桔梗皂苷D基于TGF-β/Smad通路调控上皮-间质转化对人子宫内膜癌Ishiwaka细胞迁移及侵袭的作用

Effect of platycodin D on migration and invasion of human endometrial carcinoma Ishiwaka cells by regulating epithelial mesenchymal transformation based on TGF-β/Smad pathway

目的探讨桔梗皂苷D调控上皮间质转化(EMT)对人子宫内膜癌Ishiwaka细胞迁移、侵袭的影响,及对转化生长因子β(TGF-β)/Smad信号通路的调节作用。方法取第4代对数生长期的人子宫内膜癌Ishiwaka细胞,随机分为对照组、桔梗皂苷D组、激动剂组和桔梗皂苷D+激动剂组。桔梗皂苷D组细胞加入桔梗皂苷D 18μmol/L培养24 h,激动剂组细胞加入重组人TGF-β20 ng/mL刺激24 h,桔梗皂苷D组+激动剂组细胞先加入重组人TGF-β20 ng/mL刺激24 h后,再加入桔梗皂苷D培养24 h。MTT法检测细胞存活率,Transwell实验检测细胞侵袭能力,划痕实验检测细胞迁移能力及蛋白印迹法检测TGF-β1、p-smad2/3、上皮钙粘蛋白(E-cad)和神经钙粘蛋白(N-cad)相对表达量。结果与对照组比较,桔梗皂苷D组细胞存活率及TGF-β1、p-smad2/3和N-cad蛋白相对表达量降低,细胞迁移和侵袭能力减弱,E-cad蛋白相对表达量升高,激动剂组TGF-β1、p-smad2/3和N-cad蛋白相对表达量升高,细胞迁移和侵袭能力增强,E-cad蛋白相对表达量降低(P<0.05);与桔梗皂苷组比较,桔梗皂苷D+激动剂组细胞存活率及TGF-β1、p-smad2/3和N-cad蛋白相对表达量升高,细胞迁移和侵袭能力增强,E-cad蛋白相对表达量降低(P<0.05);与激动剂组比较,桔梗皂苷D+激动剂组细胞存活率及TGF-β1、p-smad2/3和N-cad蛋白相对表达量降低,细胞迁移和侵袭能力减弱,E-cad蛋白相对表达量升高(P<0.05)。结论桔梗皂苷D可能通过抑制TGF-β1/Smad2/3信号通路调控上皮间质转化降低人子宫内膜癌Ishiwaka细胞迁移及侵袭能力。

Objective To investigate the effect of platycodin D on epithelial mesenchymal transformation(EMT)in human endometrial carcinoma Ishiwaka cell migration and invasion,and the regulation of transforming growth factor β(TGF-β)/Smad signaling pathway.Methods Ishiwaka cells in the fourth generation of logarithmically growing stage were randomly divided into control group,platycodin D group,agonist group and platycodin D+ agonist group.Cells in platycodin D group were added with platycodin D 18μmol/L and cultured for 24 h.Cells in the agonist group were added with 20 ng/mL recombinant human TGF-β for 24 h,and cells in the platycodin D+ agonist group were first added with 20 ng/mL recombinant human TGF-βfor 24 h and then cultured with platycodin D for 24 h.Cell viability was detected by MTT assay,cell invasion ability was detected by transwell assay,cell migration ability was detected by scratch assay,and TGF-β1,p-Smad2/3,epithelial cadherin(E-cad)and neural cadherin(N-cad)were detected by western blotting.Results Compared with the control group,cell survival rate and relative expression levels of TGF-β1,p-Smad2/3,N-cad and cell migration and invasion ability in platycodin D group were decreased,and the relative expression level of E-cad protein was increased.The relative expression levels of TGF-β1,p-Smad2/3 and N-cad,cell migration and invasion ability in agonist group were increased,and the relative expression level of E-cad protein was decreased(P<0.05).Compared with the platycodin D group,the cell survival rate and the relative expression levels of TGF-β1,p-Smad2/3 and N-cad protein,the cell migration and invasion ability were increased in platycodin D+agonist group,and the relative expression level of E-cad protein was decreased(P<0.05).Compared with agonist group,cell survival rate and relative expression levels of TGF-β1,p-Smad2/3 and N-cad,cell migration and invasion ability in platycodin D+ agonist group were decreased,and the relative expression level of E-cad was increased(P<0.05).Conclusion Platycodin D may regulate epithelial-mesenchymal transformation by inhibiting TGF-β1/Smad2/3 signaling pathway and reduce the invasion and migration of human endometrial carcinoma Ishiwaka cells.