冬凌草甲素调控miR-200c/EZH2轴抑制黑色素瘤细胞上皮-间质转化的机制研究

Mechanism by which oridonin regulates miR-200c/EZH2 axis to inhibit epithelial-mesenchymal transition of melanoma cells

目的 探究冬凌草甲素调控微小RNA200c(miR-200c)/组蛋白甲基化转移酶果蝇zeste基因增强子同源物2(EZH2)轴抑制黑色素瘤细胞(A375细胞)上皮-间质转化(EMT)的影响机制。方法 MTT法检测冬凌草甲素对A375细胞活力的影响;将A375细胞分为对照组、冬凌草甲素组、mimic control组、miR-200c mimics组、冬凌草甲素+inhibitor control组、冬凌草甲素+miR-200c inhibitor组,qRT-PCR检测miR-200c、EZH2 mRNA表达情况;免疫印迹法检测EZH2及EMT相关蛋白表达情况;黏附实验检测A375细胞黏附能力;Transwell实验检测A375细胞侵袭及迁移能力;荧光素酶报告基因实验检测miR-200c与EZH2的靶向关系;构建移植瘤裸鼠模型,检测肿瘤质量;免疫组化法分析EMT相关蛋白表达情况。结果 A375细胞存活率随冬凌草甲素浓度的增加以剂量依赖性显著降低(P<0.05),由于40μmol/L冬凌草甲素接近半数抑制浓度,因此选取40μmol/L冬凌草甲素进行后续实验;与对照组相比,冬凌草甲素组、miR-200c mimics组A375细胞中miR-200c、钙粘附蛋白E(E-cadherin)表达水平显著增加,EZH2 mRNA及蛋白表达水平、神经型钙黏附蛋白(N-cadherin)及波形蛋白(Vimentin)表达水平、迁移细胞数、侵袭细胞数、黏附细胞数显著降低(P<0.05);与对照组、mimic control组相比,miR-200c mimics组EZH2蛋白表达水平显著降低(P<0.05);miR-200c沉默可逆转冬凌草甲素对A375细胞的作用;与对照组相比,冬凌草甲素组肿瘤质量、EZH2 mRNA、N-cadherin及Vimentin蛋白表达水平显著降低,miR-200c、E-cadherin蛋白表达水平显著增加(P<0.05);与冬凌草甲素组相比,冬凌草甲素+miR-200c inhibitor组肿瘤质量、EZH2 mRNA、N-cadherin及Vimentin蛋白表达水平显著增加,miR-200c、E-cadherin蛋白表达水平显著降低(P<0.05)。结论 冬凌草甲素可能通过促进miR-200c/EZH2轴来抑制A375细胞的EMT及肿瘤生长。

Objective To explore the mechanism by which oridonin regulates the microRNA200 c(miR-200 c)/enhancer of zeste homolog 2(EZH2) axis to inhibit the epithelial-mesenchymal transition(EMT) of melanoma cells(A375 cells). Methods The MTT method was used to detect the effect of Rubescensine A on the viability of A375 cells. A375 cells were divided into a control group, oridonin group, mimic control group, miR-200 c mimic group, oridonin+inhibitor control group, and oridonin+miR-200 c inhibitor group. qRT-PCR was used to detect the expression of miR-200 c and EZH2 mRNA, Western blot was used to detect the expression of EZH2 and EMT-related proteins, adhesion test was used to detect the adhesiveness of A375 cells, Transwell test was used to detect the invasiveness and migration abilities of A375 cells, while a luciferase reporter gene experiment was used to detect the targeting relationship between miR-200 c and EZH2, construct a nude mouse model of transplanted tumor to detect tumor quality. Finally, the expression of EMT-related proteins was analyzed immunohistochemically. Results The survival rate of A375 cells decreased significantly with increasing oridonin concentration in a dose-dependent manner(P<0.05). Since 40 μmol/L oridonin was close to the half inhibitory concentration, 40 μmol/L oridonin was selected for follow-up experiments. Compared with those in the control group, the expression levels of miR-200 c and E-cadherin in A375 cells in the oridonin group and miR-200 c mimic group increased significantly, while the expression levels of EZH2 mRNA and protein, neural cadherin(N-cadherin), and Vimentin, number of migrating cells, number of invading cells, and number of adhering cells decreased significantly(P<0.05). Compared with the control group and the mimic control group, the expression level of EZH2 protein in the miR-200 c mimics group was significantly decreased(P<0.05), and the silencing of miR-200 c could reverse the effect of oridonin on A375 cells. Compared with the levels in the control group, the tumor quality and expression levels of EZH2 mRNA, N-cadherin, and Vimentin were significantly decreased in the oridonin group, while the expression levels of miR-200 c and E-cadherin were significantly increased(P<0.05). Moreover, compared with those in the oridonin group, the tumor quality and expression levels of EZH2 mRNA, N-cadherin and Vimentin in the oridonin+miR-200 c inhibitor group were significantly increased, while the expression levels of miR-200 c and E-cadherin were significantly decreased(P<0.05). Conclusions Oridonin may inhibit the EMT of A375 cells and tumor growth by promoting the miR-200 c/EZH2 axis.