Dioscin Inhibits Epithelial-mesenchymal Transition In Lung Cancer By Regulating MAP3K3 And Its Downstream Signaling Pathway

PurposeLung adenocareinoma(LADC),the most common type of histologic lung cancer,constitutes the deadliest human cancer,killing 650,000 people worldwide each year and increasing incidence among smokers,quitters and nonsmokers.Moreover,LADC is most comnon among women and young people who never smoke,so it is imperative to understand and treat the disease.This experiment mainly studied the effect of dioscin(Dio)on the proliferation,migration and invasion ability of human lung adenocarcinoma cells and the effect of dioscin on epithelial mesenchymal transformation(EMT),so as to provide certain theoretical basis for the clinical anti-lung cancer of dioscin.Methods(1)MAP3K3 promotes epithelial mesenchymal transformation of human lung adenocarcinoma cells A549 and H12991.Adenovirus transfection constructed stable strains that overexpressed MAP3K3.2.Cloning experiment was used to detect the changes of cell proliferation caused by overexpression of MAP3K3 gene.3.Changes in migration and invasion of human lung adenocarcinoma cells after overexpression of MAP3K3 were detected in the scratch experiment and Transwell chamber experiment.4.Western Blot assay was performed to detect the emt-related protein expression after the overexpression of MAP3K3 gene,and the influence of AKT/p-AKT,mTOR/p-mTOR,GSK3 β/p-GSK3 β,JNK/p-JNK,ERK/p-ERK.(2)Study on the effect and mechanism of dioscin in inhibiting EMT of human lung adenocarcinoma cells A549 and H12991.CCK8 assay was used to detect the effect of dioscin on the growth and proliferation activities of human lung adenocareinoma cells A549 and H1299,so as to determine the concentration of diosgenin in subsequent experiments.2.Scratch experiment and Transwell chamber experiment were conducted to detect the effect of dioscin on the migration and invasion of human lung adenocarcinoma cells.3.Western Blot and Immunofluorescence were used to detect the expressions of EMT-related proteins such as E-eadherin,N-cadherin,Vimentin,ZEB1,ZO-1,Snail and Slug,Claudin-1.4.The expressions of MAP3K3 and its downstream signaling pathway proteins AKT/p-AKT,mTOR/p-mTOR,GSK3 β/p-GSK3 β,JNK/p-JNK,ERK/p-ERK were detected by Western Blot.Results(1)MAP3K3 promotes the epithelial-mesenchymal transformation of human lung adenocarcinoma cells A549 and H12991.Cloning showed that the monoclonal area increased significantly after the overexpression of MAP3K3 gene.2.Scratch healing experiment and Transwell chamber showed that the scratch healing rate and transmembrane number of cells significantly increased after the overexpression of MAP3K3.3.Western Blot analysis showed that the overexpression of MAP3K3 significantly reduced the expression of E-cadherin in A549 and H1299 cells,and increased the expression of N-cadherin and Vimentin.The overexpression of MAP3K3 gene significantly increased the expression of p-AKT,p-mTOR,p-GSK3β,p-JNK in A549 and H1299 cells.(2)The effect and mechanism of diosgenin on A549 and H1299 EMT of human lung adenocarcinoma cells1.CCK8 showed that the proliferation of human lung adenocarcinoma cells A549 and H1299 was significantly inhibited by the drug action,and showed a time-concentration-dependent relationship.2.Scratch experiments and Transwell chamber experiments showed that migration and invasion of A549 and H1299 could be significantly inhibited in a concentration-dependent manner.3.Immunofluorescence and Western Bolt detection showed that drug interference for 24h significantly increased the expression of emt-related proteins such as ZO-1,Claudin-1 and E-cadherin and reduced the expression of emt-related proteins such as-cadherin、Vimentin、ZEB1、MMP9 in A549 and H1299 cells.4.Western Bolt assays showed down-regulated expression of MAP3K3,p-AKT,p-mTOR,p-GSK3 β and up-regulated expression of p-JNK,p-ERK.Conclusion1.MAP3K3 gene can promote the epithelial-mesenchymal transformation of human lung adenocarcinoma cells A549 and H1299.2.Dioscin can significantly inhibit the invasion,metastasis and epithelial-mesenchymal transformation of A549 and H1299 cells,possibly through the regulation of MAP3K3/AKT/mTOR/GSK3 β signaling pathway.JNK and ERK signaling pathways were activated after the intervention of dioscin,but phosphorylated JNK and ERK were significantly up-regulated,suggesting that drugs may not mediate the activation of JNK and ERK signaling pathways through MAP3K3,there may be other signal transduction,and the up-regulation or down-regulation of phosphorylated JNK and ERK have been reported in the literature,which need to be further explored.