Yu Xiao, Zhang Wendi, Ning Qin, Luo Xiaoping
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2012 Jun;32(3):370-374. doi: 10.1007/s11596-012-0064-0. Epub 2012 Jun 9.
The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.
本研究探讨了微小RNA-34a(miR-34a)调控的Notch1基因对人胶质瘤细胞系U87增殖和凋亡的影响。根据不同处理,将U87细胞分为miR-34a模拟物组、阴性对照组、模拟转染组和空白对照组。在miR-34a模拟物组中,使用脂质体2000将miR-34a模拟物转染到人U87胶质瘤细胞中。将转染无义微小RNA的细胞设为阴性对照组。仅用脂质体2000处理的细胞指定为模拟转染组。在空白对照组中,细胞常规培养,不进行处理。采用实时RT-PCR检测miR-34a和Notch1的表达。用蛋白质免疫印迹法监测Notch1蛋白的变化。通过CCK-8和流式细胞术检测细胞增殖和凋亡。结果显示,与对照组相比,miR-34a模拟物组U87细胞的增殖能力显著降低,凋亡细胞增加。与对照组相比,模拟物组中miR-34a的表达显著上调(P<0.05)。此外,与对照组相比,miR-34a模拟物组中Notch1蛋白水平显著降低(P<0.05),但Notch1的mRNA表达在这些组之间无显著差异。结论是,miR-34a可能通过降低靶基因Notch1的表达来抑制U87细胞的增殖并诱导其凋亡,提示miR-34a可能成为脑胶质瘤有前景的基因治疗靶点。