桃叶珊瑚苷调节RhoA/ROCK信号通路对胶质母细胞瘤细胞活力和上皮间质转化的影响

Impacts of aucubin on viability and epithelial mesenchymal transformation of glioblastoma cells by regulating RhoA/ROCK signaling pathway

[目的]研究桃叶珊瑚苷(AU)对胶质母细胞瘤(GBM)细胞活力和上皮间质转化(EMT)的影响,并对其作用机制进行探讨。[方法]将U87细胞随机分为对照组、AU低浓度组、AU中浓度组、AU高浓度组、Y-27632组、AU高浓度+Y-27632组。细胞计数器试剂盒(CCK-8)法检测细胞活力,流式细胞术检测细胞凋亡,Transwell小室实验检测细胞迁移和侵袭,蛋白免疫印迹法(Western Blot)检测基质金属蛋白酶(MMP)2、MMP9、波形蛋白(Vimentin)、上皮钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)、Ras同源基因家族成员A(RhoA)、Rho相关卷曲螺旋蛋白激酶(ROCK)1、ROCK2表达。构建GBM裸鼠模型,随机分为裸鼠对照组、AU组、Y-27632组、AU+Y-27632组,测量肿瘤质量与体积,免疫组化法检测移植瘤组织RhoA、ROCK1、ROCK2蛋白表达。[结果]GBM细胞活力随着AU浓度的升高而逐渐降低(P<0.05),选择U87作为后续实验细胞,选择10、25、50μmol/L浓度作为AU后续实验浓度。与对照组比较,AU低、中、高浓度组和Y-27632组细胞活力、迁移和侵袭细胞数、MMP2、MMP9、N-cadherin、Vimentin、RhoA、ROCK1、ROCK2表达显著下降,凋亡率、E-cadherin表达显著升高(P<0.05),其中高浓度AU和Y-27632组共同处理的细胞变化更显著(P<0.05)。AU和Y-27632均能抑制移植瘤质量和体积,降低RhoA/ROCK信号通路蛋白表达(P<0.05)。[结论]AU能抑制GBM细胞活力、迁移侵袭和EMT,促进细胞凋亡,其作用机制可能与抑制RhoA/ROCK信号通路有关。

[Objective]To investigate the impacts of aucubin(AU)on the viability and epithelial-mesenchymal transition(EMT)of glioblastoma(GBM)cells,and to explore its mechanism of action.[Methods]U87 were grouped into control group,low concentration group,medium concentration group,high concentration group,Y-27632 group,and high concentration+Y-27632 group.Cell counting kit-8(CCK-8)method was applied to detect cell viability;flow cytometry was applied to detect cell apoptosis;Transwell cell experiment was applied to detect cell migration and invasion;Western blot was applied to detect the expression of matrix metalloproteinase(MMP)2,MMP9,Vimentin,E-cadherin,N-cadherin,RAS homologous gene family member A(RhoA),Rho-related coiled protein kinase(ROCK)1,ROCK2.The nude mouse model of GBM was constructed,and was grouped into nude mice control group,AU group,Y-27632 group,and AU+Y-27632 group;the mass and volume of tumors were measured,immunohistochemical method was applied to detect the expression of RhoA,ROCK1,and ROCK2 proteins in transplanted tumor tissue.[Results]The viability of GBM cells gradually decreased with the increase of AU concentration(P<0.05);U87 was selected as the follow-up experimental cells,and 10,25,50μmol/L was selected as the follow-up experimental concentration of AU.Compared with the control group,the cell viability,migration and invasion cell counts,MMP2,MMP9,N-cadherin,Vimentin,RhoA,ROCK1,and ROCK2 expression in the low,medium,and high concentration AU groups and Y-27632 group were obviously decreased,the apoptosis rate and E-cadherin expression were obviously increased(P<0.05);the changes in high concentration AU and Y-27632 group were more obvious(P<0.05).Both AU and Y-27632 were able to inhibit the mass and volume of transplanted tumors,and reduce the expression of RhoA/ROCK signaling pathway proteins(P<0.05).[Conclusion]AU can inhibit the viability,migration,invasion and EMT of GBM cells and promote cell apoptosis,which may be related to the inhibition of RhoA/ROCK signaling pathway.