异泽兰黄素通过EZH2/H3k27me3信号通路抑制非小细胞肺癌增殖和侵袭转移OA

Objective:To investigate the mechanisms by which eupatilin(Eup)inhibits proliferation,invasion,and metastasis of non-small cell lung cancer(NSCLC)through the enhancer of zeste homolog 2/histone H3 lysine 27 trimethylation(EZH2/H3K27me3)signaling pathway.Methods:In vivo,a subcutaneous xenograft tumor model was established in nude mice using H1299 cells to evaluate the anti-NSCLC effects of Eup.Immunohistochemistry(IHC-P)was used to detect the expression of proliferation-and invasion/metastasis-related proteins,including proliferating cell nuclear antigen(PCNA),matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),and vascular endothelial growth factor A(VEGFA).In vitro,cell counting kit-8(CCK-8)assays were performed to determine the viability of H1299 cells treated with different concentrations of Eup(0-200 μmol·L-1)and to select appropriate concentrations.Colony formation and 5-ethynyl-2'-deoxyuridine(EdU)assays were used to evaluate cell proliferation.Wound healing and invasion assays were conducted to assess cell migration and invasion.Human umbilical vein endothelial cell(HUVEC)angiogenesis assays were used to evaluate the effects of Eup on angiogenesis.Transcriptomic analysis was performed to identify the targets of Eup in H1299 cells and to explore its major functions.Molecular docking and molecular dynamics simulations were conducted to predict the binding affinity and interaction stability between Eup and its target proteins.Western blot was used to detect the effects of Eup on the expression levels of EZH2/H3K27me3 pathway-related proteins and proliferation-and invasion/metastasis-related proteins,including PCNA,MMP-2,MMP-9,and VEGFA.Results:In the subcutaneous xenograft model,compared with the model group,Eup treatment dose-dependently inhibited the growth of H1299 xenograft tumors,and the tumor inhibition rate was significantly increased(P＜0.05).IHC-P results showed that,compared with the model group,high-dose Eup significantly reduced the expression levels of PCNA,MMP-2,MMP-9,and VEGFA in vivo(P＜0.05).In vitro,compared with the control group,Eup inhibited the proliferation,invasion,and metastasis of NSCLC cells in a concentration-dependent manner.Transcriptomic analysis further showed that,compared with the control group,Eup significantly downregulated EZH2 expression,and its functional effects were associated with inhibition of tumor metastasis.Molecular docking and molecular dynamics simulations indicated that Eup exhibited strong binding affinity with EZH2 and stable interactions.Western blot results demonstrated that,compared with the model group,Eup significantly inhibited,in a dose-dependent manner,the expression levels of EZH2,H3K27me3,and proliferation-and invasion/metastasis-related proteins(PCNA,MMP-2,MMP-9,and VEGFA)in both in vivo and in vitro experiments(P＜0.05).In vitro,compared with the control group,overexpression of EZH2 via plasmid transfection partially reversed the inhibitory effects of Eup on the expression of key proteins involved in proliferation and invasion/metastasis in H1299 cells.Conclusion:Eup effectively inhibits the proliferation,migration,and invasion of H1299 cells both in vivo and in vitro.The underlying mechanism may be related to inhibition of the EZH2/H3K27me3 signaling pathway and downregulation of proliferation-and invasion/metastasis-related proteins,including PCNA,MMP-2,MMP-9,and VEGFA.Eup may serve as a potential therapeutic agent for suppressing proliferation and invasion/metastasis in NSCLC.