| BackgroundGastric cancer(gastric cancer,GC)is one of the most common malignant tumors at present,characterized by high recurrence probability,fast invasive rate and rather strong diffusion transfer,and low probability of complete cure in clinical treatment.China is one of the nations with the highest incidence of the gastric cancer ailments.Most patients are often at progressive stages or advanced stages,regardless of the diagnosis,treatment and intervention measures been relatively limited,patients have a shorter life cycle.However more studies have shown that the phenomenon of apoptosis resistance plays a vital role in tumor immersion metastasis,but there are no relevant reports on whether this is regulated by coagulation factors.Among them,clotting factor Xa(FXa)is the key enzyme in clotting processes.In this study,we explore whether it is involved in gastric cancer.ObjectivesTo explore the phenomenon and mechanisms of FXa mediated anoikis resistance in gastric cancer.Methods1.The relationship between FXa and gastric cancer was analyzed by TCGA and geo database.The biological characteristics and clinical prognosis of gastric cancer regulated by FXa were predicted by go and KEGG analysis.Immunohistochemical staining was used to detect the expression of FXa in gastric cancer tissues and its relationship with clinicopathological features of gastric cancer patients.2.The expression of FXa in gastric cancer cell line was studied by q PCR and WB.The model of suspension cells was constructed by the utilization of a polyhema glue;the expression of FXa regulated apoptosis and invasion and metastasis related protein was verified by WB.FXa used to detect the apoptosis phenotype of gastric cancer cells.Trans well experiments,scratch test and CCK8 test were used to verify that FXa regulated the invasion,proliferation and metastatic phenotype of gastric cancer cells.The expression and clinical significance of pyruvate dehydrogenase kinase(PDK),reactive oxygen species(ROS)and lactate dehydrogenase(LDH)in energy metabolism pathway regulated by FXa in gastric cancer cells were studied by utilizing WB and ELISA.3.The downstream target GOSR1 protein of FXa was identified by protein mass spectrometry to verify its phenotype in cells;the relationship between GOSR1 and FXa in cells was studied by knocking down GOSR1,at the same time,the expression level of GOSR1 gene in tumor cell adhesion and suspension was investigated.Flow cytometry was used to detect apoptosis of tumor cells.Trans well assay was used to detect the invasion ability of tumor cells,scratch assay was used to detect the metastasis and proliferation ability of tumor cells,and CCK8 assay was used to detect cell proliferation.The expressions of lactate dehydrogenase(LDH),pyruvate dehydrogenase kinase(PDK)and reactive oxygen species(ROS)in the FXA glycolysis pathway were detected by WB and ELISA methods,and the mitochondrial oxidative phosphorylation pathway was detected.4.The model of subcutaneous tumor formation of gastric cancer cells in vivo verified the regulatory effect of FXa and GOSR1 in gastric cancer.Results1.The TCGA tumor database was used to analyze 375 tumor samples and 32 normal samples,and 62 coagulation related genes were obtained,which confirmed that FXa m RNA expression was related to the progression of gastric cancer.The analysis of online website and TCGA data showed that the expression level in paracancerous tissues was higher than that in paracancerous tissues,and it was negatively correlated with the poor prognosis of gastric cancer.Univariate regression analysis showed that age,UICC stage(Ⅲ,Ⅳ),macrophages and FXa were associated with the prognosis of gastric cancer patients(HR = 1.350,95% CI = 0.956-1.543,P =0.020).However,in multivariate Cox regression analysis,FXa was significantly correlated with the prognosis of gastric cancer patients(HR = 1.050,95% CI =0.905-1.217,P = 0.004);GEO database(gse22237)verified that FXa was associated with poor prognosis of gastric cancer patients,which was consistent with TCGA data.These results suggest that FXa can be used as an independent prognostic factor of gastric cancer compared with other factors.Go and KEGG were enriched in the pathways of matrix degradation,steroid metabolism and energy metabolism.2.The The expression levels of FXa m RNA and protein in gastric cancer cell lines were detected by q RT PCR and WB.These results show that the expression of FXa in HGC27 cells were significantly higher than that in GES-1 cells(P<0.001),but decreased in MKN45,NCI-N87 and SNU216 cells,no significant difference in AGS cells.Then,in order to verify whether FXa plays a role in anoikis resistance of gastric cancer cells,MKN45 and NCI-N87 cells were suspended for 24 hours.Post suspension,FXa expression of MKN45 and NCI-N87 cells in adherent and suspended state were detected respectively.Furthermore,results showed that FXa expression of MKN45 and NCI-N87 cells decreased after 24 hours of suspension.Compared with adherent cells,the apoptosis probability of MKN45 and NCI-N87 cells is greatly increased(P<0.01),the data of cells penetrating the Trans well chamber is greatly reduced(P<0.001),and the expression level of FXa is increased.The invasion ability of cells was greatly weakened(P<0.001),the range of tumor cell metastasis and spread was on the other hand greatly reduced(P<0.001),and the proliferation and growth ability of tumor cells was significantly reduced(P<0.05).Compared with the cells in the control group,the expression levels of Caspase-3 and caspase-7 genes in MKN45 cells increased significantly(P<0.001),while the expression levels of the apoptosis-inhibiting protein Bcl-2 decreased drastically(P<0.001).In the NCI-N87 cell family/ species,24 hours after the completion of cell resuspension,the expression levels of the two genes Caspase-3 and Caspase-7 in the cells with up-regulated FXa expression levels increased significantly,but inhibited apoptosis.The expression level of Bcl-2 did not change significantly in the cells in the two different states of adherence and suspension(P>0.05).3.The LDH activity of gastric cancer cells after FXa overexpression was detected by ELISA.In MKN45 cells,compared with the control group,the LDH activity of gastric cancer cells after FXa overexpression and suspension for 24 hours was significantly increased(P<0.001),but it was not significantly increased in the adherent state(P<0.001).Similarly,in NCI-N87 cell line,the LDH activity significantly increased after FXa overexpression and suspension for 24 h(P<0.001).The same method was used to detect ROS activity in gastric cancer cells.In MKN45 cells,FXa overexpression significantly decreased ROS activity after 24 hours of suspension compared with control cells(P<0.001);similarly,in NCI-N87 cells,ROS activity in control group decreased after 24 hours of suspension(P<0.05),and compared with the up-regulation of the FXa expression level,the activity of the ROS declined significantly after 24 hours of cell resuspension(P<0.001).Compared with the control group,after 24 hours of resuspension,the expression levels of the two proteins PDK1 and PDK4 increased significantly(P<0.001),while the expression levels of the two proteins PDK2 and PDK3 increased significantly after 24 hours of resuspension.There was no significant change(P>0.05).4.Results of protein mass spectrometry also revealed that the expression of GOSR1 was up-regulated after overexpression of FXa and suspension,and the interaction between FXa and GOSR1 was confirmed by Co-IP.5.WB was used to detect the expression of GOSR1 protein in normal gastric epithelial cells GES-1 and gastric cancer cell lines SNU216,AGS,NCI-N87,MKN45 and HGC27.These results also showed that compared with normal gastric epithelial cells GES-1,the expression of GOSR1 protein in NCI-N87 and MKN45 cell lines was significantly increased(P<0.05),but decreased in AGS and HGC27 cells(P<0.05).There was no significant difference in SNU216 cells(P<0.05).MKN45 and NCI-N87 cells were suspended for 24 hours.The expression of GOSR1 increased significantly(P <0.01),and decreased after 48 hours(P<0.05),but still significantly higher than that in adherent cells.6.Flow cytometry was used to detect the apoptotic rate of cells in suspension for 24 hours after transfection with plasmid knockdown GOSR1.The results showed that compared with knockdown GOSR1 control group,the apoptotic rate of cells in suspension for 24 hours after knockdown GOSR1 also significantly increased(P<0.05).Compared with MKN45 cells without GOSR1 knockdown,the migration distance of MKN45 cells in GOSR1 knockdown group and FXa + knockdown group decreased significantly at 24 hours(P<0.05).Compared with FXa non expression group,the expression of Bcl-2 was decreased in FXa overexpression group and GOSR1 knockdown group(P<0.05).Meanwhile,the expression of Bcl-2 was significantly lower in FXa overexpression group and GOSR1 knockdown group than in GOSR1 knockdown group(P<0.01).Compared with FXa non expression group,the expression of ROS in FXa overexpression and GOSR1 knockdown group was significantly decreased(P<0.001),while the expression of ROS in FXa overexpression and GOSR1 knockdown group was significantly lower than that in GOSR1 knockdown group(P<0.01).7.Objectives to establish the subcutaneous tumor models of OE-NC-FXa,OE-FXa and sh-NC-GOSR1,sh-GOSR1 MKN45 cells were also revealed.In addition,results also revealed that: the growth rate of tumors in the over expression group was significantly lower than that in the un-overexpression group;compared with the un-knockdown GOSR1 group,the growth trend in the knockdown GOSR1 gastric cancer cell line was the same as that in the former FXa overexpression group.In MKN45 cells,compared with NC group,the expression of Bcl-2 and ROS in OE-FXa and sh-GOSR1 group were decreased(P<0.05).Conclusion1.This study confirmed the positive expression of FXa in gastric cancer tissues and gastric cancer cell lines.The expression level of FXa in gastric cancer tissues was significantly lower than that in adjacent tissues and correlated with tumor size and TNM stage.2.FXa promotes the invasion,proliferation and migration of gastric cancer cells.3.FXa regulates the anoikis resistance of tumor cells through GOSR1/ROS/Bcl-2 signal axis,so as to promote the invasion and metastasis of gastric cancer. |
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