[摘 要] 目的：探讨丹皮酚（Pae）通过调控果蝇zeste基因增强子同源物2/神经外营养蛋白4抗体/细胞周期蛋白依赖性激酶抑 制因子2A（EZH2/NXPH4/CDKN2A）轴对肺癌A549细胞增殖、迁移和侵袭能力的影响。方法：以梯度浓度（0、6.25、12.5、25、50、 100、200 μg/mL）的Pae处理肺癌A549细胞，采用CCK-8法确定干预浓度；将A549细胞分为对照组（未经任何处理的A549细胞）、 Pae 组（12.5 μg/mL Pae）、Pae + siEZH2组（转染siEZH2 + 12.5 μg/mL Pae）、Pae+siNC组（转染siNC + 12.5 μg/mL Pae）、Pae + vector NC组（转染vectorNC + 12.5 μg/mL Pae）、Pae + vectorEZH2组（转染vector EZH2+12.5 μg/mL Pae），予以相应的处理后，采 用克隆形成实验检测细胞克隆数，流式细胞术检测细胞周期分布，Transwell实验检测细胞侵袭能力的变化，划痕实验检测细胞迁 移能力的变化，流式细胞仪检测细胞凋亡情况，WB法检测EZH2、NXPH4、CDKN2A、Bcl-2和caspase-3蛋白表达量。在A549细 胞中单独转染siEZH2或siNC，采用流式细胞仪测量细胞凋亡，WB法检测Bcl-2和caspase-3蛋白表达。建立A549细胞裸鼠移植 瘤模型，评估Pae的体内抗肿瘤作用及其对相关蛋白表达的影响。结果：选择12.5 μg/mL为后续实验干预浓度。与对照组相比， Pae组细胞克隆数、S期细胞比例、迁移、侵袭能力以及EZH2、NXPH4和Bcl-2蛋白表达量显著降低，G0/G1期细胞比例、细胞凋亡 率以及CDKN2A和caspase-3蛋白表达量明显升高（均P < 0.05）；与Pae + siNC组相比，Pae + siEZH2组细胞克隆数、S期细胞比 例、迁移侵袭能力以及EZH2、NXPH4和Bcl-2蛋白表达量下降幅度更大，G0/G1期细胞比例、细胞凋亡率以及CDKN2A和 caspase-3蛋白表达量升高幅度更大（P < 0.05）；与Pae+vectorNC组相比，Pae + vectorEZH2组细胞克隆数、S期细胞比例、迁移侵 袭能力以及EZH2、NXPH4和Bcl-2蛋白表达量显著升高，G0/G1期细胞比例、细胞凋亡率以及CDKN2A和caspase-3蛋白表达量 明显下降（P < 0.05）。敲低EZH2后A549细胞凋亡率和caspase-3表达明显上升，Bcl-2表达明显下降（P < 0.05）。体内实验表明 Pae可显著降低肿瘤体积和质量且抑制EZH2/NXPH4/CDKN2A通路的活性。结论：Pae通过抑制EZH2/NXPH4/CDKN2A通路 抑制A549细胞增殖、迁移和侵袭，诱导细胞凋亡。

[Abstract] Objective: To investigate the effects of paeonol (Pae) on the proliferation, migration, and invasion abilities of lung cancer A549 cells by regulating the enhancer of zeste homolog 2/neurexophilin 4 /cyclin dependent kinase inhibitor 2A (EZH2/NXPH4/ CDKN2A) axis. Methods: A549 lung cancer cell lines were treated with Pae at gradient concentrations (0, 6.25, 12.5, 25, 50, 100, 200 μg/mL). CCK-8 assay was used to screen for suitable Pae concentrations. A549 cells were assigned into the control group (untreated A549 cells), the Pae group (12.5 μg/mL Pae), the Pae + siEZH2 group (transfected with siEZH2+12.5 μg/mL Pae), the Pae + siNC group (transfected with siNC+12.5 μg/mL Pae), the Pae + vectorNC group (transfected with vector NC + 12.5 μg/mL Pae), and the Pae + vectorEZH2 group (transfected with vector EZH2 + 12.5 μg/mL Pae). After the indicated treatments, the colony formation assay was applied to detect the number of cell clones. Cell cycle distribution was measured by flow cytometry. Transwell assay was used to detect changes in cell invasion ability. Scratch assay was employed to detect changes in cell migration ability. Flow cytometry was applied to detect cell apoptosis. WB assay was used to detect the protein expression levels of EZH2, NXPH4, CDKN2A, Bcl-2, and caspase-3. Only siEZH2 or only siNC were transfected into A549 cells. Cell apoptosis was measured by flow cytometry, and the expressions of Bcl-2 and caspase-3 proteins were detected by WB assay. AnA549-cell nude-mouse transplant model was established to evaluate the in vivo antitumor efficacy of Pae and its effects on the expressions of relevant proteins. Results: 12.5 μg/mL was chosen as the concentration for subsequent experiments. Compared with those in the control group, the cell clone number, the proportion of S phase cells, the migration ability, the invasion ability, and the expression levels of EZH2, NXPH4, and Bcl-2 proteins were significantly reduced in the Pae group, while the proportion of G0/G1 phase cells, the apoptosis rate and the expression levels of CDKN2A and caspase-3 proteins were significantly increased (all P < 0.05). Compared with those in the Pae + si NC group, the cell clone number, the proportion of S phase cells, the migration ability, the invasion ability, and the expression levels of EZH2, NXPH4, and Bcl-2 proteins decreased more markedly in the Pae + siEZH2 group, while the proportion of G0/G1 phase cells, the apoptosis rate and the expression levels of CDKN2A and caspase-3 proteins increased more markedly (all P < 0.05). Compared with those in the Pae+vector NC group, the cell clone number, the proportion of S phase cells, the migration and invasion abilities, and the expression levels of EZH2, NXPH4, and Bcl-2 proteins increased significantly in the Pae + vectorEZH2 group, while the proportion of G0/G1 phase cells, the apoptosis rate and the expression levels of CDKN2A and caspase-3 proteins decreased significantly (P < 0.05). After EZH2 knockdown, the apoptosis rate and caspase-3 expression of A549 cells increased significantly, while Bcl-2 expression decreased significantly (both P < 0.05). In vivo experiments showed that paeonol significantly reduced tumor volume and mass, and inhibited the activity of the EZH2/NXPH4/ CDKN2A pathway. Conclusion: Pae inhibits the proliferation, migration and invasion of A549 cells and induces apoptosis by down regulating the EZH2/NXPH4/CDKN2Apathway.

天津市自然科学基金（No. 23JCYBJC01350）