摘要
目的在U87人脑胶质瘤细胞中过表达miR-497,探讨miR-497对人脑胶质瘤细胞增殖的影响。方法包装阴性对照(NC)及miR-497慢病毒;构建U87-NC及U87-miR-497细胞系;荧光素酶报告基因实验检测miR-497在U87细胞中与神经调节蛋白受体降解蛋白1(Nrdp1)的靶向关系;集落形成实验检测细胞增殖能力、流式细胞术检测细胞周期变化;Western blot法检测Nrdp1、AKT及磷酸化的AKT(p-AKT)蛋白表达水平。结果成功包装p GLV3/H1-NC及p GLV3/H1-miR-497慢病毒;构建稳定的U87-NC及U87-miR-497细胞系;过表达miR-497的U87-miR-497细胞较对照组U87-NC细胞集落形成能力增强;荧光素酶报告基因实验证实miR-497在U87细胞中靶向作用于Nrdp1;在稳定感染细胞中,过表达miR-497后Nrdp1蛋白水平降低,p-AKT蛋白水平增加,而AKT蛋白未见明显改变。结论过表达miR-497通过靶向作用于Nrdp1促进U87胶质瘤细胞的增殖。
Objective To investigate the effect of miR-497 over-expression on the proliferation of U87 human glioma cells. Methods We packaged both p GLV3/H1-NC lentivirus as a negative control group and p GLV3/H1-miR-497 lentivirus as an experimental group,and then constructed U87-NC and U87-miR-497 cell lines,respectively. The relationship between miR-497 and neuregulin receptor degradation protein 1（ Nrdp1） was analyzed by luciferase reporter assay in U87 cells; cell colony formation assay was used to detect cell proliferation and flow cytometry to detect cell cycle; the expressions of Nrdp1,AKT and phosphorylated AKT（ p-AKT） were determined by Western blotting. Results We successfully packaged p GLV3/H1-NC and p GLV3/H1-miR-497 lentivirus,and obtained stable U87-NC and U87-miR-497 cell lines. When miR-497 was over-expressed in U87 cells,the cell colony formation ability was enhanced compared with the control group U87-NC. The luciferase reporter assay confirmed that miR-497 targeted Nrdp1 in U87 cells. In the stable infected cells,the level of Nrdp1 protein decreased and p-AKT protein increased,while the AKT protein did not change significantly after over-expression of miR-497. Conclusion Over-expression of miR-497 promotes the proliferation of glioma cells U87 by targeting Nrdp1.
作者
高永
孙来生
吴自成
陈新成
GAO Yong SUN Laisheng WU Zicheng CHEN Xincheng(Department of Neurosurgery, Xinyi People＇s Hospital, Xinyi 221400, China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第8期1051-1055,1061,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
江苏省卫生计生委科研项目(YG201514)
