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目的探讨非小细胞肺癌中磷脂酰肌醇-3-激酶(PI3K)/AKT和丝裂原活化蛋白激酶MAPK/ERK信号通路对Forkhead转录因子(Fox蛋白家族)FOXO1活性的影响及分子机制。方法体外培养人非小细胞肺癌系A549,分别选用PI3K/AKT信号通路特异性抑制剂LY294002和MAPK/ERK信号通路特异性抑制剂UO126及两种抑制剂联合处理细胞之后,MTT比色法检测其对细胞增殖的影响;Western blot检测蛋白FOXO1、p-FOXO1及信号下游蛋白Bim表达的变化;免疫荧光法检测FOXO1蛋白在A549细胞中亚细胞的定位的变化。结果与对照组相比,LY294002和UO126都能明显地抑制A549细胞的增殖,且具有时间依赖性。Western blot结果显示,FOXO1的蛋白水平未见显著性变化,而FOXO1的磷酸化水平明显下降,Bim的表达相应增加。免疫荧光结果显示,FOXO1在A549细胞中的核转位增多。LY294002和UO126联合处理A549细胞时,较药物单独作用效果明显增加。结论 PI3K/AKT和MAPK/ERK信号通路能调节FOXO1磷酸化水平,且具有协同作用,抑制其转录活性。FOXO1通过激活Bim蛋白,促进细胞凋亡,抑制细胞的增殖。
Objective To investigate the effect of phosphatidylinositol 3-kinase (AKT) / AKT and mitogen-activated protein kinase MAPK / ERK signaling pathway on FOXO1 activity in Forkhead transcription factor (Fox protein family) and its molecular mechanism in non-small cell lung cancer. Methods Human non-small cell lung cancer cell line A549 was cultured in vitro. The cells were treated with LY294002, a specific inhibitor of PI3K / AKT signaling pathway and UO126, a MAPK / ERK signaling pathway inhibitor, and the two inhibitors. MTT assay The changes of FOXO1, p-FOXO1 and Bim expression were detected by Western blot. The changes of FOXO1 protein subunit localization in A549 cells were detected by immunofluorescence. Results Compared with the control group, both LY294002 and UO126 could significantly inhibit the proliferation of A549 cells in a time-dependent manner. Western blot results showed that there was no significant change in the protein level of FOXO1, but the phosphorylation of FOXO1 was significantly decreased and the expression of Bim was increased correspondingly. Immunofluorescence results showed that FOXO1 nuclear translocation in A549 cells increased. LY294002 and UO126 combined treatment of A549 cells, compared with the drug effect alone significantly increased. Conclusion The PI3K / AKT and MAPK / ERK signal pathways can regulate FOXO1 phosphorylation and have a synergistic effect and inhibit their transcriptional activity. FOXO1 through the activation of Bim protein, promote apoptosis, inhibit cell proliferation.