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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 overexpress miR-497 in U87 human glioma cells and investigate the effect of miR-497 on the proliferation of human glioma cells. Methods U87-NC and U87-miR-497 cell lines were packaged by negative control (NC) and miR-497 lentivirus. Luciferase reporter assay was used to detect the expression of miR-497 in U87 cells compared with neuregulin receptor- (Nrdp1). The colony formation assay was used to detect the proliferation of cells. Flow cytometry was used to detect cell cycle changes. Western blot was used to detect the expression of Nrdp1, AKT and phosphorylated AKT (p-AKT). RESULTS: The pGLV3 / H1-NC and pGLV3 / H1-miR-497 lentivirus were successfully packaged; the stable U87-NC and U87-miR-497 cell lines were constructed; U87-miR-497 cells overexpressing miR- The luciferase reporter assay confirmed that miR-497 targets NDRp1 in U87 cells. In stable infected cells, the level of Nrdp1 protein is decreased after overexpression of miR-497. The expression of p-AKT Protein levels increased, while no significant changes in AKT protein. Conclusion Overexpression of miR-497 enhances proliferation of U87 glioma cells by targeting Nrdp1.