芍药苷通过影响EGFR/RhoA通路在非小细胞肺癌A549细胞中的抗肿瘤活性

The antitumor activity of paeoniflorin in non-small cell lung cancer A549 cells via EGFR/RhoA pathway

目的:探究基于生信靶点和基础实验研究芍药苷(PF)对人非小细胞肺癌细胞(A549)的潜在抗肿瘤活性机制。方法:分别采用CCK-8实验、平板克隆实验、细胞划痕实验及流式细胞术实验检测PF对NSCLC细胞系A549生长、迁移和凋亡的影响;利用SwissTargetPrediction、PharmMapper和ChEMBL数据库检索PF的相关靶点,通过GeneCards、DisGeNET和OMIM数据库中检索NSCLC的靶点,使用STRING和DAVID数据库进行蛋白质-蛋白质分析与富集分析,运用SYBYL软件对排名前10靶点对应的蛋白质与PF进行分子对接,应用Western blot检测PF对p-AKT和p-EGFR和RhoA蛋白表达。结果:CCK-8和平板克隆实验表明PF能有效抑制肺癌细胞生长。50μmol/L和100μmol/L的PF在24 h对A549细胞的迁移率分别为22.89%、13.78%,低于空白组的55.85%(P<0.05)。PF在24 h对A549细胞的凋亡率高于对照组(P<0.05)。基因靶点预测与验证结果显示:PF与NSCLC的核心靶点为32个,对NSCLC富集分析发现,包含PI3K-AKT、MAPK、Ras、Rap1、雌激素、FoxO和VEGF信号通路等7条潜在作用通路。分子对接结果显示,与PF作用排名前三的蛋白质分别为RhoA、AKT和EGFR。与对照组相比,PF干预A549细胞后p-AKT和p-EGFR和RhoA的表达水平降低,差异有统计学意义(P<0.05)。结论:PF可能通过调控EGFR/RhoA通路抑制非小细胞肺癌A549细胞生长、迁移并促进其凋亡。

Objective:To explore the potential antitumor mchanism of Paeoniflorin(PF)induced human non-small cell lung cancer(A549)cells by combination with basic experiments and bioinformation methods.Methods:The effects of PF on the viability,migration and apoptosis of NSCLC cell line A549 were detected by CCK-8 assay,colony-formation assay,wound healing assay and flow cytometry respectively.The related targets of PF were collected from SwissTargetPrediction,PharmMapper and ChEMBL databases.The NSCLC targets were collected from GeneCards,DisGeNET and OMIM databases.STRING and DAVID databases were used to conduct protein-protein analysis and enrichment analysis of common targets.SYBYL software was used to simulated the interaction of PF with the proteins corresponding to the top 10 targets,and Western blot was used to detect the expression of p-AKT,p-EGFR,and RhoA protein treated by PF on A549 cells.Results:The CCK-8 and colony-formation experiment showed that PF could inhibit cell growth effectively.The migration rates of A549 cells treated with 50μmol/L and 100μmol/L PF at 24 h was 22.89%and 13.78%,respectively,which was lower than that of the 0μmol/L group(55.85%,P<0.05).The apoptosis rate of A549 cells treated with PF at 24 h was higher than that of the control group(P<0.05).The results of gene target prediction and verification showed 32 core targets of PF and NSCLC were identified by topological analysis.KEGG enrichment analysis revealed 7 pathways including PI3K-AKT,MAPK,Ras,Rap1,estrogen,FoxO and VEGF signaling pathways.SYBYL showed that RhoA,AKT and EGFR were the top 3 proteins with the highest binding scores to PF.Compared with the control group,the expression of core proteins including p-AKT,p-EGFR and RhoA was decreased after PF intervention,which were statistically significant(P<0.05).Conclusion:PF could inhibit the proliferation,migration and promote apoptosis of NSCLC A549 cells by regulating the EGFR/RhoA pathway.