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 FOXM1 siRNA transfection group was 37.3 ± 8.6,significantly lower than that of the blank control (135.3 ± 7.0) and 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.FOXM1siRNA transfection induced cell cycle arrest at G2/M phase with a percentage of (55.6 ± 4.83) ％,significantly higher than that of the blank control [(24.30 ± 1.95) ％]and negative control group [(21.3 ± 2.06) ％,P ＜ 0.05].Additionally,the FOXM1 siRNA transfection significantly increased the chemosensitivity of A549 cells to AG1478 (P ＜ 0.05).Besides,AG1478 induced expression and nuclear relocation of FOXO3a.After the FOXO3a siRNA transfection,the expression of FOXM1 protein was significantly up-regulated,and resulted in a reduction of AG1478-induced inhibition of FOXM1.Conclusions The expression of FOXM1 is down-regulated by AG1478 via FOXO3a in the NSCLC cell lines,and then increases the chemosensitivity of A549 cells to AG1478.It suggests that FOXM1 could be a potential target for the therapy and drug exploitation for NSCLC.%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叉头转录因子M1 (FOXM1)、叉头转录因子O3a(FOXO3a)的表达调控,以及RNA干扰技术下调FOXM1、FOXO3a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Western blot法检测肺腺癌细胞株A549中FOXM1和FOXO3a mRNA和蛋白的表达情况；瞬时转染FOXM1和FOXO3a小干扰RNA (siRNA)后,RT-PCR和Western blot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FOXM1 mRNA和蛋白的表达(均P＜0.05).转染FOXM1 siRNA后,FOXM1 mRNA和蛋白及其细胞周期蛋白B1(cyclin B1)、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FOXM1 siRNA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FOXM1 siRNA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).FOXM1 siRNA转染组的G2/M期细胞比例为(55.60±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FOXM1 siRNA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FOXO3a分子表达并促进其核定位；转染FOXO3a siRNA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FOXO3a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.
展开▼
