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目的 :研究沉默迁移诱导基因7(migration-inducing gene-7,Mig-7)表达对人脑胶质瘤细胞株U87体外血管生成拟态(vasculogenic mimicry,VM)形成能力和侵袭能力的影响,及其可能的作用机制。方法 :采用慢病毒感染系统将特异性针对Mig-7基因的Mig-7-shRNA转入人脑胶质瘤U87细胞中,并观察感染效率;用携带有Mig-7-shRNA和阴性对照-shRNA(negative control-shRNA,NC-shRNA)的慢病毒感染U87细胞后,分别采用实时荧光定量PCR和蛋白质印迹法检测各组细胞中Mig-7、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,PBK,AKT)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)和MMP-9 mR NA的表达水平以及PI3K、AKT、磷酸化AKT[phospho-AKT(ser473),p-AKT]、MMP-2和MMP-9蛋白的表达水平;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U87细胞VM形成能力和侵袭能力的影响。结果:携带有Mig-7-shRNA和NC-shRNA的慢病毒成功感染U87细胞,并获得稳定低表达Mig-7基因的U87细胞株;与感染NC-shRNA慢病毒和未感染病毒的U87细胞相比,Mig-7-shRNA感染组U87细胞中Mig-7、PI3K、AKT、MMP-2和MMP-9 mRNA的表达水平以及PI3K、AKT、p-AKT1、MMP-2和MMP-9蛋白的表达水平均显著降低(P值均<0.01);与空白对照组和NC-shRNA感染组相比,Mig-7-shRNA感染组U87细胞的侵袭抑制率分别为75.67%和75.08%(P值均<0.01),并且Mig-7-shRNA感染组U87细胞VM形成能力分别下降了79.35%和78.90%(P值均<0.05)。结论 :沉默Mig-7基因的表达降低U87细胞的VM形成及侵袭能力,这可能与Mig-7基因沉默后下调PI3K、AK,p-AKT、MMP-2和MMP-9的表达有关,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。
Objective: To investigate the effect of migration-inducing gene-7 (Mig-7) on the formation and invasion of vasculogenic mimicry (VM) in human glioma cell line U87 Possible mechanism of action. Methods: Mig-7-shRNA specific to Mig-7 gene was transfected into human glioma U87 cells by lentivirus infection system and the infection efficiency was observed. Cells harboring Mig-7-shRNA and negative control-shRNA (Negative Control-shRNA, NC-shRNA) were transfected into U87 cells. The expression of Mig-7, phosphatidylinositol 3-kinase (MAPK) in each group were detected by real-time fluorescence quantitative PCR and Western blotting. PI3K, PBK, AKT, MMP-2 and MMP-9 mR NA, as well as PI3K, AKT, phospho- AKT (ser473), p-AKT], MMP-2 and MMP-9 protein levels in vitro. The effects of Mig-7 gene silencing on VM formation and invasion of U87 cells were observed by in vitro three-dimensional culture and Transwell chamber invasion assay . RESULTS: U87 cells were successfully infected with lentivirus carrying Mig-7-shRNA and NC-shRNA, and U87 cell line stably expressing Mig-7 gene was obtained. U87 cells infected with NC-shRNA lentivirus and uninfected virus The expression of Mig-7, PI3K, AKT, MMP-2 and MMP-9 mRNA and the protein expressions of PI3K, AKT, p-AKT1, MMP-2 and MMP-9 in U87 cells infected with Mig-7-shRNA (P <0.01). The invasion inhibition rates of U87 cells in Mig-7-shRNA infected group were 75.67% and 75.08%, respectively, compared with the blank control group and NC-shRNA infected group (P < 0.01). The VM formation capacity of U87 cells in Mig-7-shRNA infected group decreased by 79.35% and 78.90%, respectively (all P <0.05). Conclusion: The silencing of Mig-7 gene can reduce the VM formation and invasion in U87 cells, which may be related to the down-regulation of the expression of PI3K, AK, p-AKT, MMP-2 and MMP-9 after Mig-7 gene silencing, -7 gene plays an important role in the VM and invasion of human glioma cells.