Yang Jian, Zhang Jian-Nan, Chen Wei-Lin, Wang Gui-Song, Mao Qing, Li Shan-Quan, Xiong Wen-Hao, Lin Ying-Ying, Ge Jian-Wei, Li Xiao-Xiong, Gu Zhao, Zhao Chun-Run
Department of Neurosurgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, P. R. China.
Operation Room, Shanghai Children's Medical Center, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, P. R. China.
Oncotarget. 2017 Jun 13;8(24):38444-38455. doi: 10.18632/oncotarget.16461.
We investigated the effects of aquaporin 5 (AQP5) gene silencing on the proliferation, migration and apoptosis of human glioma cells through regulating the EGFR/ERK/p38MAPK signaling pathway. qRT-PCR was applied to examine the mRNA expressions of AQP5 in five human glioma cell lines. U87-MG, U251 and LN229 cells were selected and assigned into blank, vector, AQP5 siRNA and FlagAQP5 groups. MTT assay was used to measure cell proliferation. Flow cytometry (FCM) with AnnexinV-FITC/PI double staining and PI staining were employed to analyze cell apoptosis and cell cycle respectively. Scratch test was used to detect cell migration. Western blotting was performed to determine the EGFR/ERK/p38 MAPK signaling pathway-related proteins. Results showed that the positive expression of AQP5 in primary glioblastoma was associated with the tumor size and whether complete excision was performed. The mRNA expressions of AQP5 in cell lines of U87-MG, U251 and LN229 were significantly higher than in U373 and T98G. The proliferation rates of U87-MG, U251 and LN229 cells in the AQP5 siRNA group were lower than in the vector and blank groups. The apoptosis rate increased in the AQP5 siRNA group compared with the vector group. Scratch test demonstrated that AQP5 gene silencing could suppress cell migration. Compared with the vector and blank groups, the AQP5 siRNA group showed decreased expressions of the ERK1/2, p38 MAPK, p-ERK1/2 and p-p38 MAPK proteins. AQP5 gene silencing could inhibit the cell proliferation, reduce cell migration and promote the cell apoptosis of U87-MG, U251 and LN229 by suppressing EGFR/ERK/p38 MAPK signaling pathway.
我们通过调节表皮生长因子受体（EGFR）/细胞外信号调节激酶（ERK）/p38丝裂原活化蛋白激酶（p38MAPK）信号通路，研究了水通道蛋白5（AQP5）基因沉默对人胶质瘤细胞增殖、迁移和凋亡的影响。应用实时定量聚合酶链反应（qRT-PCR）检测5种人胶质瘤细胞系中AQP5的信使核糖核酸（mRNA）表达。选取U87-MG、U251和LN229细胞，分为空白组、载体组、AQP5小干扰RNA（siRNA）组和FlagAQP5组。采用噻唑蓝（MTT）比色法检测细胞增殖。分别采用膜联蛋白V-异硫氰酸荧光素（AnnexinV-FITC）/碘化丙啶（PI）双染法和PI染色法，通过流式细胞术（FCM）分析细胞凋亡和细胞周期。采用划痕试验检测细胞迁移。通过蛋白质印迹法检测EGFR/ERK/p38 MAPK信号通路相关蛋白。结果显示，原发性胶质母细胞瘤中AQP5的阳性表达与肿瘤大小及是否进行完整切除有关。U87-MG、U251和LN229细胞系中AQP5的mRNA表达显著高于U373和T98G细胞系。AQP5 siRNA组中U87-MG、U251和LN229细胞的增殖率低于载体组和空白组。与载体组相比，AQP5 siRNA组的凋亡率升高。划痕试验表明，AQP5基因沉默可抑制细胞迁移。与载体组和空白组相比，AQP5 siRNA组中ERK1/2、p38 MAPK、磷酸化ERK1/2（p-ERK1/2）和磷酸化p38 MAPK（p-p38 MAPK）蛋白的表达降低。AQP5基因沉默可通过抑制EGFR/ERK/p38 MAPK信号通路，抑制U87-MG、U251和LN229细胞的增殖，减少细胞迁移，促进细胞凋亡。
