科研之友
微信
新浪微博
Facebook
分享链接
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=0.000,0.000,0.000,0.000,0.001,0.002），经高浓度（640、320、160、80、40μmol/L）替莫唑胺处理后TMZ+Compound 3k组U251MG细胞存活率亦低于TMZ组（P=0.000,0.000,0.000,0.000,0.003），而经低浓度（10、5、2.50μmol/L）替莫唑胺处理后TMZ+Compound 3k组U251MG细胞存活率高于TMZ组（P=0.000,0.000,0.006）。（2）胶质母细胞瘤动物模型显示，荷瘤鼠存在替莫唑胺耐药。（3）生物信息学分析，F3-T3融合蛋白的生物学功能显著富集于DNA修复通路（P=0.000）。TCGA数据库中胶质瘤患者PKM2基因高表达组生存率和总生存期均低于低表达组（P <0.05）。（4）Western blotting法显示，经替莫唑胺处理48 h再更换培养基后24、36和48 h,F3-T3转染组U87MG(P=0.000,0.000,0.004)和U251MG(P=0.000,0.007,0.005)细胞p-H2AX蛋白相对表达量均低于空载体转染组；经替莫唑胺处理后F3-T3转染组U87MG和U251MG细胞均可见明显的PKM2入核，而空载体转染组细胞均未见这一现象；si-PKM2-1009和si-PKM2-1377分别敲低U87MG(P=0.000,0.001,0.006)和U251MG(P=0.000,0.000,0.000)细胞PKM2基因表达的效果最显著。结论 F3-T3融合基因可促进PKM2入核，激活DNA损伤修复相关通路，进而介导胶质母细胞瘤对替莫唑胺耐药，不同细胞株对替莫唑胺的耐药浓度不一致，PKM2抑制剂可逆转这种耐药。
-
单位天津医科大学总医院
