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、40 μmol/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、5 μmol/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2.50 μmol/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、5 μmol/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、20 μmol/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、40 μmol/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、2.50 μmol/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。

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

Abstract:

Objective: 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, 40 μmol/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 higher than that in the empty vector transfected group after TMZ 640, 320, 160, 80, 40, 20, 5 μmol/L (P=0.000, 0.000, 0.000, 0.002, 0.001, 0.002); the survial rate of U87MG cells in the si-PKM2-1009 transfected group was lower than that of F3-T3 transfected group after TMZ 640, 320, 160, 80, 40, 20, 10, 5, 2.50 μmol/L, the survival rate of U251MG cells in the si-PKM2-1377 transfected group was also lower than that in F3-T3 transfected group after TMZ 640, 320, 160, 80, 40, 20, 5 μmol/L (P=0.000, 0.000, 0.002, 0.000, 0.002, 0.048, 0.042); and the survival rate of U87MG cells in TMZ + Compound 3k group was lower than TMZ group after TMZ 640, 320, 160, 80, 40, 20 μmol/L (P=0.000, 0.000, 0.000, 0.000, 0.001, 0.002), and the survival rate of U251MG cells in TMZ + Compound 3k group after treatment with high concentrations of TMZ (640, 320, 160, 80 and 40 μmol/L) was also lower than TMZ group (P=0.000, 0.000, 0.000, 0.000, 0.003), while the survival rate of U251MG cells in TMZ + Compound 3k group after treatment with low concentrations of TMZ (10, 5 and 2.50 μmol/L) was higher than that of TMZ group (P=0.000, 0.000, 0.006). 2) An animal model of glioblastoma showed the presence of TMZ resistance in homozygous mice. 3) Bioinformatic analysis showed that the biological function of F3-T3 was significantly enriched in the DNA repair pathway (P=0.000). Survival and overall survival of glioblastoma patients in the TCGA database were lower in the PKM2 high expression group than in the low expression group (P < 0.05). 4) Western blotting showed that the relative expression of p-H2AX in U87MG (P=0.000, 0.000, 0.004) and U251MG (P=0.000, 0.007, 0.005) cells in the F3-T3 transfected group were lower than those in the empty vector transfected group after TMZ treatment for 48 h and then medium change for 24, 36 and 48 h. PKM2 protein incorporation into the nucleus was observed in both U87MG and U251MG cells in the F3-T3 transfected group after TMZ treatment, whereas it was not observed in cells in the empty vector transfected group; si-PKM2-1009 and si-PKM2-1377 knocked down the relative expression of p-H2AX in U87MG (P=0.000, 0.001, 0.006) and U251MG (P=0.000, 0.000, 0.000) cells, respectively. Conclusions: In the presence of TMZ, F3-T3 promotes PKM2's nuclear translocation and activates DNA damage repair pathways, which in the eventually results resistance of glioblastoma cell to TMZ. PKM2 inhibitors can compromise the resistance glioblastoma cells stably expressing F3-T3 fusion gene to TMZ.

Key words: Glioblastoma, Receptor, fibroblast growth factor, type 3, Gene fusion, Pyruvate kinase, DNA repair, Temozolomide, Drug resistance, neoplasm, Cell proliferation, Immunoblotting, Tumor cells, cultured, Disease models, animal