Effect Of PD-L1 Expression On Biological Behavior Of Gastric Cancer Cells And Its Mechanism

Objective:To investigate the effect of PD-L1 expression on the migration,invasion and adhesion of gastric cancer cells MKN45 and SGC7901,and to explore the effect of PD-L1 expression on epithelial-mesenchymal transformation（EMT）related proteins and Wnt/β-catenin signaling pathway related proteins.Methods:Firstly,Western blot and qRT-PCR were used to identify the expression of PD-L1 in MKN45 and SGC7901 gastric cancer cells.Then three kinds of PD-L1-si RNA sequences were screened by Western blot and qRT-PCR methods,and the best PD-L1-si RNA sequence was selected for follow-up experiments.The best PD-L1-si RNA and NC-si RNA were used to interfere with the two kinds of gastric cancer cells,and the interference was verified by Western blot and qRT-PCR.The experiment was divided into control group,si-NC group and si-PD-L1 group.Transwell migration test,Transwell invasion test and MTT test were used to detect the migration,invasion and adhesion of the two kinds of gastric cancer cells.Western blot and qRT-PCR were used to detect the protein and mRNA expression of EMT-related proteins（E-cadherin,Vimentin,Snail）in each group of two kinds of gastric cancer cells.Western blot and qRT-PCR were used to detect the protein and mRNA expression of Wnt/β-catenin signal pathway related protein（GSK-3β,β-catenin,c-myc）in each group of two kinds of gastric cancer cells.Results:PD-L1 was highly expressed in both MKN45 and SGC7901 gastric cancer cells.The gastric cancer cell model with silent expression of PD-L1 was successfully constructed by using the best PD-L1-si RNA selected.The results of Transwell migration test showed that the migration rate of two kinds of gastric cancer cells in si-PD-L1 group was significantly lower than that in the other two groups（F=84.631 and 301.046,respectively,P<0.001）.The results of Transwell invasion test showed that the invasion rate of two kinds of gastric cancer cells in si-PD-L1 group was significantly lower than that in the other two groups（F=19.371 and 47.001,respectively,P<0.05）.The results of MTT assay showed that the adhesion rate of two kinds of gastric cancer cells in si-PD-L1group was significantly lower than that in the other two groups（F=79.869 and 42.101,respectively,P<0.01）.Western blot was used to detect the expression of EMT-related proteins E-cadherin,Vimentin and Snail in two kinds of gastric cancer cells:control group,si-NC group and si-PD-L1 group,the results showed that there was significant difference in protein expression between si-PD-L1 group and the other two groups（FMKN45-E-cadherin protein=127.708,FMKN45-Vimentin protein=79.625,FMKN45-Vimentin protein=85.259,FSGC7901-Vimentin protein=59.608,FMKN45-Snail protein=57.170,FSGC7901-Snail protein=149.758,all P<0.05）;qRT-PCR was used to detect EMT-related proteins E-cadherin,Vimentin and Snail from the point of view of mRNA,the results showed that the expression of mRNA in si-PD-L1 group was significantly different from that in the other two groups（FMKN45-E-cadherin mRNA=477.171,FSGC7901-E-cadherin mRNA=292.801,FMKN45-Vimentin mRNA=65.965,FSGC7901-Vimentin mRNA=64.091,FMKN45-Snail mRNA=145.091,FSGC7901-Snail mRNA=25.551,all P<0.05）;among them,the protein and mRNA expression of E-cadherin in si-PD-L1group was higher than that of the other two groups,while the protein and mRNA expression of Vimentin and Snail was lower than that of the other two groups（all P<0.05）.Western blot was used to detect the expression of GSK-3β,β-catenin and c-myc related to Wnt/β-catenin signaling pathway in two kinds of gastric cancer cells:control group,si-NC group and si-PD-L1 group,the results showed that there was significant difference in protein expression between si-PD-L1 group and the other two groups(F_{MKN45-GSK-3β}protein=264.262,FSGC7901-GSK-3βprotein=15.557,FMKN45-β-catenin protein=323.557,FSGC7901-β-catenin _protein=115.931,F_{MKN45-c-myc protein}=121.703,F_{SGC7901-c-myc protein}=163.183,all P<0.05);qRT-PCR was used to detect Wnt/β-catenin signal pathway related proteins GSK-3β,β-catenin and c-myc from the point of view of mRNA,the results showed that there was significant difference in mRNA expression between si-PD-L1 group and the other two groups(FMKN45-GSK-3βmRNA=41.693,FSGC7901-GSK-3βmRNA=64.132,FMKN45-β-catenin mRNA=88.069,FSGC7901-β-catenin mRNA=103.791,FMKN45-c-myc mRNA=208.368,FSGC7901-c-myc _mRNA=73.055,all P<0.05);among them,the protein and mRNA expression of GSK-3βin si-PD-L1 group was higher than that in the other two groups,while the protein and mRNA expression ofβ-catenin and c-myc was lower than that in the other two groups（all P<0.05）.Conclusion:This study confirmed that the changes of migration,invasion and adhesion of two kinds of gastric cancer cells were regulated by the expression of PD-L1.The expression of PD-L1 affects the expression of proteins related to EMT and Wnt/β-catenin signal pathway.The expression of PD-L1 promotes the migration,invasion and adhesion of the above two gastric cancer cells and other biological behaviors,and its preliminary mechanism may be related to the regulation of EMT related proteins and Wnt/β-catenin signal pathway in gastric cancer.