Effect And Mechanism Of Ursolic Acid Mediated β-catenin Nuclear Metastasis On Non-small Cell Lung Cancer

[Objective]The aim of this study was to clarify the inhibitory effect of UA on proliferation,migration and invasion of non-small cell lung cancer A549 and H1299 cells,and to explore the effect of UA on Wnt/β-catenin signaling pathway,thereby further revealing the molecular mechanism of proliferation,migration and invasion of UA against NSCLC.So as to provide a new research idea and direction for the subsequent study of UA anti-NSCLC,and provide experimental basis for clinical search for new targets of NSCLC.[Methods]This study intends to use two human lung adenocarcinoma A549,H1299 cell in vitro model.In the first part,the proliferation of A549 and H1299 cell lines was observed by CCK-8 experiment and clone formation experiment,and the relative inhibition rate of cells was detected.Then,the transwell chamber experiment and wound-healing test were used to observe the migration and invasion effect of A549 and H1299 cells in vitro.The second part mainly used Western blot experiment to detect the expression of core proteins β-catenin,p-β-catenin and the upstream and downstream GSK3β,Axin,cyclin-D1,c-Myc in the Wnt/β-catenin signaling pathway in different dose groups.In addition,we also extracted nuclear protein to test the the expression of β-catenin protein in the nucleus.Meanwhile,the effect of UA on the nuclear import of β-catenin was observed by immunofluorescence assay.In the third part,we over-expressed the core protein β-catenin of Wnt pathway through lentiviral infection technology.On the basis of this,a negative virus control group,β-catenin over-expression group and over-expression plus UA group were established,and then we observed the changes in proliferation,migration and invasion levels in each group.The expression of protein levels in Wnt/β-catenin signal pathway was also observed by Western blot.[Results]The first part:UA could inhibit the proliferation of A549 and H1299 cells in a concentration-time dependent manner,and the IC50 at 24h was 24.53±0.39 μM and 27.12±0.89 ∑M,respectively.According to the results of CCK-8 experiment,10,20,and 30 μM were selected as the concentration groups of the subsequent experiments,and the action time was 24h.Cloning formation experiments,wound-healing experiments and transwell chamber invasion experiments showed that UA could inhibit the number of clone formation,migration distance and trans-welling cells in a concentration-dependent manner,compared with the untreated control group.All of this suggested that UA has a certain inhibitory effect on the proliferation,migration and invasion of NSCLC cells.The second part:Western blot results showed that UA can up-regulate the expression of GSK3β,Axinl and down-regulate the expression of cyclin-D1 and c-Myc in Wnt/β-catenin signaling pathway.Then,we extracted the nuclear protein of the two cells,and discovered that UA could inhibit the expression of β-catenin both in cytoplasm and nucleus.Immunofluorescence experiments showed that UA could inhibit the nuclear motility of β-catenin,suggesting that UA can inhibit the Wnt/β-catenin pathway activity through regulating β-catenin nucleus movement.The third part:After lentivirus over-expressed β-catenin,both the negative virus control group and the over-expressed drug-added group were established,and it was found that UA can reversibly inhibit the proliferation,migration and invasion of NSCLC cells and inhibit the expression of cyclin-D1 and c-Myc,which were the downstream target gene of the Wnt/p-catenin pathway.[Conclusions]1.UA could inhibit the proliferation,migration and invasion of NSCLC cells in a concentration-dependent manner;2.UA could inhibit the Wnt/β-catenin pathway activity by mediating β-catenin nuclear translocation;3.UA inhibited the proliferation,migration and invasion of NSCLC cells might by inhibiting the nuclear translocation of β-catenin in the Wnt/β-catenin pathway.