DOK3在胶质母细胞瘤替莫唑胺耐药中的作用及机制研究

Role and mechanism of DOK3 in temozolomide resistance in glioblastoma

目的 研究停靠蛋白3(DOK3)在胶质母细胞瘤替莫唑胺(TMZ)耐药中的作用及可能机制。方法 使用CCK8法测定细胞活力和TMZ的半数抑制浓度(IC_(50)),采用流式细胞术评价细胞凋亡。Western blot法检测DOK3、O^(6)-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)、磷脂酰肌醇3-激酶(PI3K)、p-PI3K、AKT和p-AKT蛋白的表达。Transwell法测定细胞的迁移能力,免疫荧光测定核转录因子(NF-κB)的核转位。结果 DOK3在TMZ抗性的U251细胞中的表达水平低于其亲本细胞,而MGMT、p-PI3K以及p-AKT的表达水平高于其亲本细胞。DOK3的过表达降低了TMZ耐药细胞对TMZ的IC_(50)值,也加速了TMZ诱导的细胞凋亡和细胞迁移抑制,降低了MGMT、p-PI3K和p-AKT的蛋白表达以及抑制了NF-κB的核转位。结论 DOK3通过PI3K/AKT/NF-κB途径调控MGMT,导致神经胶质瘤细胞对TMZ产生耐药性。

Objective To determine the effect and possible mechanisms of docking protein 3(DOK3)in temozolomide resistance in glioblastoma.Methods Cell counting kit-8 was used to measure the cell viability and the half-inhibitory concentration(IC_(50))of temozolomide,and the flow cytometry was used to evaluate the cell apoptosis.Western blot was used to detect the expression level of DOK3,O^(6)-methylguanine-DNA methyltransferase(MGMT),phosphatidylinositol 3-kinase(PI3K),p-PI3K,AKT,and p-AKT proteins.Transwell was used to measure the migration ability of cells,and immunofluorescence was used to measure the nuclear translocation of nuclear transcription factor-κB(NF-κB).Results The expression level of DOK3 in temozolomide-resistant U251 cells was lower than its parent cells,while the expression levels of MGMT,p-PI3K,and p-AKT were higher than their parent cells.Overexpression of DOK3 reduced the IC_(50) value of temozolomide-resistant cells and accelerated temozolomide-induced cell apoptosis and cell migration inhibition.Meanwhile,overexpression of DOK3 reduced the expression of MGMT,p-PI3K,and p-AKT and inhibited nuclear translocation of NF-κB.Conclusion DOK3 regulates MGMT through the inactivated PI3K/AKT/NF-κB pathway,leading to drug resistance of glioma cells to temozolomide.