The Expression And Significance Of EphB6 In Gastric Cancer

Background and objectiveGastric cancer(GC)is one of the most common malignancies in the world.Its morbidity and mortality rank second in China for malignant tumors.In recent years,some advances have been made in the diagnosis and treatment of GC,but postoperative recurrence rate remains high and the prognosis is still not optimistic.Therefore,it is necessary to further explore the mechanisms of occurrence and development of GC and find new types of GC biomarkers.This may be new breakthroughs in the diagnosis and treatment,and it is expected to improve the current level of diagnosis and treatment of GC.EphB6 is a member of the Eph receptor family and belongs to a kinase-deficient type receptor among tyrosine kinase receptors.Numerous studies suggest that down-regulation or deletion of EphB6 expression is closely related to the occurrence and prognosis of tumors.These data indicated that EphB6 as a malignancy-suppressing factor play an important role in development and tumorigenesis.However,this role of tumor suppressor factor is still controversial.At present,the expreesion and significance of EphB6 in GC has not been well investigated.In our pre-experiment,high-throughput sequencing of ctDNA in peripheral blood samples from patients with GC revealed high-frequency mutations in the EphB6.Based on the research progress and the results of previous studies,we used EphB6 as a research object to elucidate the expression of EphB6 in GC tissues and GC cell lines,construct EphB6 stably overexpressed GC cell lines,observe its significance for the biological behavior of GC cells,and initially explore the relevant molecular mechanisms.Methods1.In this study,we detected expression of EphB6 protein in a normal gastric epithelial cell line(GES-1)and four GC cell lines(MGC-803?MKN-45?AGS?SGC-7901)by using Western blot,observed the localization of EphB6 protein in cells by immunocytochemistry,detected the expression of EphB6 protein in GC tissues and corresponding adjacent normal tissues by immunohistochemistry,analyzed the relationship between EphB6 expression and clinicopathological parameters.2.We constructed the human EphB6 lentivirus overexpression vector and packaged and identified the virus.Lentivirus was transfected into human GC cell lines with low expression of EphB6 in vitro.The cell lines stably overexpressing EphB6 protein was confirmed by Puromycin screening and Western blot.3.CCK-8 assay,Transwell invasion assay,flow cytometry PI staining and Annexin V staining were used to detect the changes of cell proliferation,cell cycle distribution,apoptosis,invasion and metastasis before and after overexpression of EphB6.4.We detected the effect of EphB6 on its ligand EFNB2 by qRT-PCR.Results1.The expression of EphB6 protein was detected in all cell lines tested,in which MGC-803,AGS and SGC-7901 were significantly lower than GES-1(P<0.01),the expression in AGS cell was the lowest.The positive staining of EphB6 protein was brownish-yellow and coarse-like in the cytoplasm.The strongest expressions were located in the basal stem cells of the tissue glands.The expression of EphB6 protein in GC tissues was significantly lower than that in the adjacent tissues(P<0.001).The correlation between the down-regulation of EphB6 expression and the clinicopathological parameters of GC showed that the down-regulation of EphB6 protein was not significantly associated with the patient's gender,age,tumor location,depth of invasion,nerve invasion,and lymph node metastasis,but was related to the degree of tumor differentiation(P=0.03<0.05),vascular tumor thrombus(P=0.018<0.05)and clinical stage(P=0.016<0.05).2.The recombinant plasmid was successfully constructed and the sequencing results were consistent.The recombinant EphB6 Lentiviral recombinant expression vector was successfully obtained through lentivirus packaging.The titer of the virus satisfies the following experimental requirements.The level of EphB6 protein in AGS and SGC-7901 cells transfected with lentivirus was significantly increased(P<0.01).3.The CCK-8 assay showed that: With the progress of the experiment(48h,72 h,96h),the OD value of EphB6 overexpressing group was significantly lower than that of empty vector control group in AGS and SGC-7901 cells(P<0.05);After the data was transformed into the proliferation rate,it also showed that: With the progress of the experiment,the fold increase of cells in the control group was significantly greater than the experimental group(P <0.05);Flow cytometry analysis of the cell cycle showed that: AGS and SGC-7901 experimental group cells compared to the control group,G1 phase cell ratio increased significantly(P <0.001),S phase cell ratio was significantly reduced(P <0.01);Flow cytometry Apoptosis results showed that: AGS and SGC-7901 cells in the experimental group had no statistical difference in apoptosis index compared with the control group;Transwell assay detected the invasion and metastasis ability showed that: AGS and SGC-7901 experimental group compared to the control group,the number of cells transferred significantly reduced(P<0.001).4.The expression level of EFNB2 mRNA in the experimental group was also significantly higher than that in the control group(P<0.001).Conclusion1.The expression level of EphB6 protein is significantly down-regulated in GC cell lines and GC tissues,and its down-regulation in GC tissues is closely related to vascular tumor thrombus,tumor differentiation and clinical stage.This suggests that EphB6 may inhibit the proliferation and metastasis of GC,participate in the early occurrence,development and progression of GC.EphB6 protein may be used as a reference indicator to assess the extent of progression of GC and prognosis.2.We successfully constructed EphB6 Lentiviral recombinant overexpression vector,and successfully obtained stable AGS and SGC-7901 GC cell lines with EphB6 overexpression,which laid a foundation for subsequent studies.3.Overexpression of EphB6 significantly inhibited the proliferation and invasion in AGS and SGC-7901 cells,but had no effect on the apoptosis of cells.The study also shows that the inhibition of cell proliferation may be caused by blocking the transition of cells from the G1 phase to the S phase.These data further confirmed that EphB6 has the function of inhibiting proliferation,invasion and metastasis of GC.4.Overexpression of EphB6 can upregulate the expression of its ligand EFNB2.This phenomenon may be a self-protection mechanism of tumor.Without this self-protection mechanism,the tumor suppressor function of EphB6 may be more powerful.