表皮生长因子受体酪氨酸激酶抑制剂AG1478通过叉头转录因子O3a对非小细胞肺癌细胞中叉头转录因子M1表达的影响  

Effects of AG1478 on the expression of FOXM1 gene via FOXO3a in non.small cell lung cancer cells

作　　者：公小迪[1] 袁海花[1] 王炯轶[1] 郭跃辉[1] 时婧[1] 姜斌[1] 

机构地区：[1]上海交通大学医学院附属第三人民医院肿瘤科,201900

出　　处：《中华肿瘤杂志》2013年第8期572-578,共7页Chinese Journal of Oncology

摘　　要：目的 探讨表皮生长因子受体酪氨酸激酶抑制剂AG1478对非小细胞肺癌细胞中又头转录因子M1（FOXM1）、叉头转录因子O3a（FOX03a）的表达调控，以及RNA干扰技术下调FOXM1、FOX03a的表达后对肺癌细胞增殖及其对AG1478药物敏感性的影响。方法采用逆转录聚合酶链反应（RT-PCR）和Westernblot法检测肺腺癌细胞株A549中FOXM1和FOX03amRNA和蛋白的表达情况；瞬时转染FOXM1和FOX03a小干扰RNA（siRNA）后，RT—PCR和Westernblot法检测转染效率和相关蛋白的表达；采用CCK-8法、集落形成实验和流式细胞仪分别检测细胞增殖、集落形成能力及细胞周期分布的改变。结果AG1478呈时间依赖性抑制FOXM1mRNA和蛋白的表达（均P〈0．05）。转染FOXM1siRNA后，FOXM1mRNA和蛋白及其细胞周期蛋白B1（eyelinB1）、c-Myc、Bcl-2蛋白的表达明显下降，p21和剪切后多腺苷二磷酸核糖聚合酶（cleaved—PARP）蛋白的表达则明显上调（均P〈0．05）。空白组、阴性对照组和FOXMIsiRNA转染组的集落数分别为（135．3±7．0）个、（125．3±7．5）个和（37．3±8．6）个，阴性对照组和FOXMlsiRNA转染组的集落形成抑制率分别为（7．40±0．94）％和（72．40±6．09）％（P〈0．05）。FOXMlsiRNA转染组的G2／M期细胞比例为（55．604-4．83）％，明显高于空白组[（24．30±1．95）％]和阴性对照组[（21．30±2．06）％，P〈0．05]。转染FOXM1siRNA后，可明显增加A549细胞对AG1478的药物敏感性（P〈0．05）。AG1478可诱导活化的FOX03a分子表达并促进其核定位；转染FOX03asiRNA后，FOXM1蛋白水平明显上调，并通过活化的AKT削弱AG1478对FOXM1表达的抑制。结论AG1478通过诱导活化的FOX03a表达及胞核重定位，下调FOXM1的表达，进而抑制肺癌细胞增殖，增加肺癌细胞对AG1478的敏感性。Objective To explore the effects of EGFR-TKI AG1478 on the expression of FoxM1 and FOXO3a genes in non-small cell cancer （NSCLC） cell lines, and explore the effect on cell proliferation and drug sensitivity to AG1478 after down-regulation of FOXM1 and FOXO3a expression by RNAi technique. Methods Human lung cancer cells were treated with AG1478 at different concentrations. RT-PCR and Western blot were used to examine the expression of P-EGFR, FOXM1, FOXO3a mRNA and protein. After transient transfection of FOXM1 and FOXO3a siRNA, RT-PCR and Western blot were employed to determine the transfection efficiency and expression of the related proteins. CCK-8 assay, colony formation assay and flow cytometry were performed to evaluate the cell proliferation, colony formation ability and the changes in cell cycle distribution. Results The expressions of FOXM1 mRNA and protein were inhibited by AG1478 in a dose-dependent manner （both P 〈 0.05）. After transfection with FOXM1 siRNA, the expressions of FOXM1 mRNA and protein, and proteins of cyclin B1, c-Myc, and Bcl-2 were significantly down-regulated,and the expressions of p21 and cleaved-PARP proteins were significantly up-regulated （all P 〈0.05）. The colony number of FOXM 1 siRNA transfection group was 37.3± 8.6, significantly lower than that of the hlank control （ 135. 3± 7.0） anti negative control group （ 125. 3 ± 7.5, P 〈 0. 05）. The colony formation inhibition rate was （7.40 ± 0.94）% in the negative control group and （72.4± 6.09 ）% in the FOXM1 siRNA transfection group. FOXMIsiRNA transfection induced cell cycle arrest at GJM phase with a percentage of （55.6 ± 4.83 ）%, significantly higher than that of the hlank control [ （24.30 ±1.95 ）% ] and negative control group [ （21.3 ± 2.06） %, P 〈 0.05 ]. Additionally, the FOXMI siRNA transfection significantly increased the ehemosensitivity of A549 cells to AG1478 （P 〈0.05）. Besides, AG1478 induced expression and nuclear relocation of FOXO3a. After the FOXO3a siRNA t

关 键 词：肺肿瘤 表皮生长因子受体酪氨酸激酶抑制剂 AG1478 叉头转录因子M1 叉头转录因子O3a 细胞增殖 药物敏感性 

分 类 号：R734.2[医药卫生—肿瘤]