The analysis of invasion associated gene in glioblastoma

我们揭示了以下两点。1)基质金属蛋白酶（matrix metalloproteinases，MMPs）是一种重要的蛋白酶，通过降解细胞外基质在肿瘤的发生、发展过程中发挥重要作用。我们研究了MMP-2（明胶酶A）和膜型1-MMP（MT 1-MMP，MMP-2=proMMP-2的酶原的激活剂）及其抑制剂，金属蛋白酶组织抑制剂（TIMP-1和TIMP-2）在人星形细胞肿瘤侵袭中的作用。采用夹心酶联免疫分析系统、定量逆转录聚合酶链反应（RT-PCR）、免疫组化、细胞转染等方法进行研究。结果表明，MT 1-MMP可能有助于胶质母细胞瘤细胞的侵袭和CSF传播的基础上，TIMP-2的失衡。2)Ets转录因子与肿瘤恶性程度相关。我们分析了Ets-DN-表达对细胞粘附、迁移和粘着斑激酶（FAK）磷酸化的影响。表达Ets-DN的U251细胞（U251-DN）在纤连蛋白包被的培养皿上显示出细胞粘附减少，肌动蛋白应力纤维的伸展和延伸，但在胶原包被的培养皿上没有。在用纤连蛋白包被的培养皿上，U251-DN细胞中FAK的磷酸化水平也减弱。半定量RT-PCR分析显示U251-DN细胞中整合素α5亚基的表达水平降低。荧光素酶报告基因检测显示，Ets DN的表达下调了整合素α5启动子的转录。半定量RT-PCR结果显示，胶质瘤中Ets-1 mRNA和整合素α5 mRNA的表达水平存在相关性。这些结果表明，Ets-1通过上调整合素α5亚基的表达，介导细胞内信号传导和随后的侵袭过程的加速，包括细胞粘附和迁移，从而促进胶质瘤恶性。

We revealed two points as follows. 1)Among proteinases, matrix metalloproteinases(MMPs)are thought to play a key role in the tumor progression through the degradation of extracellular matrix. We examined th role of MMP-2(gelatinase A)and membrane type 1-MMP(MT1-MMP, an activator of the zymogen of MMP-2=proMMP-2)together with their inhibitors, tissue inhibitors of metalloproteinases(TIMP-1 and TIMP-2), in the invasion of human astrocytic tumors. The investigations were performed using sandwich enzyme immunoassay systems, quantitative reverse transcription polymerase chain reaction(RT-PCR), gymography, Immunohistochemistry and cell transfection. The results suggest that MT1-MMP may contribute to the invasion and CSF dissemination of glioblastoma cells on the basis of an imbalance to TIMP-2. 2)Ets transcription factors are associated with tumor malignancy. We analyzed effects of Ets-DN-expression on cell adhesion, migration and phosphorylation of focal adhesion kinase(FAK). U251 cells expressing Ets-DN(U251-DN)showed reduced cell adhesion, spreading and extension of actin stress fibers on dishes coated with fibronectin but not on dishes coated with collagen. Phosphorylation levels of FAK in U251-DN cells were also attenuated on dishes coated with fibronectin. Reduced expression level of integrin α5 subunit in U251-DN cells was demonstrated by semi-quantitative RT-PCR analysis. Furthermore, down-regulation of transcription from the integrin α5 promoter by expression of Ets-DN was shown by luciferase reporter assay. Semi-quantitative RT-PCR of surgical samples of brain tumors revealed that the expression level of Ets-1 mRNA correlated with that of integrin α5 mRNA in glioma. These results suggest that Ets-1 contributes to glioma malignancy by upregulating expression of the integrin α5 subunit, which composes integrin α5β1, mediates intracellular signaling and the subsequent acceleration of the invasive process including cell adhesion and migration.