Mechanism Of Huatan Tongyu Jiedu Decoction In Regulating Wnt/β-catenin Signaling Pathway To Inhibit TGF-β1-Mediated Human Gastric Cancer Cell Invasion And Migration And Epithelial-mesenchymal Transition

Objective:To observe the effect of Huatan Tongyu Jiedu Decoction（HTJD）on invasion and metastasis and epithelial-mesenchymal transformation（EMT）of human gastric cancer cell line MGC-803,MKN-45 and SGC-7901,and explore the specific mechanism of HTJD on Wnt/β-catenin signaling pathway.Method:1.Effects of HTJD on invasion and migration of human gastric cancer cells:MGC-803,MKN-45 and SGC-7901 cells were treated with transforming growth factor-β1（TGF-β1）to establish a cell invasion and metastasis model.The experiment was divided into five groups:blank group,model group,HTJD low,medium and high dose(10,20,40μg·mL^-1)group.Except blank group,the other four groups were all induced by TGF-β1 with concentration of 10 ng·mL^-1 After the completion of modeling,the blank group and the model group continued to culture cells with complete medium.HTJD group was treated with complete medium containing HTJD(the final concentration was 10,20,40μg·mL^-1)respectively,and continued to culture for 24 hours.The invasion and migration ability of human gastric cancer cell lines MGC-803,MKN-45 and SGC-7901 were detected by Transwell chamber test and scratch healing test;the inhibition ability of HTJD on invasion and migration of three kinds of cells was compared,and the best cell line was selected for follow-up test.2.The effects of htjd on EMT and Wnt/β-catenin signaling pathway of MGC-803cells:the methods of grouping and modeling were the same as above,and the expression of fibronectin（FN）and collagen Ⅳ（ColⅣ）were detected by ELISA, Western blot,Western blot E-cadherin,N-cadherin and Vimentin were detected,And the expression of Wnt,β-Catenin,GSK-3β,Axin and APC in the Wnt/β-catenin signaling pathway.3.The specific regulatory mechanism on Wnt/β-catenin signaling pathway of MGC-803 cells:10 mmol·L^-1 of Wnt/β-catenin signaling pathway specific activator（LiCl）was used to treat MGC-803 cells for 24 hours to establish the Wnt/β-catenin signaling pathway activation model.The optimal dose of HTJD(40μg·mL^-1),Wnt/β-catenin signaling pathway specific inhibitor XAV939(8μmol·L^-1)and HTJD+ XAV939 were used to intervene MGC-803 cells for 24 hours respectively.The invasion and migration ability of human gastric cancer cell lines MGC-803 were detected by Transwell chamber test and scratch healing test.WB method was applied to detect the relative expression of E-cadherin,Vimentin Wnt1,β-catenin（total-protein and nucleoprotein）,phospho-β-Catenin,GSK-3β,phospho-GSK-3β,Axin and APC.Results:1.Effects on invasion and migration of human gastric cancer cells:the results of Transwell chamber experiment and scratch healing experiment showed that compared with the blank group,the number of MGC-803,MKN-45 and SGC-7901 cells in the model group increased significantly,and the scratch healing rate increased significantly（P<0.01）;compared with the model group,HTJD(10,20,40μg·mL^-1)group,the number of cell penetrating membrane and the rate of scratch healing were significantly reduced（P<0.05,P<0.01）.2.Effects on EMT and Wnt/β-catenin signaling pathway of MGC-803 cells:The results of ELISA and WB showed that compared with the blank group,the EMT related protein E-cadherin protein of MGC-803 cells in the model group decreased significantly,and N-cadherin,Vimentin,FN,ColⅣ.The expression of GSK-3β,Axin and APC in Wnt/β-catenin signaling pathway decreased significantly（P<0.01）,and the expression of Wnt1,β-catenin total protein and nuclear protein increased significantly（P<0.01）.Compared with the model group,the expression of EMT related protein E-cadherin in MGC-803 cells in HTJD(10,20,40μg·mL^-1)group increased significantly,while the expression of N-cadherin,Vimentin,FN,ColⅣ increased significantly.The expression of GSK-3β,Axin and APC in Wnt/β-catenin signaling pathway was significantly increased（P<0.05,P<0.01）.3.Regulation mechanism of Wnt/β-catenin signaling pathway in MGC-803 cells:the results of Transwell chamber experiment and scratch healing experiment showed that compared with the blank group,the number of MGC-803 cells in the model group increased significantly,and the scratch healing rate increased significantly（P<0.01）;compared with the model group,HTJD,XAV939 and the combination group,the number of cell penetrating membrane and the rate of scratch healing were significantly reduced（P<0.05,P<0.01）.WB results showed that LiCl could significantly down-regulate the expression of E-cadherin,p-β-catenin,GSK-3β,Axin and APC in MGC-803 cells compared with the blank group（P<0.01）,and up-regulate the expression of Vimentin,total protein and nuclear protein of Wnt1,p-GSK-3β,β-catenin（P<0.01）;compared with the LiCl group,HTJD and XAV939 could significantly up-regulate the expression of E-cadherin,p-β-catenin,GSK-3β,Axin and APC in MGC-803 cells（P<0.01）,down-regulate the expression of Vimentin,Wnt1,p-GSK-3β,total protein ofβ-catenin and nuclear protein（P<0.05,P<0.01）.Compared with the HTJD group,there were significant differences in the expression of all proteins except Axin protein in the combination group.Conclusion:1.HTJD can effectively inhibit the invasion,metastasis and EMT of human gastric cancer cell line MGC-803,MKN-45 and SGC-7901,of which the strongest inhibitory effect is on MGC-803.2.HTJD may inhibit the invasion and metastasis of MGC-803 cells and EMT via inhibiting the activation of Wnt/β-catenin signaling pathway.The specific mechanism may be via inhibit the phosphorylation of GSK-3β,stabilize the expression of GSK-3β,Axin and APC,promote the phosphorylation degradation ofβ-catenin,inhibit the nuclear import ofβ-catenin,and reduce the expression of downstream EMT target proteins.