甘草素通过调控miRNA抑制人黑色素瘤A375细胞的侵袭转移

Liquiritigenin inhibits migration and invasion of human melanoma A375 cells via regulating miRNA expression

目的 :探讨甘草素对人黑色素瘤A375细胞侵袭转移的抑制作用及与miRNA相关调控机制。方法 :不同浓度甘草素处理24 h后,采用噻唑蓝(MTT)法测定细胞存活情况,确定甘草素对细胞无毒作用剂量;通过划痕试验和Transwell小室迁移、侵袭试验分别观察细胞的非定向迁移力和定向迁移侵袭力;miRNA芯片筛选差异表达的miRNA,q PCR方法验证hsa-miR-4534和hsa-miR-4487的下调表达;Western blot检测PTEN、p-AKT、AKT、MMP2和TIMP2蛋白表达。结果:10~100μmol/L剂量甘草素处理A375细胞24 h对细胞无明显损伤作用,划痕试验、Transwell小室迁移试验、侵袭试验显示甘草素能抑制A375细胞侵袭转移;甘草素可下调hsa-miR-4534和hsa-miR-4487的表达,上调PTEN、TIMP2表达水平,降低p-AKT、MMP2的表达水平。结论:甘草素通过下调hsa-miR-4534和hsa-miR-4487表达,进而上调PTEN和TIMP2靶基因的表达水平,阻碍p-AKT信号通路并降低MMP2蛋白表达,从而发挥抑制人黑色素瘤A375细胞侵袭和转移的作用。

Objective：To investigate the effect of liquiritigenin （LQ） on cell migration and invasion by monitoring regulation of miRNA and its downstream target gene expression in human melanoma A375 cells. Methods：MTl＇ assay was used to detect the cell viability of A375 cells treated with different concentrations of LQ. The effect of LQ on cells migration and invasion was measured by wound healing method and Transwell migration/invasion assay. Decreased expression of hsa-miR-4487 and hsa-miR-4534 was screened by miRNA Expression Array, and confirmed by quantitative PCR. The expression levels of PTEN,p-AKT,AKT,MMP2, and TIMP2 were examined by Western blot. Results：Ten to 100 p^mol/L of LQ treatment on A375 cells for 24 h had no obvious damage to cells. LQ inhibited cell migration and invasion in a dose-dependent manner. Hsa-miR-4534 and hsa-miR-4487 expression were downregulated by LQ. Meanwhile,LQ increased the expression level of VFEN and TIMP2,and decreased p-AKT and MMP2 expression in A375 cells. Conclusion：LQ inhibits migration and invasion of A375 cells,probably via regulating miRNA expression and its downstream target genes.