慢病毒介导的Cullin3沉默对人胶质瘤细胞的影响

Effects of Lentivirus-mediated Cullin3 Silencing on Human Glioma Cells

目的:探讨慢病毒介导特异性短发夹RNA(sh RNA)干扰Cullin3表达对人胶质瘤细胞的影响。方法:RT-q PCR及Western blot检测Cullin3在人胶质瘤细胞系SW1783,U251及U87MG中的表达;构建靶向Cullin3的sh RNA重组慢病毒,转染U251和U87MG细胞并测定转染效率。实验分为3组:Blank组(阴性对照组)、NC-sh RNA组(空载慢病毒组)及Cullin3-sh RNA组(慢病毒介导Cullin组)。MTT法和Brd U实验检测细胞活力和增殖能力;Transwell实验检测细胞迁移能力;流式细胞术检测细胞周期的变化。结果:与正常人星形胶质细胞相比,Cullin3在人胶质瘤细胞系SW1783,U251及U87MG中高表达。Cullin3-sh RNA转染U251和U87MG细胞后,与Blank组及NC-sh RNA组相比,Cullin3-sh RNA组Cullin3表达水平下降(P<0.05),细胞活力和细胞增殖能力显著降低,侵袭能力下降,细胞周期被阻滞于G0/G1期。结论:Cullin3在胶质瘤细胞增殖和侵袭过程中发挥重要作用,提示Cullin3有望成为人脑胶质瘤基因治疗的候选靶点。

Objective： To investigate the effects of Cullin3 gene silencing on the biological characteristics of human glioma cells. Methods： Cullin3 mRNA and protein levels in human glioma cells SW1783, U251 and U87MG were detected by RT-qPCR and Western blot. A lentiviral vector expressing Cullin3 shRNA was constructed and transfected into the U251 and U87MG cells and the transfection efficiency was confirmed. Cells were divided into three groups： the blank （negative control group）, NC-shRNA （blank vector group） and the Cullin3-shRNA（Cullin3-shRNA vector group）. The cell viability and cell proliferation were determined by MTT assay and BrdU incorporation assay respectively. Meanwhile, the cell migration was analyzed by TransweU assay. Flow cytometry was used to monitor the changes of cell cycle distribution. Results： The Cullin3 protein levels in SW1783 ,U25.1 and U87MG glioma cells were significantly higher than that in the normal astrocytes. Transfection of Cullin3-shRNA significantly abolished Cullin3 expression. Compared with the Blank and NC-shRNA groups, Cullin3-shRNA significantly inhibited the growth and proliferation of glioma cells U25 land U87MG. , In addtion, the migration rate was significantly decreased in Cullin3-shRNA group. Flow cytometry indicated that the cell cycle was arrested at G0/G1 phase in Cullin3-shRNA group. Conclusion： Cullin3 plays a critical role in gliomagenesis and development of glioma and it maybe a potential target for human glioma therapy.