In vitro Investigation on Effect of Lentiviral Vector-mediated RNA Interference Inhibiting VASH1 Expression on Proliferation and Apoptosis of Human Glioma Cells U-87MG
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摘要: 目的 探讨运用慢病毒载体介导的RNA干扰技术抑制血管生成抑制因子1（VASH1）的表达对人脑胶质瘤U-87MG细胞增殖和凋亡的影响。方法 应用pGCL-GFP质粒构建针对VASH1的慢病毒shRNA载体，转染入工具细胞293T，筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞；通过RT-PCR和Western blot评价其对U-87MG细胞内VASH1表达的影响；用四甲基偶氮唑蓝（MTT）法观察U-87MG细胞增殖活性的改变；用流式细胞仪（FCM）检测U-87MG细胞凋亡的变化。结果 与转染阴性对照组和空白组相比，转染pGCL-GFP-VASH1慢病毒载体的U-87MG细胞VASH1的mRNA 蛋白表达水平明显降低（P<0.01）；细胞的增殖活性显著上调（P<0.01），细胞凋亡明显减少（P<0.01）。结论 VASH1 shRNA慢病毒载体可抑制VASH1在人脑胶质瘤U-87MG细胞中的表达，并增加肿瘤细胞的增殖活性，抑制细胞凋亡。Abstract: Objective To investigate the effect of lentiviral vector-mediated RNA interference inhibiting VASH1 gene expression on the proliferation and apoptosis of human glioma cells U-87MG. Methods A lentiviral vector carrying short hairpinRNA(shRNA) of human VASH1 gene (pGCL-GFP-VASH1) was constructed and used to transfect 293T cells. The transfection efficiency was evaluated and then the lentiviral vector with suitable concentration was transfected into the human glioma cells U-87MG; RT-PCR and Western blot were used to detect the expression of VASH1 in U-87MG cells. MTT assay was used to observe the inhibion ratio of U-87MG cells growth. FCM analysis was used to observe the apoptosis. Results RT-PCR and Western blot analyses demonstrated that pGCL-GFP-VASH1 could significantly inhibit the expression of VASH1 mRNA and protein in U-87MG cells(P<0.01); MTT results showed that it could increase the growth of U-87MG cells(P<0.01). FCM results showed that the occurrence of apoptosis was suppressed significantly in U-87MG cells(P<0.01). Conclusion Lentiviral vector-mediated RNA interference targeting against VASH1 could effectively inhibit the expression of VASH1, induce the cell proliferation and suppress the apoptosis in U-87MG cells.
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Key words:
- VASH1 /
- RNA interference(RNAi) /
- Lentiviral vector /
- Glioma
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