RNA干扰介导EphB4基因表达下调对胶质瘤细胞系U251生长的影响  

The effect of RNA interference-mediated down-regulation of EphB4 on the growth of malignant glioma cell line U251

作　　者：张艳荣[1] 董万利[1] 胡锦[2] 惠国桢[1] 金由辛[3] 史毅[3] 

机构地区：[1]苏州大学附属第一人民医院神经内科,苏州215006 [2]上海交通大学附属第六人民医院神经外科,上海200233 [3]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200032

出　　处：《肿瘤》2008年第12期1042-1046,共5页Tumor

基　　金：国家自然科学基金资助项目(编号:30770824);上海科学技术发展基金资助项目(编号:034047)

摘　　要：目的:探讨靶向EphB4基因的小干扰RNA(small interference RNA,siRNA)对胶质瘤细胞EphB4 mRNA表达及细胞生长的影响。方法:体外化学合成针对人EphB4基因的小干扰RNA并转染恶性胶质瘤U251细胞系后,RT-PCR法检测EphB4 mRNA表达的变化,CCK-8检测法观察RNA干扰对肿瘤细胞增殖活性的影响,FCM检测细胞周期变化,划痕实验观察细胞的迁移能力,采用Transwell小室穿膜细胞的数量来反应细胞的侵袭能力。结果:U251细胞转染siRNA-EphB4 100nmol/L后,EphB4 mRNA的表达水平下降了75.0%,细胞增殖抑制表现为剂量依赖性,FCM检测与阴性对照相比,细胞周期不同程度阻滞于亚G1期;并且细胞的迁移能力与对照组比较有所下降,Transwell小室穿膜细胞与对照组比较明显减少。结论:siRNA-EphB4能够明显靶向并抑制U251细胞中EphB4基因的表达,而EphB4基因表达下调可使U251细胞增殖受到影响,细胞周期出现凋亡峰。转染siRNA-EphB4后U251细胞的迁移和侵袭能力受到不同程度的抑制。提示抑制EphB4基因表达有可能成为胶质瘤治疗的新方法。Objective：To determine the interfering effects of EphBd-targeted short interfering RNA （siRNA） on EphB4 mRNA expression and its effect on the growth of glioma U251 cell line. Method： EphB4-targeted siRNA was designed and synthesized, and then was transfected into U251 cells. The inhibition of EphB4 mRNA expression was detected by RT-PCR. The effect of EphB4-targeted siRNA on cell growth rate was measured by CCK-8 method. Cell apoptosis was tested by flow eytometry （FCM）. Wound healing test was used to observe the migration ability of cells. The invasiveness of tumor cells was evaluated by counting the number of cells passing the Transwell membrane. Results：EphB4 mRNA transcription level was decreased by 75.0% after transfection of malignant glioma U251 cells with 100 nmol/L siRNA-EphlM. The inhibition of cell proliferation was in a dose-dependent manner. FCM analysis showed that cells were arrested at sub-G1 phase at different degrees and the migration capacity decreased after transfection with 100 nmol/L siRNA-EphB4 compared with the negative control. The number of cells permeating the matrigel membrane significantly were decreased in the siRNA-EphB4 transfection group compared with the control group. Conclusion： siRNA-EphB4 markedly targetes and knocks down EphB4 gene transcription. Down-regulation of EphB4 affects cell proliferation and induces apoptosis of cells. Transfection of siRNh-EphB4 into U251 cells inhibits the migration and invasion abilities of cells at various degrees. It indicates that silencing EphB4 expression might become a noval approach in the treatment of glioma.

关 键 词：神经胶质瘤 受体 EPHB4 RNA 小分子干扰 基因疗法 

分 类 号：R739.41[医药卫生—肿瘤]