Differential Expression Of Esophageal Cancer Four Syndromes Serum Proteins And Their Regulation Of Removing Heat And Phlegm Formula

Objective:In this study,protein chip was used to investigate the expression of differential proteins in serum of esophageal cancer patients and screen the differential proteins of different syndromes,and further explore the regulatory mechanism of removing heat-phlegm formula and its two separated prescriptions(removing heat prescription and removing phlegm prescription)on the proliferation of EC cell and EC associated proteins` expression,so as to provide basis for the molecular pathogenesis of EC syndromes and clinical medication.Methods:1.Establish the serum of esophageal cancer patients and the corresponding clinical data base.A total of 21 patients with different syndromes of esophageal cancer and 7 healthy normal people were selected according to distribution of their sex and age.2.Use antibody-chip AAH-BLG-1(containing 507 kinds of cells important functional protein)to screen the expression of differential proteins in serum of esophageal cancer patients.Then compare the differential proteins in 11 groups: the esophageal cancer patients group and the normal group;the esophageal cancer patients with phlegm and Qi stagnation syndrome(P)group and the normal group;the esophageal cancer patients with body fluid insufficiency and heat agglomeration syndrome(F)group and the normal group;the esophageal cancer patients with blood stasis syndrome(B)group and the normal group;the esophageal cancer patients with qi and yang insufficiency syndrome(Q)group and the normal group;the F group and the P group;the B group and the F group;the B group and the Q group;the B group and the P group;the F group and the Q group;the Q group and the Pgroup.3.According to the above 11 groups of differentially expressed proteins,the primary function of proteins and their involved signaling pathway was analyzed.4.Effects of removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on the proliferation of EC-1cell,Eca109 cell,EC9706 cell and TE-1 cell was detected by MTT method.5.Effects of removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on the cell cycle of EC-1cell,Eca109 cell,EC9706 cell and TE-1 cell was detected by flow cytometry method.6.Effects of removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on MMP1?MMP14?OPG?FBLN5 and PECAM-1 proteins expression of EC-1 cell,Eca109 cell,EC9706 cell and TE-1cell was detected by Western blot method.Results:1.There were 39 differentially expressed proteins in esophageal cancer patients and normal controls;100 differentially expressed proteins in normal controls and P group;22differentially expressed proteins in normal controls and F group;16 differentially expressed proteins in normal controls and B group;25 differentially expressed proteins in normal controls and Q group;29 differentially expressed proteins in F group and P group;6differentially expressed proteins in B group and F group;14 differentially expressed proteins in B group and Q group;8 differentially expressed proteins in B group and P group;7differentially expressed proteins in F group and Q group;31 differentially expressed proteins in Q group and P group;2.The screened proteins were classified by GO analysis,and the difference between the esophageal cancer patients and the normal control group was the positive regulation of MAPK cascade and leukocyte migration;the difference between the normal control group and the P group was the positive regulation of cell locomotion and motility;the difference between the normal control group and the F group was the regulation of cell-cell adhesion and regulation of T cell activation;the difference between the normal control group and the B group was the positive regulation of response to external stimulus,the leukocyte and mononuclear cell proliferation;the difference between the normal control group and the Q group was the regulation of interleukin-12 production,the leukocyte and mononuclear cell proliferation;3.Pathways were analyzed for the screened differential proteins.In the esophageal cancerpatients and the normal control group,Cytokine-cytokine receptor interaction,PI3K-Akt signaling pathway,Jak-STAT signaling pathway and Chemokine signaling pathway were the main pathways;In the P group and the normal control group,Cytokine-cytokine receptor interaction,PI3K-Akt signaling pathway,Jak-STAT signaling pathway and Pathways in cancer were the main pathways;In the F group and the normal control group,Cytokine-cytokine receptor interaction,PI3K-Akt signaling pathway,Jak-STAT signaling pathway and Chemokine signaling pathway were the main pathways;In the B group and the normal control group,Cytokine-cytokine receptor interaction,Jak-STAT signaling pathway,PI3K-Akt signaling pathway and Toll-like receptor signaling pathway were the main pathways;In the Q group and the normal control group,Cytokine-cytokine receptor interaction,PI3K-Akt signaling pathway,Rap1 signaling pathway and Ras signaling pathway were the main pathways.4.Removing heat and phlegm formula and its two separated prescriptions(removing heat prescription and removing phlegm prescription)all had inhibitory effects on the proliferation of EC-1,EC9706,Eca109 and TE-1 cells,and the removing heat and phlegm formula had the strongest inhibitory effect,then the removing heat group,the removing phlegm group.5.Removing heat and phlegm formula and its two separated prescriptions(removing heat prescription and removing phlegm prescription)all had effects on the cell cycle of EC-1,EC9706,Eca109 and TE-1 cells: Compared with the blank control group,cells of S phase in the removing heat group,the removing phlegm group and the whole formula group of EC-1,Eca109 and TE-1 cells decreased,and the removing phlegm group changed the most;EC9706 cells of removing heat group in S phase increased significantly,G2 phase cells decreased significantly,removing phlegm group and the whole formula group cells in G1 phase decreased significantly.6.Removing heat and phlegm formula and its two separated prescriptions(removing heat prescription and removing phlegm prescription)could down regulate MMP1?MMP14?OPG ? FBLN5 and PECAM-1 expression in EC-1,EC9706,Eca109 and TE-1 cells.Compared with the blank control group,the expression of MMP1?MMP14?OPG?FBLN5and PECAM-1 reduced in the four cells.Conclusion:1.Protein chip could detect the expression of protein in wade range and high-throughput.It can be used as a new method of screening tumor molecular markers.2.There are differentially expressed proteins in patients with different syndromes of esophageal cancer,there main functions are different.3.Removing heat and phlegm formula and its two separated prescriptions(removing heat prescription and removing phlegm prescription)all have inhibitory effects on the proliferation of EC9706,Eca109,TE-1 and EC-1 cells,and present dose-dependent and time-dependent relationship.4.Removing heat and phlegm formula and its two separated prescriptions(removing heat prescription and removing phlegm prescription)all have effects on the cell cycle of EC9706,Eca109,TE-1 and EC-1 cells.5.The inhibitory effects of removing heat and phlegm formula and its two separated prescriptions(removing heat prescription and removing phlegm prescription)on the proliferation of Eca109,EC9706,TE-1 and EC-1 cells have relationship with regulating the expression level of MMP1?MMP14?OPG?FBLN5 and PECAM-1,which provide molecular basis for the treatment of EC with removing heat-phlegm formula.