Objective: To investigate the inhibitory effects of silencing migration-inducing gene-7 (Mig-7) on vasculogenic mimicry formation, migration and invasion of human glioma cells in vitro and whether MEK/ERK signaling pathway mediates these effects.
Methods: Human glioma U251 cells were infected by lentiviral vectors carrying a small interfering RNA targeting Mig-7 gene (sh-Mig-7) or a negative control shRNA (sh-NC), and real-time quantitative PCR was used to detect the expression level of Mig-7 mRNA in the cells. Three-dimensional culture and Transwell chamber invasion assay were used to observe the effect of Mig-7 gene silencing on vasculogenic mimicry formation and invasion ability of the U251 cells. Western blotting was performed to detect the changes in the protein expression levels of MEK/ERK in the infected cells.
Results: We successfully obtained a U251 cell line with stable low expression of Mig-7 gene using RNA interference technique. Compared with the cells infected with sh-NC lentivirus and the non- infected cells, U251 cells infected with the lentiviral vector carrying sh-Mig-7 showed significantly decreased expression level of Mig-7 (P < 0.01) with obviously lowered vasculogenic mimicry formation and invasion abilities (P < 0.05). Mig-7 silencing also significantly lowered the expressions of MEK and ERK proteins in U251 cells (P < 0.05).
Conclusions: Silencing of Mig-7 gene inhibits vasculogenic mimicry formation and invasion of U251 cells possibly by suppressing MEK/ERK signaling, suggesting the important role of Mig-7 gene in vasculogenic mimicry formation and invasion of human glioma cells.
目的: 研究迁移诱导基因7（Mig-7）通过MEK/ERK信号通路抑制人脑胶质瘤细胞株U251体外血管生成拟态（VM）形成能力和迁移、侵袭能力的影响。
方法: 采用RNA干扰技术将特异性针对Mig-7基因的sh-Mig-7转入人脑胶质瘤U251细胞中，并观察感染效率；用携带有sh-Mig-7和阴性对照（sh-NC）的慢病毒感染U251细胞后，采用实时荧光定量PCR法检测各组细胞中Mig-7的表达水平；采用体外三维培养和Transwell小室侵袭实验观察Mig-7基因沉默对各组U251细胞VM形成能力和侵袭能力的影响。采用western-blot检测各组细胞中MEK/ERK的蛋白表达水平。
结果: 携带有sh-Mig-7和sh-NC的慢病毒成功感染U251细胞，并获得稳定低表达Mig-7基因的U251细胞株；与感染sh-NC慢病毒和未感染病毒的的细胞相比，sh-Mig-7感染组U251细胞中Mig-7的表达水平以显著降低（P均 < 0.01）；与空白对照组和sh-NC感染组相比，sh-Mig-7感染组U251细胞的侵袭能力明显下降（P < 0.01），并且sh-Mig-7感染组U251细胞VM形成能力明显下降（P < 0.05）。与空白对照组和sh-NC感染组相比，sh-Mig-7感染组U251细胞的MEK、ERK蛋白的表达水平均显著降低（P < 0.05）。
结论: 沉默Mig -7基因的表达，可能通过MEK/ERK信号通路抑制U251细胞的VM形成及侵袭能力，提示Mig -7基因在人脑胶质瘤细胞的VM和侵袭中发挥重要的作用。
Keywords: Mig-7; RNA interference; glioma; tumor invasion; vasculogenic mimicry.