EGFR信号通路调控人胶质瘤U87细胞侵袭转移的分子机制

摘要

Objective To study the relationship between EGFR signal pathway and invasion and metastasis of U87 glioma cells, and discuss the molecular mechanism. Methods U87 glioma cells were cultured in medium that contained epidermal growth factorrn(EGF 100 ng/mL) or epidermal growth factor inhibitor－－AG1478(10μmol/L) or combination,then the methyl thiazolyl tetrazo-rnlium (MTT) assay and transwell chamber were used to detect the proliferation and invasive ability of U87 glioma cells;Expression levels of matrix metalloproteinases-2(MMP-2) ,matrix metalloproteinases-9(MMP-9) were determined by gelatinase zymography e-lectrophoresis; Western blot was used to determine the expression levels of phosphorylation of the epidermal growth factor receptor (P-EGFR) ,and phosphorylation of protein kinase B (P-AKT). Results Exogenous EGF(100 ng/mL)increased the expression lev-els of P-EGFR、P-AKT, the growth ratio increased 19. 25% ,22. 32%(P<0. 05)at 24 ,48 h respectively after treated, increased the number of invasion cell from (27 ± 4) cells to (126 ± 3) cells(P<0. 05),and increased the expression levels of MMP-2, MMP-9 ; AG1478 could block the effects of EGF increased the expressions of P-EGFR,P-AKT in time-independent(P<0. 05) ,decreased the invasive ability of U87 glioma(P<0. 05) , inhibited the expression of MMP-2 , MMP-9 (P<0. 05). Conclusion The EGFR-PI3K/ AKT signaling pathways involved in the regulation of U87 glioma cells proliferation and invasion and metastasis. Its mechanism is possible that after the EGFR-PI3 K/AKT signaling pathways activated, caused the high expression of MMP-2, MMP-9 and increased the damage to the exracellular matrix(ECM).%目的 探讨表皮生长因子受体(EGFR)信号通路与人胶质瘤细胞(U87细胞)增殖和侵袭转移的关系及调控分子机制.方法 表皮生长因子(EGF,100 ng/mL)、EGFR 抑制剂--AG1478(10 μmol/L)单独和联合处理U87细胞,采用MTT法和Transwell小室体外侵袭实验检测U87细胞增殖和体外侵袭能力;明胶酶谱法检测基质金属蛋白酶-2(MMP-2)、MMP-9的表达水平;Western blot法检测磷酸化EGFR (P-EGFR)、磷酸化AKT(P-AKT)蛋白表达.结果 EGF(100 ng/mL)增加P-EGFR和P-AKT蛋白表达,使加药后24 h和48 h的细胞生长率分别提高了19.25%、22.32%(P<0.05),使12 h过膜细胞数由(27±4)个上升到(126±3)个(P<0.05),促进U87细胞的MMP-2、MMP-9蛋白表达(P<0.05);AG1478(10 μmol/L)可以阻断EGF增加P-EGFR和P-AKT蛋白表达的作用(P<0.05),并具有时间依赖性(P<0.05),减弱U87细胞体外侵袭能力(P<0.05),抑制MMP-2、MMP-9蛋白的表达(P<0.05).结论 EGFR-PI3K/AKT信号通路参与调节U87细胞增殖和侵袭转移过程,其机制可能是EGFR-PI3K/AKT信号通路活化后,导致MMP-2和MMP-9蛋白的表达增加,对细胞外基质的破坏增强.