胶质母细胞瘤FGFR3-TACC3融合基因介导丙酮酸激酶M2入核促进DNA损伤修复基础研究  

The study of FGFR3-TACC3 fusion gene mediating pyruvate kinase M2 nuclear translocation to promote DNA damage repair in glioblastoma

作　　者：任修德 李涛[1] 范吉康 王希森 贾晓丹 杨学军 REN Xiu-de;LI Tao;FAN Ji-kang;WANG Xi-sen;JIA Xiao-dan;YANG Xue-jun(Department of Neurosurgery,Tianjin Medical University General Hospital,Tianjin 300052,China;Department of Neurosurgery,Beijing Tsinghua Changgung Hospital,Beijing 102218,China)

机构地区：[1]天津医科大学总医院神经外科,300052 [2]北京清华长庚医院神经外科,102218

出　　处：《中国现代神经疾病杂志》2023年第8期745-757,共13页Chinese Journal of Contemporary Neurology and Neurosurgery

基　　金：国家自然科学基金青年科学基金资助项目(项目编号:82102951)。

摘　　要：目的探讨胶质母细胞瘤FGFR3-TACC3(F3-T3)融合基因介导丙酮酸激酶M2(PKM2)入核激活DNA损伤修复致替莫唑胺(TMZ)耐药的作用机制。方法慢病毒转染构建稳定表达F3-T3融合基因和空载体的胶质母细胞瘤细胞系U87MG和U251MG,构建稳定表达F3-T3融合基因的胶质母细胞瘤裸鼠模型,小动物活体成像系统观察荷瘤鼠肿瘤荧光信号强度;采用生物信息学分析基因芯片转录组数据分析F3-T3融合基因的生物学功能,并分析肿瘤基因组学图谱计划(TCGA)数据库中胶质瘤患者生存期与PKM2基因表达的关系;瞬时转染小干扰RNA(siRNA)敲低PKM2基因表达;CCK-8细胞增殖实验观察经梯度浓度替莫唑胺处理后、转染siRNA后、替莫唑胺联合PKM2抑制剂Compound 3k处理后U87MG和U251MG细胞增殖活性;提取核质蛋白并观察经替莫唑胺处理后总蛋白提取物、胞质提取物和胞核提取物PKM2蛋白表达情况;Western blotting法检测稳定表达F3-T3融合基因的U87MG和U251MG细胞PKM2蛋白相对表达量、磷酸化组蛋白H2AX(p-H2AX)相对表达量、siRNA敲低PKM2基因p-H2AX相对表达量。结果(1)CCK-8细胞增殖实验显示,经替莫唑胺640、320、160、80、40μmol/L处理后F3-T3转染组的U87MG细胞存活率均高于空载体转染组(P=0.000,0.000,0.000,0.004,0.010),经替莫唑胺640、320、160、80、40、20、5μmol/L处理后F3-T3转染组的U251MG细胞存活率亦均高于空载体转染组(P=0.000,0.000,0.000,0.000,0.002,0.001,0.002);然而,经替莫唑胺640、320、160、80、40、20、10、5和2.50μmol/L处理后si-PKM2-1009转染组的U87MG细胞存活率均低于F3-T3转染组(P=0.000,0.000,0.000,0.012,0.006,0.030,0.000,0.007,0.025),经替莫唑胺640、320、160、80、40、20、5μmol/L处理后si-PKM2-1377转染组U251MG细胞存活率亦低于F3-T3转染组(P=0.000,0.000,0.002,0.000,0.002,0.048,0.042);经替莫唑胺640、320、160、80、40、20μmol/L处理后TMZ+Compound 3k组U87MG细胞存活率低于TMZ组(P=0.000,0.000,0Objective:To explore the mechanism of FGFR3-TACC3(F3-T3)fusion gene mediating DNA damage repair through promoting pyruvate kinase M2(PKM2)'s nuclear translocation in glioblastoma.Methods:The lentiviral transfection technology was used to constructed stable transitional cell lines U87MG cells and U251MG cells that stably expressing F3-T3 and empty vector.In vivo glioblastoma model was constructed by intracranial in situ tumor implantation in athymic mice,and the tumorigenic ability of F3-T3 transfected cells in athymic mice of each treatment group was observed by small-animal in vivo imaging system.Bioinformatics analysis was performed to analyze gene microarray data exploring the possible biological functions of F3-T3 mediating chemoresistance and identify the relationship between survival expectations and PKM2 expression levels in glioblastoma patients from The Cancer Genome Atlas(TCGA)database.Transient transfection of small interference RNA(siRNA)was used to knock down the expression of PKM2 in the F3-T3 transfected cells.Proliferative activity of U87MG and U251MG cells treated with different concentrations of temozolomide(TMZ),transfected with siRNA,and TMZ in combination with Compound 3k,a PKM2 inhibitor,was observed in CCK-8 cell proliferation assays.Nuclear and cytoplasmic proteins were extracted separately and PKM2 protein expression was observed in whole cell extract,cytoplasmic extract and cytosolic extract after TMZ treatment.Relative expression of PKM2,relative expression of cytosolic phosphorylated histone H2AX(p-H2AX)and relative expression of siRNA knockdown PKM2 gene in U87MG and U251MG cells stably expressing the F3-T3 fusion gene,detected by Western blotting.Results:1)CCK-8 cell proliferation assay showed that the survival rate of U87MG cells in the F3-T3 transfected group was higher than that in the empty vector transfected group after treatment with TMZ 640,320,160,80,40μmol/L(P=0.000,0.000,0.000,0.000,0.004,0.010),and the survival rate of U251MG cells in the F3-T3 transfected group was also

关 键 词：胶质母细胞瘤 受体 成纤维细胞生长因子 3型 基因融合 丙酮酸激酶 DNA修复 替莫唑胺 抗药性 肿瘤 细胞增殖 免疫印迹法 肿瘤细胞 培养的 疾病模型 动物 

分 类 号：R739.4[医药卫生—肿瘤]