of ZBTB7A are significantly increased in recurrent GBM. High expression of ZBTB7A is associated with shorter survival. ZBTB7A protein could be induced by radiation and might contribute to the radioresistance of GBM.