Inhibitory Effect Of Recombinant Human Sema3A On Angiogenesis Of Gastric Cancer And The Associated Mechanisms

Semaphorin 3A(Sema3A),was originally considered to be a negative regulatory protein of nervous system development and axon guidance.Recent studies had shown that Sema3 A was underexpressed in a variety of cells such as endothelial cells,bone marrow cells,and tumor cells,and had received increasing attention in the study of angiogenesis and tumorigenesis.However,its specific regulatory mechanisms for tumor angiogenesis were not fully explained.This study found that Sema3 A was lowly expressed in tissues and serum of gastric cancer patients and various human gastric cancer cell lines,and its receptor neuropilin-1(NRP-1)was highly expressed in gastric cancer tissues and human gastric cancer cell MKN-28.Sema3 A was negatively correlated with microvessel density(MVD)of gastric cancer tissues.These results suggest that Sema3 A may participate in and negatively regulate the occurrence and development of angiogenesis in gastric cancer.Therefore,this study hypothesized that the artificial addition of recombinant human Sema3 A can inhibit the angiogenesis of gastric cancer,thereby reversing the progression of tumor development.In order to verify this hypothesis,we established a " gastric cancer cell-human umbilical vein endothelial cell(HUVEC)" in vitro co-culture system to mimic the angiogenic microenvironment of gastric cancer in human body,and then exogenously added recombinant human Sema3 A.Afterwards,the tube formation ability and the expression of vascular endothelial growth factor receptor 2(VEGFR2)protein of HUVEC were significantly decreased,while the expression level of NRP-1 protein was not significantly changed.The results suggest that Sema3 A may inhibit the growth and metastasis of gastric cancer by down-regulating the expression of VEGFR2 and inhibiting VEGF-induced tumor angiogenesis.Therefore,in the implementation of anti-VEGF gastric cancer treatment regimen,the addition of Sema3 A protein exogenously may play a role in inhibiting angiogenesis of gastric cancer,which may provide a new idea for clinical treatment of gastric cancer.Objective:To study the expression of Sema3 A and its receptor NRP-1 in gastric cancer,and the correlation between Sema3 A and microvessel density.To explore the effect of recombinant human Sema3 A on angiogenesis of gastric cancer and its related mechanisms.Methods:1.SP method of immunohistochemistry was used to detect the expression of Sema3 A and NRP-1 between gastric cancer tissues and normal gastric mucosa,and the microvessel density in gastric cancer tissues.2.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion level of Sema3 A protein in the serum of gastric cancer patients.3.Western blotting was used to detect the protein expression of Sema3 A and its receptor NRP-1 in normal gastric mucosal cells(GES-1)and five gastric cancer cell lines(MGC-803,HGC-27,MKN-28,SGC-7901,MKN-45).4.The "normal gastric mucosal cells-human umbilical vein endothelial cells" and "gastric cancer cells-human umbilical vein endothelial cells" in vitro co-culture system were established to simulate the normal and gastric angiogenesis microenvironment in human body.Recombinant human Sema3 A protein was added exogenously into the co-culture system,and the tube formation experiment was used to detect the effect of different concentrations of Sema3 A on the tube formation ability of HUVEC in the co-culture system.5.Western blotting was used to detect the effect of different concentrations of recombinant human Sema3 A on the expression of NRP-1 and VEGFR2 of HUVEC in co-culture system.Results:1.Expression and localization of Sema3 A and its receptor NRP-1 in gastric cancerThe results of immunohistochemistry showed that the expression of Sema3 A in gastric cancer tissues was significantly lower than that in normal gastric mucosa tissues(P<0.001),and Sema3 A was primarily located in the cytoplasm.The expression of NRP-1 in gastric cancer tissues was significantly higher than that in normal gastric mucosa tissues(P<0.001),and NRP-1 was mainly located in cytoplasm and cell membrane.There was no significant correlation between the expression of Sema3 A and NRP-1(P =0.963).2.Relationship between Sema3 A and its receptor NRP-1 in clinicopathological featuresThe results of clinicopathological analysis showed that Sema3 A and its receptor NRP-1 were associated with TNM staging of patients with gastric cancer.The positive expression rate of Sema3 A in stage III patients was significantly lower than that in stage I and II(P =0.037),the positive expression rate of NRP-1 in stage III patients was significantly higher than that in stage I and II(P =0.026).The expression of Sema3 A and NRP-1 was not correlated with age,gender,tumor diameter,degree of differentiation,and lymph node metastasis(P>0.05).3.Secretion level of Sema3 A protein in serum of patients with gastric cancerThe ELISA results showed that the secretion level of Sema3 A in the serum of patients with gastric cancer was significantly lower than that of the normal control group(P<0.001).4.Correlation between Sema3 A expression level and microvessel density in gastric cancerMVD count analysis showed that the positive expression rate of Sema3 A in gastric cancer tissues was negatively correlated with MVD values(P<0.001).5.Expression of Sema3 A and its receptor NRP-1 in human gastric cancer cell lines and normal gastric mucosal cellsWestern blotting results showed that the expression levels of Sema3 A in four human gastric cancer cell lines(HGC-27,MKN-28,SGC-7901,MKN-45)were lower than that in GES-1(P<0.05).Among them,Sema3 A had the lowest expression in MKN-28 cells.The expression level of NRP-1 in MKN-28 cells was higher than that in GES-1(P=0.007).6.Comparison of HUVEC tube formation ability in in vitro co-culture systemThe co-culture system were constructed to simulate the normal angiogenic microenvironment(GES-1+HUVEC)and the angiogenic microenvironment of gastric cancer angiogenesis microenvironment(MKN-28+HUVEC).The results of tube formation in vitro showed that the tube formation ability of HUVEC in the MKN-28+HUVEC co-culture system was significantly higher than that in the normal group(P<0.001).The ELISA results showed that the secretion level of Sema3 A in the MKN-28+HUVEC co-culture system was significantly lower than that in the normal group(P<0.001).7.Effect of different concentrations of recombinant human Sema3 A on the tube formation ability of HUVEC in MKN-28+HUVEC co-culture systemThe results of tube formation experiments showed that after exogenous addition of recombinant human Sema3 A,the number and length of tube formed by HUVEC in MKN-28+HUVEC co-culture system were significantly reduced,and the integrity of lumen structure was also significantly reduced,and the tube formation capacity of HUVEC was decreased with the increase of recombinant human Sema3 A concentration(P<0.05).8.Effect of different concentrations of recombinant human Sema3 A on the expression of VEGFR2 and NRP-1 in HUVEC of MKN-28+HUVEC co-culture systemWestern blotting experiments showed that the protein expression level of VEGFR2 was significantly decreased with the addition of recombinant human Sema3 A into the co-culture system(P<0.01),but had no significant effect on the protein expression of NRP-1.Conclusion:1.Sema3 A is lowly expressed in gastric cancer tissues,serum and cell lines,and NRP-1 is highly expressed in gastric cancer tissues and MKN-28 cells.2.Sema3 A and its receptor NRP-1 are associated with TNM staging of gastric cancer patients,and Sema3 A is negatively correlated with microvessel density.3.Recombinant human Sema3 A has the effect of inhibiting angiogenesis of gastric cancer in vitro,which may be related to down-regulation of VEGFR2 protein expression.