The role of radiotherapy-induced ZBTB7A overexpression in radioresistance of glioblastoma
Objective 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.
GlioblastomaRadiation resistanceOver expressionZinc finger and BTB domain containing 7 A
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