The analysis of invasion associated gene in glioblastoma

我们揭示了以下两点。1)在多种蛋白水解酶中，基质金属蛋白酶(MMPs)被认为通过降解细胞外基质在肿瘤进展中起关键作用。我们研究了明胶酶A(明胶酶A)和膜型1-基质金属蛋白酶(MT1-基质金属蛋白酶)及其抑制物金属蛋白酶组织抑制物(TIMP-1和TIMP-2)在人脑星形细胞肿瘤侵袭中的作用。研究采用双抗体夹心酶免疫分析系统、定量逆转录聚合酶链式反应(RT-PCR)、脑血流图、免疫组织化学和细胞转染法。结果提示，MT1-MMPs可能通过与TIMP-2的失衡参与胶质母细胞瘤细胞的侵袭和脑脊液扩散。2)Ets转录因子与肿瘤恶性程度相关。我们分析了Ets-dN表达对细胞黏附、迁移和粘着斑激酶(FAK)磷酸化的影响。表达Ets-dN(U251-dN)的U251细胞在纤维连接蛋白涂层的培养皿上的细胞黏附、铺展和肌动蛋白应激纤维的伸展减少，而在胶原涂层的培养皿上未见。涂有纤维连接蛋白的培养皿也能降低U251-DN细胞中FAK的磷酸化水平。半定量逆转录聚合酶链式反应显示U251-DN细胞整合素α5亚单位表达水平降低。此外，荧光素酶报告基因分析显示，整合素α5启动子的转录受Ets-dN表达下调。半定量逆转录聚合酶链式反应显示Ets-1m RNA在脑胶质瘤中的表达水平与整合素α-5m RNA的表达水平呈正相关。这些结果表明，Ets-1通过上调整合素α5亚单位的表达，介导细胞内信号转导，加速细胞黏附和迁移等侵袭过程，从而参与胶质瘤的恶性转化。整合素α5β1是整合素DNA 5亚基的组成部分。

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.