木犀草素通过调节PI3K/AKT/NF-κB信号通路对骨肉瘤U2OS细胞EMT转化和细胞生物学行为的影响

Luteolin affects the EMT transformation and cell biological behavior of osteosarcoma U2OS cells via the PI3K/AKT/NF-κB signaling pathway

目的:探讨木犀草素通过调节磷脂酰肌醇-3-激酶/蛋白激酶B/核转录因子-kappa B(phosphoinositide 3-kinase/protein kinase B/nuclear factor-kappaB,PI3K/AKT/NF-κB)信号通路对骨肉瘤U2OS细胞(epithelial-mesenchymal transition,EMT)作用的分子机制。方法:采用不同浓度的木犀草素(10、20、40μmol/L)处理U2OS细胞,MTT法检测木犀草素对U2OS细胞增殖的影响;Transwell实验检测木犀草素对U2OS细胞迁移的作用;qPCR检测木犀草素对Bax、Bcl-2、Caspase-2 mRNA表达的影响;Western blot检测木犀草素对p-PI3K、p-AKT、p-IKK、NF-κB等PI3K/AKT/NF-κB途径相关蛋白表达的影响;免疫荧光检测木犀草素对EMT相关蛋白E-cadherin、vimentin表达的影响。结果:木犀草素可明显抑制U2OS细胞增殖(P<0.05),且具有时间-浓度依赖性;同时,可以显著抑制U2OS细胞的迁移(P<0.05);可以上调U2OS细胞内Bax、Caspase-2 mRNA表达水平(P<0.05),下调Bcl-2 mRNA表达水平(P<0.05);明显降低U2OS细胞内p-PI3K、p-AKT、p-IKK、NF-κB蛋白表达(P<0.05),同时显著抑制E-cadherin、vimentin蛋白表达(P<0.05)。结论:木犀草素可能通过抑制PI3K/AKT/NF-κB信号通路活化从而发挥抑制U2OS细胞增殖、迁移和EMT转化作用。

Objective:To investigate the effect of luteolin on the epithelial-mesenchymal transition(EMT)of osteosarcoma cell line U2OS and its molecular mechanism by regulating phosphoinositide 3-kinase/protein kinase B/nuclear factor-kappaB(PI3K/AKT/NF-κB)signaling pathway.Methods:U2OS cells were treated in different concentrations(10,20 and 40μmol/L)of luteolin.MTT assay was used to detect the effect of luteolin on the proliferation of osteosarcoma U2OS cells and transwell chamber assay to detect the influence of luteolin on migration of U2OS cells.In addition,the influence of luteolin on the mRNA expressions of Bax,Bcl-2 and Caspase-2 in U2OS cells was evaluated via qPCR.The changes of p-PI3K,p-AKT,p-IKK and NF-κB proteins were also observed through Western blot.Immunofluorescence was applied to detect the influence of luteolin on the protein expression of E-cadherin and vimentim.Results:Luteolin inhibited significantly the proliferation(P<0.05)in a time-concentration-dependent manner and the migration of U2OS cells(P<0.05).After treatment with luteolin,the mRNA expressions of Bax and Caspase-2 were upregulated significantly(P<0.05),while Bcl-2 was downregulated significantly(P<0.05)in U2OS cells.Moreover,the protein expressions of p-PI3K,p-AKT,p-IKK,NF-κB,E-cadherin and vimentin were decreased significantly(P<0.05).Conclusion:Luteolin inhibits the proliferation,migration and EMT transformation of osteosarcoma U2OS cells,which may be related to the inhibition of PI3K/AKT/NF-κB signaling pathway.