| Objective:To explore the expression of epoxide hydrolase 2（EPHX2）in metastatic gastric cancer and its effect on the growth,migration and invasion of gastric cancer cells.Methods:1.The expression profile data of gastric cancer samples were obtained from GEO database.The data were standardized by Affy package and the differentially expressed genes were screened by limma package of R.2.The expression of EPHX2 in gastric cancer tissues and adjacent normal tissues was detected by Western blot,Real-time PCR and tissue microarray combined with immunohistochemistry.3.Stable cell lines for over-expression or knockdown in two gastric cancer cell lines AGS and HGC-27 were constructed.And protein expression of EPHX2 was validated by Western blot.4.A series of assays for function study including CCK-8 kit,plate colony formation assay,soft agar colony formation assay,cell wound-healing assay,Transwell cell migration assay in vitro and Transwell cell invasion assay in vitro,were implemented to exhibit the effect of EPHX2 on proliferation,migration and invasion of gastric cancer cells.Results:1.1136 differentially expressed genes were found in metastatic gastric cancer samples including 692 up-regulated genes and 444 down regulated genes.And the expression level of EPHX2 in gastric cancer metastasis samples was lower than that in non metastasis samples.2.The results of Western-blot and Real-time PCR showed that the protein and m RNA expression levels of EPHX2 were lower in gastric cancer tissues than those in adjacent normal tissues.The difference was statistically significant.Other than this,pathological analysis showed that the expression of EPHX2 was significantly related to the age of patients,lymph node metastasis,clinical stage and lymphatic vascular invasion,while the other pathological variables were not significantly related to the expression of EPHX2.The results of survival analysis showed that the survival time of patients with low expression of EPHX2 was significantly less than that of patients with high expression of EPHX2.3.The CCK-8 values(OD450)at different detection time points,the number and size of clones formed in the plate colony formation experiment,and the colony formation rate in the soft agar colony formation experiment were all reduced by overexpression of EPHX2,whereas were increased by knockdown of EPHX2.The difference was statistically significant.In the cell wound-healing assay,the cell scratch area of the EPHX2 overexpression group was wider than that of the control group,while the knockdown group was narrower than the.In the Transwell cell migration in vitro and invasion in vitro assay,the number of cells in the overexpression group cell microporous membrane was decreased,while the number of microporous cells in the knockdown group was increased,the difference was statistically significant.Conclusion:1.The m RNA and protein levels of EPHX2 in gastric cancer were significantly lower than those in adjacent tissues.2.EPHX2 inhibits the proliferation of gastric cancer cells.3.EPHX2 inhibits the migration and invasion of gastric cancer cells. |
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