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

关键词:  胶质母细胞瘤  细胞活力  上皮间质转化  桃叶珊瑚苷  RhoA/ROCK信号通路

Key words:  glioblastoma  cell viability  epithelial mesenchymal transformation  aucubin  RhoA/ROCK signaling pathway

摘要:

[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（WesternBlot）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。

Abstract:

[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 10,25,50 μmol/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.