人表皮生长因子受体2插入突变导致拉帕替尼耐药的机制

The mechanism of human epidermal growth factor receptor 2 insertion mutation leading to lapa tinib resistance

目的探讨人表皮生长因子受体2(ERBB2)的p.Pro780_Tyr781 insGSP插入突变对诱导Lapatinib耐药的机制研究.方法采用聚合酶链反应(PCR)、酶切、连接等方法构建对照组pHelper.CopGFP、野生组pHelper.GV358-ERBB2、突变组pHelper.GV492-ERBB2质粒转染293T细胞,收集上清并包装慢病毒,检测滴度为108,在融合率为70%~80%的MCF-7细胞中进行转染,并在1 mg/L的嘌呤霉素中进行药筛构建稳转株,通过免疫荧光及蛋白印迹法(Western blot)检测转染效率,实验分3组:ERBB2-NC、ERBB2-WT和ERBB2-MT组.通过细胞增殖细胞计数试剂盒(CCK-8)法检测拉帕替尼作用48 h后MCF-7细胞的半数最大抑制浓度(IC50),Western blot检测在拉帕替尼作用下,ERBB2相关信号通路蛋白的表达变化,并测定拉帕替尼对细胞周期变化和细胞凋亡反应的影响.两组间样本均数比较采用t检验,多组间样本采用方差分析.结果成功构建载体并且测序正确,野生型ERBB2(ERBB2-WT)及突变型ERBB2(ERBB2-MT)稳转株构建成功.细胞增殖抑制实验结果显示ERBB2-MT组的细胞对拉帕替尼的IC50值为(90.41±7.47)μmol/L,明显高于ERBB2-WT组IC50(43.94±9.84)μmol/L,ERBB2-MT组与其他两组比较,对拉帕替尼产生显著抗性(F=55.530,P<0.01),通过增加/蛋白激酶B(Akt)的磷酸化水平促进细胞的生长,引起了ERBB2-MT组[(326.00±36.16)个]细胞克隆较ERBB2-WT组[(201.00±10.03)个]增多(t=7.478,P<0.05),并通过抑制晚期细胞的凋亡(WT:22.29±1.53,MT:10.89±2.42,t=6.905,P<0.01)和增强细胞增殖能力(WT:18.95±0.73,MT:33.05±2.69,t=8.774,P<0.01)对拉帕替尼产生耐药性.结论结果表明ERBB2-MT的插入突变为促癌性突变,通过激活Akt信号通路磷酸化而对拉帕替尼的治疗产生抗性.

Objective The insertion mutation of human epidermal growth factor receptor 2(ERBB2)(p.Pro780_Tyr781 insGSP)leads to the emergence of rapatinib resistance through activating protein kinase B(Akt)signaling pathway,in order to explore the potential mechanism of the mutation.Methods Polymerase chain reaction(PCR),restriction enzyme and ligation were used to construct control group pHelper.CopGFP,wild group pHelper.GV358-ERBB2,mutant group pHelper.GV492-ERBB2 plasmid to transfect 293T cells,collect supernatant and package lentivirus.The titer was 108,transfected in MCF-7 cells with a fusion rate of 70%-80%,and the drug was sieved in 1 mg/L puromycin to construct a stable strain,by fluorescence microscopy and western blotting to detect transfection efficiency.The experiment was divided into three groups:ERBB2-NC group,ERBB2-WT group and ERBB2-MT group.The half maximal inhibitory concentration(ICjq)of MCF-7 cells treated with lapatinib for 48 h was detected by cell proliferation cell counting kit-8(CCK-8)method.The expression of ERBB2 related signaling pathway protein was detected by Western blot after lapatinib treatment.The effect of lapatinib on cell cycle and apoptosis was determined.Results The vector was successfully constructed and sequenced correctly.The wild type ERBB2(ERBB2-WT)and mutant ERBB2(ERBB2-MT)stable transformants were successfully constructed.The cell proliferation inhibition experiment showed that the ICjo in the ERBB2-MT group was(90.41±7.47)μmol/L,which was significantly higher than that of the ERBB2-WT group(43.94±9.84)μmol/L.Compared with the other two groups,the ERBB2-MT group showed significant resistance to lapatinib(F=55.530,P<0.01).Cell growth was promoted by increasing the phosphorylation level of Akt,The ERBB2-MT group(326.00±36.16)cell clones increased compared with the ERBB2-WT group(201.00±10.03)(t=7.478,P<0.05).And advanced Apoptosis(WT:22.29±1.53,MT:10.89±2.42,t-6.905,P<0.01)and enhanced cell proliferation(WT:18.95±0.73,MT:33.05±2.69,t=8.774,P<0.01)developed resistance to lapatinib.Conclusion The results indicate that the insertion mutation of ERBB2-MT is a cancer-promoting mutation that is resistant to the treatment of lapatinib by activating phosphorylation of the Akt signaling pathway.