The Effect Of SERPINA3 Low Expression On Growth And Proliferation Of GES1 Cells

Objectives: The serine protease inhibitor A3(SERPINA3)that is the down-regulated expression of in gastric carcinogenesis was screened and identified by proteomics.The function of SERPINA3 was understanded in the process of gastric mucosal carcinogenesis will provide theoretical basis for elucidating the molecular mechanism of gastric mucosal carcinogenesis.Methods: 1.The SERPINA3 interference vector was constructed and transfected into GES1 cells.After screening the low expression of SERPINA3 was established in GES1 cell line(GES1-Mi-SERPINA3).2.The expression of SERPINA3 protein was detected by immunofluorescence and Western-blot in GES1-SERPINA3 cells.3.The effects of SERPINA3 low expression on the growth and proliferation of GES1 cells were observed by cell growth curve,soft agar colony forming assay,plate cloning assay and flow cytometry.4.The expression of Wnt1 and beta –catenin proteins was detected by Western-blot in GES1-SERPINA3 cells.Results:1.The growth rate of GES1-Mi-SERPINA3 cells was significantly faster than that of control cells(GES1 and GES1-pcDNA6.2)(P<0.01).The results of cell growth curve showed that the growth ability of GES1 cells was enhanced after low expression of SERPINA3.2.The clone numbers of GES1,GES1-pcDNA6.2 and GES1-microRNA-SERPINA3 cells were respectively48.24±8.12,52.14± 10.14 and 92.32 ± 15.41.The clone size and number of GES1-microRNA-SERPINA3 cells were significantly larger than those of the control group(GES1 and GES1-pcDNA6.2)(P<0.01).The results of plate cloning showed that the proliferation ability of GES1 cells was increased after the low expression of SERPINA3.3.The number of colonies of GES1,GES1-pcDNA6.2 and GES1-microRNA-SERPINA3 cells was 144.26±12.31,153.42±16.13 and 258.28 ± 32.36.The colony size and number of GES1-Mi-SERPINA3 cells were significantly larger than those of the control group(GES1 and GES1-pcDNA6.2)(P<0.01).The results of soft agar colony forming assay also showed that the proliferation of GES1 cells was enhanced after the low expression of SERPINA3.4.The S phase cell ratios of GES1,GES1-pcDNA6.2 and GES1-Mi-SERPINA3 cells were respectively 30.54±2.54,30.02±2.45 and 51.17±3.92.The G1 phase cell ratio was 50.78±2.16,51.67±2.58 and 23.65±1.25.The cell proliferation index was respectively 49.22,48.33 and 76.35.The results of flow cytometry showed that the S phase ratio of GES1 cells significantly increased,G1 cell ratio significantly decreased and cell proliferation index increased after the low expression of SERPINA3(P<0.01).In addition,the apoptosis rates of GES1,GES1-pcDNA6.2 and GES1-miR-SERPINA3 cells were respectively 17.81±2.28,18.33±2.34 and 8.46±1.08.The results of flow cytometry showed that the apoptosis rate of GES1 cells decreased after the low expression of SERPINA3(P< 0.01).5.The expression of Wnt1 protein in GES1-miR-SERPINA3 cells was higher than that in GES1 and GES1-pcDNA6.2 cells.The expression of beta-catenin protein was also increased,and its phosphorylation level was decreased.Conclusions: The low expression of SERPINA3 increase the growth and proliferation of GES1 cells,decrease cell apoptosis,which probably related to activate Wnt1/beta catenin pathway,.