三氧化二砷通过FOXO3a/cofilin-1通路调控肺腺癌A549细胞的增殖、凋亡、迁移和侵袭能力

The regulatory roles of arsenic trioxide in inhibiting the proliferation,apoptosis,migration and invasion of lung adenocarcinoma cell A549 through FOXO3a/cofilin-1 pathway

目的:研究三氧化二砷(arsenic trioxide,ATO)对肺腺癌A549细胞的增殖、凋亡、迁移和侵袭能力的影响,及可能的作用机制。方法:用不同浓度的ATO处理肺腺癌A549细胞后,采用CCK-8法检测ATO对A549细胞增殖的影响,以及蛋白质印迹法检测ATO对A549细胞核和细胞质中FOXO3a蛋白表达水平的影响。采用si RNA干扰的方法下调A549细胞中FOXO3a m RNA和蛋白的表达水平,随后分别采用流式细胞术(flow cytometry,FCM)、细胞划痕愈合实验及Transwell小室实验检测ATO单药处理、沉默FOXO3a表达、沉默FOXO3a表达联合ATO处理以及LY294002单药处理对A549细胞凋亡、迁移及侵袭能力的影响,最后再用蛋白质印迹法检测对A549细胞中丝切蛋白1(cofilin-1)及磷酸化cofilin-1(phosphorylated cofilin-1,p-cofilin-1)蛋白表达水平的影响。结果:ATO能抑制A549细胞的增殖(P<0.05),且随着ATO浓度的升高,细胞核中FOXO3a蛋白的表达水平显著升高(P<0.01),细胞质中FOXO3a蛋白的表达水平显著降低(P<0.01)。ATO和LY294002都能显著提高A549细胞的凋亡率(P均<0.05),而抑制A549细胞的迁移及侵袭能力(P均<0.05);沉默FOXO3a表达后,A549细胞的凋亡率显著降低(P<0.01),细胞的迁移及侵袭能力则显著提高(P均<0.05);ATO能部分逆转沉默FOXO3a表达导致的A549细胞的凋亡率的降低(P<0.05),以及细胞的迁移及侵袭能力的提高(P均<0.05)。蛋白质印迹法检测结果提示,ATO和LY294002都能显著下调A549细胞中p-cofilin-1蛋白的表达水平(P均<0.05),沉默FOXO3a表达后细胞中p-cofilin-1蛋白的表达水平显著上调(P<0.01),ATO能部分逆转沉默FOXO3a表达导致的p-Cofilin-1蛋白表达水平的上调(P<0.01)。所有分组中cofilin-1蛋白的表达水平未发生变化。结论:ATO能明显抑制A549细胞的增殖、迁移及侵袭,并促进细胞的凋亡,FOXO3a可能为ATO抑制肺癌恶性进展的一个潜在靶点,且FOXO3a可能通过激活cofilin-1的磷酸化发挥抑癌作用。

Objective:To investigate the effect of arsenic trioxide(ATO)on the proliferation,apoptosis,migration and invasion of human lung adenocarcinoma cell A549 and the underlying mechanism.Methods:CCK8 assay was used to examine the inhibitory effect of ATO on the proliferation of A549 cells after treatment with different concentrations of ATO.Western blotting was used to detect the effect of ATO on the expression level of FOXO3a protein in the cell nucleus and cytoplasm.RNA interference with siRNA was used to silence the expression of FOXO3a on both mRNA and protein level.Then,flow cytometry(FCM)assay,wound healing assay and Transwell assay were used to investigate the effect on the apoptosis,migration and invasion of A549 cells,respectively,upon ATO treatment,FOXO3a silencing,FOXO3a silencing combined with ATO treatment or LY294002 treatment.Finally,Western blotting was used to examine the effect of the above treatments on the expression levels of cofilin-1 and p-cofilin-1(phosphorylated cofilin-1)protein in A549 cells.Results:ATO inhibited the proliferation of A549 cells(P<0.05).With the increase of ATO concentration,the expression of FOXO3a protein in the cell nucleus increased significantly(P<0.01),while that in the cytoplasm decreased significantly(P<0.01).Both ATO and LY294002 could significantly increase the apoptosis rate(both P<0.05)while inhibit the migration and invasion of A549 cells(all P<0.05).The apoptosis rate of A549 cells decreased significantly(P<0.01),while the migration and invasion of A549 cells was significantly enhanced(P<0.01)after FOXO3a silencing.ATO could partially reverse the inhibitory effect of FOXO3a silencing on the apoptosis rate(P<0.05)and the promotional effect of FOXO3a silencing on the migration and invasion of A549 cells(both P<0.05).Western blotting analysis results showed that both ATO and LY294002 could significantly down-regulate the expression level of p-cofilin-1 in A549 cells(both P<0.05);the expression level of p-cofilin-1 was significantly upregulated in A549 cells after FOXO3a silencing;ATO could partially reverse the up-regulatory effect of FOXO3a silencing on the expression of p-cofilin-1(P<0.01);and the expression level of cofilin-1 did not change in all treatment groups.Conclusion:ATO can obviously inhibit the proliferation,migration and invasion of A549 cells and promote the apoptosis of A549 cells.FOXO3a may be a potential molecular target of ATO for suppressing the malignant progression of lung cancer and may show its anti-cancer effect through stimulating the phosphorylation of cofilin-1.