Niweikang Inhibits The Transfer Of MC Cells By Inhibiting Epithelial-mesenchymal Transition Through AKT/GSK-3β/β-catenin Pathway

Objective:To observe the effects of Niweikang on proliferation,apoptosis,migration and invasion of MC cells,and to explore the process of Niweikang’s inhibition of MC cells’epithelial-mesenchymal transformation through AKT/GSK-3β/β-catenin pathway.Methods:In the first part:Promoting effects of MNNG（M-methyl-N-nitro-N-nitroso-uanidine）on proliferation and epithelial mesenchymal transformation of GES-1 cells.The expression of EMT-related proteinsα-SMA,E-cadherin,N-cadherin and vimentin was detected by Western blot.In the second part,CCK-8 method and colony formation experiment were used to observe the inhibitory effect of Niweikang on MC cells;Transwell method was used to observe the inhibitory effect of Niweikang on migration and invasion of MC cells;flow cytometry was used to detect the promoting effect of Niweikang on apoptosis of MC cells;Western blot was used to observe the expressions of PCNA,Caspase-3,Bax,Bcl-2,MMP-2,MMP-9,E-cadherin,N-cadherin,vimentin,P-AKT Ser⁴⁷³,β-catnin,α-SMA,GSK-3β,P-GSK-3βSer⁹ and AKT proteins of MC cells.In the third part,Western blot was used to detect the expression of E-cadherin,N-cadherin,vimentin,P-AKT Ser⁴⁷³,β-catnin,α-SMA,GSK-3β,P-GSK-3βSer⁹ and AKT proteins in MC cells,and real-time fluorescence quantitative PCR（Real-time PCR）was used to detect the expression of C-myc in MC cells.Result:In the first part MC cell model was constructed.Western blot results showed that compared with GES-1 cells,E-cadherin of MC cells was significantly down-regulated,while vimentin,α-SMA and N-cadherin of mesenchymal markers were significantly up-regulated.The second part Compared with BC group,the results of CCK8 in the second part showed that the survival rate of MC cells decreased in a dose-dependent manner（P<0.05）;the clone colony formation experiment showed that the Niweikang-containing serum inhibited the proliferation of MC cells in a dose-dependent manner（P<0.05）;the apoptotic rate of MC cells was significantly increased by flow cytometry（P<0.05）;Transwell experiment showed the number of migration and apoptosis of MC cells were significantly increased（P<0.05）.Western blot showed that the expression of PCNA,MMP-2,MMP-9 and Bcl-2（P<0.05）were lower,while the expression of Bax and Caspase-3（P<0.05）were up-regulated by Niweikang in a dose-dependent manner with statistical significance.The third part Western blot experiment showed that Niiweikang down-regulated the expression ofβ-catenin,P-AKTSer⁴⁷³,P-GSK-3βer⁹ and E-cadherin（P<0.05）,while he expression ofα-SMA,N-cadherin and vimentin increased at the protein level（P<0.05）,but the expression of GSK-3βand AKT protein was not significant;the addition of AKT inhibitor GSK690693 inhibited the EMT process of MC cells,the expression of E-cadherin protein was up-regulated（P<0.05）,while the expression of N-cadherin,vimentin andα-SMA protein was down-regulated significantly（P<0.05）;the expression of P-AKT Ser⁴⁷³,P-GSK-3βSer⁹ andβ-catenin was inhibited（P<0.05）,but the expression of GSK-3βand AKT protein had no significant change;qRT-PCR results confirmed that the Niweikang-containing serum could inhibit the expression of C-myc mRNA（P<0.05）and the inhibition was enhanced after adding the inhibitor,with statistical significance（P<0.05）.Conclusion:Niweikang can inhibit the proliferation,migration and invasion of MC cells,suggesting that Niweikang inhibits the metastasis of MC cells through AKT/GSK-3β/β-catenin pathway.