Study On The Biological Role And Molecular Mechanism Of Semaphorin4D In The Progression Of Colon Cancer

Aims:At present,there are few reports on the role and molecular mechanismof Sema4D in the progression of colon cancer.In this study,the correlation between the expression of Sema4D and the prognosis of colon cancer was analyzed;the lentiviral transfection vector was constructed to interfere with the expression of Sema4D;the effects of biological behaviors such as proliferation,invasion,migration and angiogenesis of colon cancer cells were observed in vitro and in vivo;the downstream signaling pathway was detected to explore the mechanism of Sema4D in the progression of colon cancer,all of which was used to provide a new turning point for the diagnosis and treatment of colon cancer.Methods:We analyzed the immunohistochemical staining of Sema4D in colon cancer.Meanwhile,a statistical analysis has been introduced to reveal the relationship between Sema4D and clinical factors(tumor size,location,tissue type,differentiation degree,lymphatic metastasis,TNM stage,etc.)and verify the correlation between the expression of Sema4D and the prognosis of colon cancer patients.Western blot was used to detect the expression of Sema4D in five different colon cancer cells,and then lentiviral transfection vectors were constructed.The stably transfected HCT-116 and LoVo cell of Sema4D shRNA were selected as research objects.In vitro,the effects of Sema4D on the biological behaviors such as proliferation,invasion,migration and tube formation of colon cancer cells were detected by MTT,Transwell and endothelial cell tube assay.In vivo,it was performed in nude mice to detect the effect of Sema4D on the growth of transplanted tumor(weight,volume,HE,Ki-67,etc.).Sema4D overexpressed lentivirus and AKT inhibitor LY294002 were used to detect the changes of downstream signaling pathways and to verify the molecular mechanism of Sema4D affecting the biological behavior of colon cancer cells by regulating AKT phosphorylation.Results:The immunohistochemical staining of Sema4D in colon cancer is deep,and its expression is highly correlated with tumor size,lymph node metastasis and TNM stage.The five-year survival rate of patients was related to the expression of Sema4D.The risk of death in patients with positive Sema4D expression was 2.72 times higher than that in negative patients.The survival of patients with negative expression of Sema4D protein was better than that of positive expression.Their expression of Sema4D were higher in five kinds of colon cancer cells than in normal colon cells.After being infected with Sema4D shRNA lentivirus,the Sema4D shRNA lentivirus infection group(shS4D group)was reduced in Sema4D expression level compared with the virus-free infection group(Control group)and GFP lentivirus infection group(GFP group).In vitro,the Sema4D shRNA lentiviral infection group(shS4D group)had lower ability of proliferation,invasion,migration,and tube-forming in HCT-116 and LoVo cells than the virus-free infection group(Control group)and GFP lentivirus infection group(GFP group).In vivo,compared with virus-free infection group(Control group)and GFP lentivirus infection group(GFP group),tumor weight and volume,tumor tissue necrotic percentage,Ki-67 staining ratio of nude mice infected with Sema4D shRNA lentivirus group(shS4D group)were significantly reduced.The AKT phosphorylation level in transplanted tumor was detected by Western blot,and the AKT phosphorylation level was decreased in the Sema4D shRNA lentivirus infection group(shS4D group)compared with the virus-free infection group(Control group)and the GFP lentivirus infection group(GFP group).In vitro,Sema4D was overexpressed by lentivirus,and the proliferation,invasion,migration and tube-forming ability of the S4D overexpressed group(S4D group)were higher than those of the non-viral infection group(Control group)and the control lentivirus infection group(GFP group).But this change was reversed by using the AKT inhibitor LY294002(S4D+LY group)to blockade the Akt signaling pathway.After being adjusted of Sema4D,the phosphorylation level of AKT was decreased in Sema4D shRNA lentivirus infection group(shS4D group).Adding exogenous recombinant human Sema4D(shS4D+S4D group),the phosphorylation level of AKT was increased.Using siRNA to interfere with PlexinB1,which is a high-affinity receptor of the Sema4D,we found the AKT phosphorylation level of the interference group(siPLX group)and the control group(siCon group)decreased.On contrast,there was no change in AKT phosphorylation levels after exogenous Sema4D added(siPLX+S4D group).Conclusion:1.Sema4D expression is higher in colon cancer tissues than in normal colonic mucosa,and its expression is highly correlated with tumor size,lymph node metastasis and TNM stage.The prognosis of colon cancer patients is related to the expression of Sema4D;2.Sema4D promotes biological behaviors of colon cancer such as proliferation,invasion,migration and angiogenesis;3.Sema4D affects the biological behaviors of colon cancer cells by increasing the AKT phosphorylation level and this effect is achieved by binding to the high affinity receptor PlexinB1.