EphB3通过PI3K/AKT信号通路对胶质瘤细胞侵袭及迁移的影响

EphB3 suppresses glioma cell invasion and migration via PI3K/AKT signaling pathway

目的探究促红细胞生成素产生的肝细胞受体B3(EphB3)通过PI3K/AKT信号通路对胶质瘤的侵袭、迁移的作用机制。方法通过EphB3慢病毒过表达及EphB3-siRNA感染U87MG、U251细胞系,采用细胞划痕实验、细胞侵袭(Transwell)实验检测细胞的侵袭、迁移能力,采用蛋白印迹法(Western Blot)检测EphB3过表达及siRNA干扰后磷脂酰肌醇3-激酶(PI3K)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)的蛋白表达,采用细胞多重免疫荧光检测EphB3的过表达及敲低检测EphB3、p-PI3K、p-AKT共表达情况;收集Ⅰ~Ⅳ级胶质瘤组织样本40例,采用免疫组化检测EphB3的表达,组织多重免疫荧光检测EphB3及p-PI3K、p-AKT的共表达。结果U87MG、U251细胞系过表达EphB3后细胞侵袭、迁移能力减弱(P<0.05),敲低EphB3后U87MG、U251细胞侵袭、迁移能力增强(P<0.05);EphB3过表达后p-PI3K、p-AKT表达下降(P<0.05),EphB3敲低后p-PI3K、p-AKT表达升高(P<0.05);临床胶质瘤组织样本结果显示,EphB3的表达与胶质瘤WHO分级呈负相关;多重免疫荧光结果显示,胶质瘤的级别与p-PI3K、p-AKT的荧光信号强度呈正相关。结论EphB3的表达可抑制胶质瘤的侵袭及迁移,其机制可能通过PI3K/AKT信号通路来影响肿瘤细胞的侵袭、迁移。

Objective To investigate the mechanism by which EphB3 suppresses glioma cell invasion and migration through PI3K/AKT signaling pathway.Methods U87MG and U251 cell lines were infected with lentivirus carrying EphB3 over-expression and transfected with EphB3-siRNA,respectively.Wound healing assay and Transwell assay were applied to assess the capacity of cell migration and invasion.The protein expression of PI3K,p-PI3K,AKT,and p-AKT after EphB3 overexpression and siRNA interference were detected by Western blot.Multiple immunofluorescence assay was used to detect EphB3 overexpression,EphB3 knockdown and the co-expression of EphB3,p-PI3K and p-AKT.Forty gliomas samples with gradeⅠ-Ⅳwere collected.Immunohistochemistry was used to detect EphB3 expression.Co-expression of EphB3,p-PI3K,and p-AKT was detected by tissue multiple immunofluorescence assay.Results Invasion and migration ability of U87MG and U251 cell lines were significantly decreased after overexpressing EphB3(P<0.05),while the invasion and migration of U87MG and U251 cells were significantly enhanced after EphB3 knockdown(P<0.05).EphB3 overexpression decreased the expression of p-PI3K and p-AKT(P<0.05),while EphB3 knockdown increased the expression of p-PI3K and p-AKT(P<0.05).Results from clinical tissue samples showed that EphB3 expression was negatively correlated with the WHO grade of glioma.Multiple immunofluorescence results showed that glioma grade positively correlated with fluorescent signal intensity of p-PI3K and p-AKT.Conclusions EphB3 expression can suppress glioma cell invasion and migration.Its mechanism may mediate the invasion and migration of tumor cells through PI3K/AKT signal pathway.