钙调蛋白抑制剂N-（6-氨基己基）-5-氯-1-萘磺酰胺通过逆转上皮-间充质转化抑制胶质母细胞瘤细胞的侵袭、迁移

Calmodulin antagonist W-7 inhibits migration and invasion by reversing epithelial-to-mesenchymal （-like） transition in glioblastoma cells

目的探讨钙调蛋白（CaM）抑制剂N-（6-氨基己基）-5-氯-1-萘磺酰胺（w-7）通过逆转上皮-间充质转化（EMT）抑制胶质母细胞瘤（GBM）细胞侵袭、迁移能力。方法细胞计数试剂盒（CCK-8）检测w-7对U87、U251胶质瘤细胞存活率的影响。Transwell侵袭实验检测对照组和w-7处理组穿过小室底膜的U87、U251细胞数目。划痕愈合实验检测各组U87、U251细胞相对迁移距离。Western blot法检测各组U87、U251细胞中上皮标志物（E-钙黏蛋白）和间质标志物（N-钙黏蛋白、波形蛋白）以及转录因子（ZEB1蛋白）的相对表达水平。倒置显微镜下观察各组U87、U251细胞形态变化。免疫荧光检测各组胶质瘤细胞N-cadherin的表达差异。结果w-7处理组穿过小室底膜的U87、U251细胞数少于对照组（P=0．001）；w-7处理组U87、U251细胞愈合速率低于对照组，24h后差异均有统计学意义（P=0．001、0．006）。w-7处理组中U87、U251细胞E-钙黏蛋白的相对表达水平高于对照组（P=0．001），而N-钙黏蛋白、ZEB1、波形蛋白相对表达水平低于对照组（P=0．001）。与对照组比较，w-7处理组U87、U251细胞形态呈椭圆形、圆形改变，细胞迁移能力减弱。结论CaM抑制剂w-7能够通过逆转EMT抑制GBM细胞的侵袭、迁移能力。

Objective To investigate Calmodulin （CAM） antagonist N-（6-Aminohexyl） -5-chloro-1-naphthalenesulfonamide （W-7） inhibits invasion and migration of Glioblastoma （GBM） cells by reversing Epithelial-to-mesenchymal like transition （ EMT-like）. Methods The effect of W-7 on the survival viability of U87/U251MG was detected by cell counting kit-8 （ CCK-8 ） kit. Transwell invasion assay was used to detect the number of U87 and U251 cells passing through the basement membrane of the control and W-7 treated groups, the relative wound space of U87 and U251 cells in each group was examined by scratch healing assay. The relative expression levels of epithelial marker （E-cadherin） and mesenchymal markers （N-cadherin, vimentin） and transcription factor （ZEB1 protein） in U87 and U251 cells were detected by Western blotting, the morphological changes of U87 and U251 cells in each group were observed by phase contrast microscopy. The expression of N-cadherin in glioma cells was detected by immunofluorcscence. Results The number of U87 and U251 cells passing through the basement membrane of W-7 group was less than that of the control group （ P=0. 001 ）. The healing rate of U87 and U251 cells in W-7 group was lower than that of the control group after 24 hours （ P=0. 001, P=0. 006）. The relative expression level of E-cadherin in U87 and U251 cells in W-7 treatment group was higher than that in control group （P=0.001） , while the relative expression levels of N-eadherin, ZEB1 and vimentin were lower than those in control group （ P=0.001）. Compared with the control group, the morphology of U87 and U251 cells in W-7 treatment group was oval or round, and cell migration was weakened. Conclusion CaM antagonist W-7 could inhibits invasion and migration of GBM cells by reversing EMT-like.