沉默Mig-7基因的表达可抑制人脑胶质瘤U87细胞的体外血管生成拟态形成能力及侵袭能力

Mig-7 gene-silencing inhibits vasculogenic mimicry formation and invasion ability of glioma U87cells in vitro

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 Mig-7 （migration-inducing gene-7） gene-silencing on vasculogenic mimicry （VM） formation and invasion of glioma U87 cells in vitro, and to explore their possible mechanism.Methods： Mig-7-shRNA specifically targeting Mig-7 gene was infected into U87 cells by lentivirus infection system. The infection efficiency was observed under a fluorescence microscope. After U87 cells were infected with the lentivirus carrying Mig-7-shRNA or the negative control- shRNA （NC-shRNA）, the mRNA expression levels of Mig-7, phosphatidylinositol 3-kinase （PI3K）, protein kinase B （PBK, also named as AKT）, matrix metalloproteinase 2 （MMP-2） and MMP-9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of PI3K, AKT, phospho-AKT （Ser473） （p-AKT）, MMP-2 and MMP-9 were examined by Western blotting. Three- dimentional culture and Transwell chamber experiment were used to observe the effects of Mig-7 gene-silencing on VM formation and the invasion of U87 cells. Results： The lentivirus carrying Mig-7-shRNA or NC-shRNA was infected into U87 cells successfully, and the U87 cells with stable low-expression of Mig-7 gene was successfully obtained. Compared with U87 cells infected with NC-shRNA lentivirus or without infection, the expression levels of Mig-7, PI3K, AKT, MMP-2 and MMP-9 mRNAs were significantly decreased in U87 cells infected with Mig-7-shRNA lentivirus （all P 〈 0.01）, and the expression levels of PI3K, AKT, p-AKT1, MMP-2 and MMP-9 proteins were obviously decreased （all P 〈 0.01）. Compared with U87 cells infected with NC-shRNA lentivirus or without infection, the inhibitory rate of invasion of U87 cells infected with Mig-7-shRNA was 75.67% or 75.08% （both P 〈 0.01）, and the formation ability of VM was decreased by 79.35% or 78.90% in Mig-7-shRNA infected U87 cells （both P 〈 0.05）. Conclusion： Mig-7 gene-silencing can inhibit VM formation and invasion ability of U87 cells, which may be associated with down-regulating the expressions of PI3K, AKT, p-AKT1, MMP-2 and MMP-9. It is suggested that Mig-7 gene probably plays an important role in VM formation and invasion in human brain glioma.