Role Of Epithelial Mesenchymal Transformation Mediated By Snail In The Vasculogenic Mimicry Of Esophageal Cancer And The Intervention Mechanism Of Modified Tongyou Decoction

Objectives To study the role of Snail mediated epithelial mesenchymal transformation（EMT）in the intervention mechanism（VM）of esophageal cancer,and to explore the intervention mechanism of modified Tongyou Decoction（MTD）by affecting EMT on VM of esophageal cancer.Methods 1.Preparation of MTD: alcohol extraction method was adopted,anhydrous ethanol was immersed for 5 weeks,supernatant was taken by centrifugation,condensed to thick paste in boiling water bath,and dimethyl sulfoxide（DMSO）was dissolved.2.Grouping: normoxic control group,normoxic drug group,anoxic control group and anoxic drug group.3.The hypoxia model of Eca109 cells was established by CoCl₂ induction solution,and the inhibition concentration of the drug on cells was determined by CCK-8 method（IC50）.4.PAS staining was used to observe the differences in cell morphology in each group and the effect of MTD.5.The differences of cell migration ability in each group were observed with cell scratch experiment;6.The difference of invasion ability of cells in each group was observed with Transwell chamber experiment.7.Immunofluorescence technique was used to observe the difference in the degree of correlation between the expression of Snail and HIF-1α in each group.8.Western Blot was used to observe the protein expression of each group to the cell Snail,HIF-1α,E-cadherin,Vimentin,VE-cadherin,MMP-2 and MMP-9,and image analysis software was used to semi-quantitatively analyze the protein expression differences in each group.9.Difference of m RNA expression of cell Snail factor in each group was observed by real-time fluorescence quantitative RT-PCR.Results 1 CCK-8 showed IC50=71611μg/m L of MTD on Eca109 cells.2 The PAS staining results showed that compared with the normoxic control group,the cells in the anoxic control group were obviously strip-like.After drug intervention,the cells were more dispersed and the tubular structure disappeared.3 Cell scratch test showed that after 24 h,the scratch gap of cells in the oxygen-free control group was（362.81±1.11）μm,the scratch gap of cells in the hypoxia control group was（250.86±7.88）μm,and that in the oxygen-free drug group was（404.46±5.71）μm.The drug could inhibit cell migration in both oxygen-free and hypoxia environments.4 Transwell chamber experiment showed that after 24 hours,50% field cell invasion was observed in the normoxic control group and nearly 100% field cell invasion in the anoxic control group.In both normal and hypoxic environments,drugs can inhibit cell invasion.5 Immunofluorescence results showed that the relative values of Snail fluorescence intensity in normal oxygen control group and hypoxia control group were（1.15±0.00）and（1.45±0.01）respectively,and HIF-1α was（1.02±0.01）and（1.36±0.01）respectively.The two indexes in hypoxia control group were increased,with statistically significant difference（P < 0.05）.The relative values of Snail fluorescence intensity and HIF-1α in hypoxia drug group were（0.89±0.01）and（0.83±0.01）.Compared with hypoxia control group,the two indexes were significantly reduced,and the difference was statistically significant（P < 0.05）.6 Western Blot detection: compared with the normoxic control group,Snail,HIF-1α,VE-cadherin,MMP-2,MMP-9 and Vimentin expression increased,and E-cadherin expression decreased in the anoxic control group.The differences were statistically significant（P < 0.01）.Compared with the control group,the expressions of Snail,HIF-1α,VE-cadherin,MMP-2,MMP-9 and Vimentin in the drug group were down-regulated and the expression of E-cadherin was up-regulated.The differences were statistically significant（P < 0.01）.7 RT-PCR showed that the expression activity of the cell Snail gene in the anoxic control group was significantly enhanced compared with that of the normoxic control group.The differences were statistically significant（P < 0.01）.Compared with the control group,theexpression activity of cell Snail gene in the drug group decreased under hypoxia and normoxia.All the differences were statistically significant（P < 0.01）.Conclusions 1.Anoxic microenvironment promotes the formation of VM in Eca109 cells,which is closely related to Snail mediated EMT.2.MTD can inhibit the proliferation of Eca109 cells,and then inhibit the invasion and metastasis of cells.3.The inhibition of proliferation,invasion and metastasis of Eca109 cells by MTD is closely related to the regulation of EMT and HIF-1α induced VM mediated by Snail.Figure13;Table12;Reference 183...