归芪白术方调控let-7a-5p/MDM2/p53轴促进胃癌细胞凋亡和周期阻滞的机制研究

Mechanism study of Guiqi Baizhu Formula regulating the let-7a-5p/MDM2/p53 axis to promote apoptosis and cell cycle arrest in gastric cancer cells

关键词:  归芪白术方  胃癌  let-7a-5p/MDM2/p53轴  凋亡  周期阻滞

Key words:  Guiqi Baizhu Formula  gastric cancer  let-7a-5p/MDM2/p53 axis  apoptosis  cell cycle arrest

摘要:

目的 探讨归芪白术方调控let-7a-5p/MDM2/p53轴促进胃癌细胞凋亡和周期阻滞的机制。方法网络药理学预测归芪白术方治疗胃癌的活性成分及作用靶点；筛选出let-7a-5p表达水平最低的人胃癌HGC-27细胞，分别在24h、48h和72h以不同浓度梯度归芪白术方冻干粉对其进行干预；细胞增殖与活性检测（CCK-8）法检测归芪白术方对HGC-27细胞的增殖能力的影响；流式细胞术检测归芪白术方对HGC-27细胞周期和凋亡率的影响；划痕实验检测归芪白术方对HGC-27细胞迁移能力的影响；实时荧光定量聚合酶链式反应（qRT-PCR）和蛋白免疫印迹法（Western blot）检测归芪白术方对HGC-27细胞中MDM2、p53、Cyclin D1、c-Myc、Bcl-2、Bax的蛋白及mRNA的表达水平的影响，及对let-7a-5p miRNA表达水平的影响。结果网络药理学结果显示归芪白术方药物有效成分160个，对应靶点872个，胃癌相关基因1656个，得到药物疾病共同靶点245个；药物疾病共同靶点与let-7a-5p下游靶基因交集靶点共37个；CCK8法检测结果显示归芪白术方在各时段以浓度依赖性方式对HGC-27细胞具有抑制作用；流式细胞术检测结果显示归芪白术方促进HGC-27细胞周期阻滞和凋亡；细胞划痕实验结果显示归芪白术方抑制HGC-27细胞迁移能力；qRT-PCR和Western blot实验结果显示归芪白术方降低MDM2、CyclinD1、c-Myc、Bcl-2的蛋白和基因表达水平；同时上调let-7a-5p miRNA的表达以及p53、Bax蛋白和基因表达。结论归芪白术方可通过调控let-7a-5p/MDM2/p53轴促进胃癌细胞凋亡和周期阻滞。

Abstract:

OBJECTIVE To investigate the mechanism by which Guiqi Baizhu Formula regulates the let-7a-5p/MDM2/p53 axis to promote apoptosis and cell cycle arrest in gastric cancer cells. METHODS Network pharmacology was used to predict the active components and target molecules of Guiqi Baizhu Formula in the treatment of gastric cancer; the human gastric cancer HGC-27 cells with the lowest let-7a-5p expression levels were selected, and Guiqi Baizhu Formula freeze-dried powder was administered at different concentration gradients at 24 h, 48 h, and 72 h; the CCK-8 assay was used to assess the effect of Guiqi Baizhu Formula on the proliferation capacity of HGC-27 cells; flow cytometry was used to assess the effects of the formula on the cell cycle and apoptosis rate of HGC-27 cells; a scratch assay was conducted to evaluate the effects of the formula on the migration capacity of HGC-27 cells; real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and protein immunoblotting (Western blot) were used to assess the effects of the formula on the expression levels of MDM2, p53, Cyclin D1, c-Myc, Bcl-2, and Bax proteins and their mRNA in HGC-27 cells, as well as on the expression level of let-7a-5p miRNA.RESULTS Network pharmacology results showed that Guiqi Baizhu Formula contained 160 active components, corresponding to 872 target proteins, 1,656 gastric cancer-related genes, and 245 common drug-disease targets; there were 37 common targets between the drug-disease common targets and the downstream target genes of let-7a-5p; CCK-8 assay results showed that Guiqi Baizhu Formula exhibited concentration-dependent inhibitory effects on HGC-27 cells at various time points; flow cytometry results indicated that Guiqi Baizhu Formula promoted cell cycle arrest and apoptosis in HGC-27 cells; wound healing assay results demonstrated that Guiqi Baizhu Formula inhibited the migratory capacity of HGC-27 cells; qRT-PCR and Western blot results showed that Guiqi Baizhu Formula reduced the protein and gene expression levels of MDM2, Cyclin D1, c-Myc, and Bcl-2; simultaneously, it upregulated the expression of let-7a-5p miRNA and the protein and gene expression levels of p53 and Bax. CONCLUSION Guiqi Baizhu Formula promotes apoptosis and cell cycle arrest in gastric cancer cells by regulating the let-7a-5p/MDM2/p53 axis.