The Expression Of DDAH1 In Human Gastric Cancer And Its Influence On Serum Starvation-incuced Apoptosis In MGC803 Cell Line

?Background and Objective?Dimethylarginine dimethylaminohydrolase1(DDAH1)has drawn more and more attention,as protein in recent years,which plays its part mainly by hydrolyzing asymmetrical dimethylarginine(ADMA)the endogenous inhibitor of nitric oxide synthase(NOS).DDAH1 was involved in the regulation of diverse pathophysiologic process,including angiogenesis,tumor growth etc.There was rare research of the relationship between DDAH1 and gastric carcinoma so far,this study aims to detect the expression of DDAH1 in gastric carcinoma,to explore the relationship between the expression and the clinicopathological parameters,to construct the DDAH1 over-expressed stable transfected MGC803 cell line,and to reach this conclusion that DDAH1 influence the apoptosis in MGC803 cell line.?Methods? 1.Investigating the expression of DDAH1 in eight pairs of gastric carcinoma tissues and the adjacent normal mucosal tissues by western-blot in protein level.2.Detecting the expression of DDAH1 in 170 pairs of gastric carcinoma tissues and the adjacent normal mucosal tissues by tissue microarray combined with immunohistochemistry,and analyzed the relationship between DDAH1 expression and the clinicopathological parameters,including patients' pathological stage,lymph node metastasis,differentiation,histological types etc.3.Cloning the DDAH1 gene sequence into p CDH plasmid to construct p CDH-DDAH1 recombinant plasmid,and after confirmed conformity by restriction endonuclease analysis and sequencing,stable transfecting the vector into gastric cancer cells MGC803.Then we verify expression level of DDAH1 by immunofluorescence and western-blot.4.To detect how the over-expression of DDAH1 affects MGC803 proliferation and apoptosis by CCK8 assay and flow cytometry instrument.?Results? 1.Western blot analysis indicated that compared with the adjacent normal mucosal tissues(positive expression rate is 75.3%),the expression of DDAH1 in the gastric carcinoma was significantly lower(about 25.9%),(p<0.001).Statistical analysis revealed that the expression of DDAH1 was related to patients' TNM stage,differentiation,perineural invasion,histological types(P<0.05).2.p CDH-DDAH1 recombinant plasmid was constructed successfully.The expression of DDAH1 was obviously higher in the MGC803-DDAH1 cell line stable transfected by the recombinant plasmid.3.CCK-8 assay show that over-expressed DDAH1 protein won't affect proliferation,but can accelerate the apoptosis of MGC803 incurred by serum starvation,this difference was statistically significant(P<0.05).4.Flow cytometry instrument assay show that the apoptosis rate has no obvious difference between MGC803-DDAH1 and MGC803-Vector after treatment 72 hours with whole culture medium(P>0.05),while the apoptosis rate of MGC803-DDAH1 was obviously higher than the control group after treatment 72 hours with serum starvation,this difference was statistically significant(P<0.01).?Conclusions? 1.The expression of DDAH1 in the gastric carcinoma was significantly lower,Statistical analysis revealed that the expression of DDAH1 was related to patients' TNM stage,differentiation,perineural invasion,histological types.2.Stable transfected cell line MGC803-DDAH1 that can over-express DDAH1 was constructed successfully.3.The over-expressed protein of DDAH1 has no obvious influence on proliferation,but can accelerate the apoptosis of MGC803 incurred by serum starvation.