| Objectives: In order to obtain the proteins related to gastric mucosal carcinogenesis,Annexin A1 protein was screened and identified by early study using isotope labeling quantitative proteomics.The role and mechanism of Annexin A1 protein will be clarified in the growth and proliferation of GES1 cells,and it will provide the experimental basis for further elucidating the molecular mechanism of gastric cancer.Methods: 1.The expression vector of pCDNA3.1-Annexin A1 was transfected into GES1 cells(pCDNA3.1 empty vector was used as negative control).After screening by 400 ug/mL G418,GES1 cell line with stable and overexpression of Annexin A1 was established(GES1-Annexin A1 cell,GES1-pCDNA3.1 as control).Then the expression of Annexin A1 protein was detected with immunofluorescence and Western-blot in GES1-Annexin A1 cells.2.The the growth and proliferation of GES1 cells of Annexin A1 overexpression were observed by cell growth curve,plate cloning,soft agar colony forming assay and flow cytometry.3.The expression of Wnt1,beta-catenin and its phosphorylated protein was detected by Western-blot in GES1-Annexin A1 cells.Results:1.The growth curve was drawn and the trend showed that compared with GES1 and GES1-pCDNA3.1 cells,the growth of GES1-Annexin A1 cells was significantly accelerated after Annexin A1 was overexpressed(P< 0.01).2.The results of plate cloning showed that the clones of GES1,GES1-pCDNA3.1 and GES1-Annexin A1 cells were respectively 54.04±6.12,57.23±9.16 and 162.22±12.52.Compared with GES1 and GES1-pCDNA3.1 cells,the number of clones formed increased significantly and the volume of clones formed increased in GES1-Annexin A1 cells.The results showed that the growth and proliferation ability of GES1 cells increased after Annexin A1 was overexpressed(P<0.01).3.The results of soft agar colony formation experiment showed that the colony formation of GES1,GES1-pCDNA3.1 and GES1-Annexin A1 cells were respectively 106.34±21.54,112.65±26.36 and 265.14±48.36.Compared with GES1 and GES1-pCDNA3.1 cells,the number of colonies formed by GES1-Annexin A1 cells increased and their volume was significantly larger.The results showed that the growth and proliferation of GES1 cells were enhanced after Annexin A1 overexpression(P< 0.01).4.The results of flow cytometry showed that the S-phase ratios of GES1,GES1-pCDNA 3.1 and GES1-Annexin A1 cells cells were respectively 25.17±1.65,36.26±1.82 and 60.04±3.84.The G1-phase ratios were respectively 58.30±3.28,56.13±2.38 and 37.95±1.45,and the cell proliferation indices of the three cells were respectively 41.70,43.88 and 62.05.The results showed that after Annexin A1 was overexpressed,the S-phase proportion of GES1 cells increased significantly,the G1-phase proportion decreased,and the cell proliferation index increased(P< 0.01).In addition,the apoptotic rates of GES1,GES1-pCDNA3.1 and GES1-Annexin A1 cells were respectively 12.62±1.23,11.67±1.06 and 1.89±0.12.The results showed that the apoptosis rate of GES1 cells decreased after Annexin A1 overexpression(P<0.01).5.The results of Western-blot showed that the expression of Wnt1 protein protein increased in GES1-Annexin A1 cells and beta-catenin protein expression increased,while phosphorylation of beta-catenin protein significantly decreased compared with control GES1 and GES1-pCDNA3.1 cells(P< 0.01).Conclusions: Annexin A1 can inhibit the apoptosis and promote the growth and proliferation of GES1 cells by activating Wnt1/beta-catenin pathway. |
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