薯蓣皂苷对小鼠恶性黑色素瘤细胞侵袭、迁移及黏附的抑制作用及机制

Inhibitory effects of dioscin on invasion,migration and adhesion of mouse malignant melanoma cells

目的探讨薯蓣皂苷对小鼠恶性黑色素瘤细胞(B16-F10细胞)侵袭、迁移及黏附的抑制作用及机制。方法常规培养B16-F10细胞,用CCK-8法检测不同浓度(20、10、5、2.5、1.25、0.625、0.3125μmol/L)薯蓣皂苷干预48 h后B16-F10细胞存活率,并筛选薯蓣皂苷的最佳实验浓度。将细胞随机分为空白对照组、薯蓣皂苷组、JAK2过表达组、薯蓣皂苷+JAK2过表达组,分别加入阴性对照病毒液、含5μmol/L薯蓣皂苷的DMEM培养基、过表达JAK2病毒液、过表达JAK2病毒液+5μmol/L薯蓣皂苷的DMEM培养基,继续培养24 h。用Transwell实验检测细胞侵袭能力,用划痕实验检测细胞迁移能力,用黏附实验检测细胞黏附情况,用Western blotting法检测细胞内上皮-间充质转化[E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)及波形蛋白(Vimentin)]及Janus激酶(JAK2)/转录激活因子-3(STAT3)信号通路[JAK2、磷酸化JAK2(p-JAK2)、STAT3、磷酸化STAT3(p-STAT3)]相关蛋白表达。结果经不同浓度薯蓣皂苷干预后B16-F10细胞存活率降低,根据薯蓣皂苷48 h半数抑制浓度选择5μmol/L作为后续实验浓度。与空白对照组比较,薯蓣皂苷组细胞迁移率、侵袭率、黏附数量均低(P均<0.05);E-cadherin表达高,N-cadherin、Vimentin表达低(P均<0.05);p-JAK2、p-STAT3蛋白表达低(P均<0.05)。与薯蓣皂苷组比较,JAK2过表达组细胞迁移率、侵袭率、黏附数量均高(P均<0.05);E-cadherin表达低,N-cadherin、Vimentin表达高(P均<0.05);JAK2、p-JAK2、p-STAT3蛋白表达高(P均<0.05)。与JAK2过表达组比较,薯蓣皂苷+JAK2过表达组细胞迁移率、侵袭率、黏附数量均低(P均<0.05);E-cadherin表达高,N-cadherin、Vimentin表达低(P均<0.05);p-JAK2、p-STAT3蛋白表达低(P均<0.05)。结论薯蓣皂苷能够抑制黑色素瘤细胞侵袭、迁移及黏附,其作用机制可能与调控JAK2/STAT3信号通路抑制上皮-间充质转化有关。

Objective To investigate the inhibitory effects and mechanism of dioscin on invasion,migration and adhesion of mouse malignant melanoma cells(B16-F10 cells).Methods B16-F10 cells were routinely cultured,and the survival rates of B16-F10 cells intervened with 20,10,5,2.5,1.25,0.625,and 0.3125μmol/L dioscin were detected by CCK-8,and the optimal experimental concentration of dioscin was selected.The cells were randomly divided into the blank control group,dioscin group,JAK2 overexpression group,and dioscin+JAK2 overexpression group,which were added with the negative control viral solution,DMEM medium containing 5μmol/L dioscin,JAK2 viral solution,and DMEM medium overexpressed JAK2 virus solution+5μmol/L dioscin to culture for 24 h.Cell invasion capacity was detected by Transwell assay,cell migration ability was detected by Scratch assay,and cell adhesion was analyzed.The expression levels of intracellular epithelial-mesenchymal transformation(E-cadherin,N-cadherin and Vimentin)and Janus kinase(JAK2)/transcriptional activator-3(STAT3)signaling pathway-related proteins[JAK2,phosphorylation JAK2,(p-JAK2),STAT3,and phosphorylated STAT3(p-STAT3)]were detected by Western blotting.Results The survival rate of B16-F10 cells decreased after treatment with different concentrations of dioscin,and 5μmol//L was selected as the subsequent experimental concentration according to the half inhibitory concentration of dioscin for 48 h.Compared with the blank control group,the cell migration rate,invasion rate and cells adhesion number decreased(all P<0.05),the expression of E-cadherin increased,while the expression of N-cadherin and Vimentin decreased(both P<0.05),and the expression of p-JAK2 and p-STAT3 proteins decreased in the dioscin group(both P<0.05).Compared with the dioscin group,the cell migration,invasion rate and cells adhesion number increased(all P<0.05),E-cadherin expression decreased,N-cadherin and Vimentin expression increased(all P<0.05),and the expression of JAK2,p-JAK2,p-STAT3 proteins increased in the JAK2 overexpression group(all P<0.05).Compared with the JAK2 overexpression group,the cell migration,invasion rate and cells adhesion number decreased(all P<0.05),E-cadherin expression increased,while the expression of N-cadherin,Vimentin decreased(all P<0.05),and the expression of p-JAK2 and p-STAT3 proteins decreased in the dioscin+JAK2 overexpression group(both P<0.05).Conclusion Dioscin can inhibit melanoma cell invasion,migration and adhesion,and its mechanism may be related to the regulation of JAK2/STAT3 signaling pathway to inhibit epithelial-mesenchymal transformation.