Effect Of PD-1/PD-L1 On Proliferation,Migration And Invasion Of Esophageal Cancer Cells Via AKT Pathway

Objective:Firstly,to explore the role of PD-1 and PD-L1 in regulating invasion and metastasis of high differentiation Eca109 and low differentiation EC9706 cell lines.Then to seek the relationship between PD-1/PD-L1 and AKT pathway.Meanwhile to explore whether they will affect the expression of P21 and PLK4 which are the cell cycle related proteins.Methods:1.The Eca109 and EC9706 esophageal squamous cell cancer(ESCC)cell line was divided into negative control group,blank load transfection group and experimental group(transfect PD-1 overexpression plasmid and PD-L1 SiRNA).PD-L1-siRNA(siPD-L1)and PD-1 overexpression plasmid as experimental group,the siRNA-Control(SiCtr)and the empty vector plasmids as blank load transfection group were transiently transfected into Eca109 and EC9706 cells by liposome method;2.After transfection was observed by fluorescence microscope,the transfection was stopped at the right time;3.The PD-1 and PD-L1 protein expressions of PD-1 over-expression and PD-L1down-expression in Eca109 and EC9706 cell lines were tested by Western Blot at the protein level;4.The effect of PD-1 over-expression and PD-L1 down-expression on AKT pathway and cell cycle related protein P21 and PLK4 were analyzed in Eca109 and EC9706 cell lines was tested by Real-time PCR at the gene level;5.CCK8 value-added assay was used to detect the effect of over-expression of PD-1and down-expression of PD-L1 on the value-added ability of Eca109 and EC9706 cellline;6.The effect of PD-1 over-expression and PD-L1 down-expression on metastasis of Eca109 and EC9706 cell line Cell were used to detect by scratch assay and Transwell chamber migration experiments;7.Transwell chamber invasion experiments were used to detect the effect of PD-1over-expression and PD-L1 down-expression on invasion of Eca109 and EC9706 cell line;Results:1.Western blot results showed that the protein expression of PD-1 proteins in the PD-1 over-expression group were higher than those in the negative control group and the blank load transfection group(P<0.01),and the protein expression of PD-L1 proteins in the PD-L1 down-expression group were lower than those in the negative control group and the blank load transfection group(P<0.01)and there was no significant difference between the latter two groups.2.Real-time PCR results showed that the expression of PD-1 mRNA in the PD-1over-expression group were higher than those in the negative control group and the blank load transfection group(P<0.01),and the expression of PD-L1 mRNA in the PD-L1 down-expression group were lower than those in the negative control group and the blank load transfection group(P<0.01)and there was no significant difference between the latter two groups.Meanwhile,there was a significant difference(P<0.05)in the expression of AKT mRNA between the PD-1 over-expression group and the PD-L1 down-Expression group,as compared with the blank group and the blank load transfection group.However,there was no significant difference in the expression of PLK4 and P21 mRNA between the PD-1 over-expression group and the PD-L1down-Expression group,as compared with the blank group and the blank load transfection group.3.the value-added ability,metastasis ability,and the invasion ability in the PD-1over-expression group were higher than those in the negative control group and the blank load transfection group(P<0.01)and the value-added ability,metastasis ability,and the invasion ability in the PD-L1 down-expression group were lower than those in the negative control group and the blank load transfection group(P<0.01)by CCK8value-added assay,Transwell chamber migration and invasion experiments and cell scratch assay.There was no significant difference between the latter two groups.Conclusions:1.The results of western Blot and Real-time PCR showed that over-expression of PD-1 in ESCC cell line Eca109 and EC9706 can active the AKT signaling pathway,but it can not affect the expression of cell cycle related proteins P21 and PLK4 and down-expression of PD-L1 in ESCC cell line Eca109 and EC9706 can antagonize the AKT signaling pathway,similarly,it can not affect the expression of cell cycle related proteins P21 and PLK4.2.Over-expression of PD-1 promote proliferation,migration and invasion of the ESCC cells.Down-expression of PD-L1 inhibit proliferation,migration and invasion of the ESCC cells.