The Expression Of Spns2 In Gastric Cancer Tissues And Its Effect On The Proliferation And Migration Of Gastric Cancer Cells

BackgroundGastric cancer is one of the most common form of malignancies in the world.In China,as the process of aging population accelerates and changes of the pressure of life and dietary habits,the incidence rate remains high and patients become younger.In most patients with gastric cancer,cancer cells infiltrate and metastasize during treatment,eventually leading to death.Therefore it has important significance to study of the occurrence and development of gastric cancer In-depth.Sphingosine-1-phosphate(S1P)is a breakdown and metabolite of membrane phospholipids.It is produced from the phosphorylation of sphingosine by sphingosine kinase(SPHK).By acting either intracellularly or extracellularly in an autocrine or paracrine manner,S1P produce a series of immune effects,also involved in tumor-related activities,including cell proliferation,migration.In addition to the ABC family,Spinster homolog 2(Spns2)also acts a unique membrane transporter to secrete SIP.Spns2 is a protein with 12 transmembrane regions(containing 504 amino acid residues).It was initially reported in zebrafis to regulate myocardial precursor migration and causes to appear two hearts when absent.Spns2 was considered mainly associated with the immune system before.Shigetomo Fukuhara[21]found that knocking out of Spns2 caused a significant decrease in the concentration of S1P in the circulation of mice,leading to the accumulation of mature T and B lymphocytes in the thymus,and a significant decrease in lymphocytes in the blood and peripheral lymphoid organs.Tumor-related studies about Spns2 were rarely reported.In 2016,Eric Bradley et al.[23]found that Spns2 is associated with NSCLC.Knockout of Spns2 can accelerate the migration of cancer cells.It was revealed for the first time that Spns2 has a regulatory role in non-small cell lung cancer cells.The purpose of this study was to investigate the expression of Spns in gastric cancer tissues,and the effects of over-expression and down-regulation of Spns2 on the biological function of gastric cancer SGC-7901 cells.Methods1.20 cases of gastric cancer and adjacent tissues after surgery in Jiangsu Cancer Hospital during February 2016 and August 2016(male 12,female 8)were colected.Expression of Spns2 in gastric cancer and adjacent tissues was determined by immunohistochemistry method.2.Eukaryotic expression vector pEX-1(pGCMV/MCS/EGFP/Neo)-Spns2 and siRNA were constructed and transfected to human gastric cancer cells line SGC-7901 respectively.The experimental groups were named pEX-1-Spns2 group/siRNA-Spns2 group,and the negative control pEX-1 group/siRNA-NC group.Transfection efficiency was observed by fluorescence microscopy.3.Reverse transcription-polymerase chain reaction(RT-PCR)and western blotting were used to detect Spns2 mRNA and protein levels respectively in order to confirm the up-regulation and down-regulation consequence of Spns2.4.The effects on migration,clone formation and proliferation of SGC-7901 after Spns2 over-expressed and down-regulated were evaluated by cell counting kit-8(CCK-8)assay,clone formation assay,transwell experiment and wound healing scratch assays.Results1.Spns2 expression in gastric cancer and the adjacent tissues was(0.39±0.04)and(0.45±0.02)respectively(p<0.05).2.Eukaryotic vector pEX-1(pGCMV/MCS/EGFP/Neo)and siRNA to regulate Spns2 were successfully constructed and the transfection efficiency were approximately 60%and 75%respectively.3.After transfected with pEX-1-Spns2,both Spns2 mRNA and protein levels in cell SGC-7901 were increased,and siRNA decreased correspondingly.4.Compared with their own negative group,CCK-8 assay showed that pEX-1-Spns2 supressed the proliferation of SGC-7901(p<0.05),and siRNA-Spns2 enhanced proliferation of SGC-7901(p<0.05);Clone formation assay showed that pEX-1-Spns2 decreased clone formation rate,and siRNA-Spns2 increased clone formation rate;Migrated cell number counted of the transwell experiment showed that pEX-1-Spns2 supressed migration of SGC-7901,and siRNA-Spns2 enhanced migration of SGC-7901;Migration rate of pEX-1-Spns2 group was lower than control group,and migration rate of siRNA-Spns2 group was higher than control group.ConclusionSpns2 is low expressed in gastric cancer.Up-regulation of Spns2 in SGC-7901 cell could reduce its ability of proliferation,clony formation and migration.Down-regulation of Spns2 could enhance its ability of proliferation,clony formation and migration conversely.Spns2 may play a role in inhibiting the development of gastric cancer.Spns2 is likely to be a new therapy target for gastric cancer.