薯蓣皂苷元通过调控miR-34a及其靶基因发挥抗胃癌作用

目的探讨薯蓣皂苷元对人胃癌细胞AGS的增殖、克隆形成、迁移及侵袭的抑制作用及可能机制。方法采用细胞增殖抑制率法检测薯蓣皂苷元对胃癌细胞AGS的增殖抑制率,平板克隆形成实验检测其对AGS细胞克隆形成能力的影响,划痕实验观察薯蓣皂苷元对胃癌细胞迁移作用的影响,细胞侵袭实验检测其对AGS细胞侵袭作用的影响。采用小分子核糖核酸(miRNAs)靶基因预测方法预测miRNA-34a(miR-34a)靶基因,实时定量聚合酶链反应(qPCR)方法观察薯蓣皂苷元对AGS细胞miR-34a及其靶基因转录因子E2F1、E2F3、细胞周期蛋白D1(CCND1)表达水平的影响,并分析其与胃癌患者预后。结果 24、30、36、42μmol/L及48μmol/L浓度的薯蓣皂苷元均可显著抑制胃癌AGS细胞增殖,其作用与时间及浓度呈正相关;克隆形成实验显示24、30、36、42μmol/L及48μmol/L浓度的薯蓣皂苷元可抑制AGS细胞克隆形成能力(P<0.01);24、30、36、42μmol/L及48μmol/L浓度的薯蓣皂苷元处理后的胃癌细胞迁移及侵袭能力均显著降低(P<0.01),且其作用与时间、浓度呈正相关。36μmol/L浓度的薯蓣皂苷元作用于AGS细胞后,可显著提高miR-34a表达水平,并降低miR-34a靶基因E2F1、E2F3及CCND1表达水平;E2F1、E2F3及CCND1基因表达与胃癌患者预后呈显著负相关。结论薯蓣皂苷元可能通过调控miR-34a,下调E2F1、E2F3及CCND1基因表达,发挥抑制胃癌AGS细胞的增殖、克隆形成、迁移及侵袭功能。

Anti-cancer effect of diosgenin through regulating miR-34a and its target genes in gastric cancer

Objective To investigate the inhibitory effect and its possible mechanism of diosgenin on the proliferation, colony formation, migration and invasion of human gastric cancer cell(AGS). Methods The cell proliferation assay was used to detect the inhibition effect of diosgenin on AGS proliferation. The colony formation assay was performed to detect the effect of diosgenin on the colony formation efficiency of AGS cells. The wound healing test and transwell chamber assay were used to detect the effect of diosgenin on the migration and invasion of AGS. The MicroRNA(MiRNA) Target Prediction was used to predict the target genes of miR-34 a. The Quantitative real-time PCR(qPCR) method was performed to observe the effect of diosgenin on the expression of miR-34 a, E2 F1, E2 F3 and CCND1 in AGS cells, and analyze the correlation between their expression and the prognosis of gastric cancer patients.Results Diosgenin with different concentrations(24 μmol/L,30 μmol/L,36 μmol/L,42 μmol/L,48 μmol/L)could significantly inhibit the proliferation of AGS cells, and there was a positive correlation between the inhibiting effect and the dose and time. In different concentrations of diosgenin(24 μmol/L,30 μmol/L,36 μmol/L,42 μmol/L,48 μmol/L), the colony formation efficiency of AGS cells was significantly decreased(P<0.01), and the migration and invasion of gastric cancer cells were significantly decreased(P<0.01), with the effect positively correlated with time and concentration.Diosgenin(36 μmol/L) significantly increased the expression of miR-34 a in AGS cells and decreased the expression of E2 F1, E2 F3 and CCND1, which were target genes of miR-34 a. The expression of E2 F1, E2 F3 and CCND1 genes was significantly negatively correlated with the prognosis of gastric cancer patients.Conclusion Diosgenin seems to inhibit the proliferation, migration and invasion of AGS cells partly by regulating miR-34 a and down-regulating the expression of E2 F1, E2 F3 and CCND1 genes.