MiR-140 Reverse Gastric Mesenchymal Stem Cell Induced Gastric Cancer Cell Immune Escape By Targeting PD-L1

objectiveTo observe the expression of miR-140 and PD-L1 in gastric cancer tissues and analyzed the correlation with clinicopathological characteristics.To up-regulating miR-140 in gastric cancer mesenchymal stem cells and observe the effect on proliferation,apoptosis and PD-L1 in gastric cancer cells.To construct immune circumstance of gastric cancer cells by PBMC and observe the effect of miR-140 mimics GCMSC on immune escape,discuss the mechanisms,to provide basic dates for gastric diagnosis,prognosis evaluation,immune therapy,stem cell therapy and target therapy.Methods1.MiR-140 and PD-L1 expression in gastric cancer tissues and paired para cancer normal gastric tissues was detected by qRT-PCR and Western blot,the correlation with tumor diameter and TNM stage was observed;2.Gastric cancer mesenchymal stem cells and gastric mesenchymal stem cells were extracted from gastric cancer or gastric tissues,miR-140 expression was detected;3.GCMSC was transfected with miR-140 mimics and CCK-8 was used to detect proliferation,flow cytometry assay was used to detect apoptosis in GCMSC intervened HGC-27 cells.4.GCMSC induced HGC-27 immuneescape and miR-140 mimics GCMSC reversed immune escape interfered by PBMC was observed,cytokines secreted by PBMC was detected with ELISA.5.PD-L1 was detected in miR-140 mimics/GCMSC HGC-27 cells.6.The target modulating relationship of miR-140 and PD-L1 was forecasted and luciferase reporter was used to identify the directed targeting relation.Results1.MiR-140 was lower in gastric tissues than in para cancer normal gastric tissues(P<0.05),negative correlated with tumor diameter and TNM stage(P<0.05);PD-L1 m RNA and protein were higher in gastric tissues(P<0.05),positively correlated with tumor diameter and TNM stage(P<0.05).2.MiR-140 in GCMSC was higher than in NGMSC(P<0.001).3.Compared with HGC-27,the survival rate of GCMSC/HGC-27 cell was increased(P<0.001),apoptosis rate was decreased(P<0.05);Compared with HGC-27 or GCMSC/HGC-27,the survival rate of miR-140 mimics GCMSC/HGC-27 cell was decreased(P<0.01;P<0.001),apoptosis rate was increased(P<0.05;P<0.01).4.When GCMSC/HGC-27 cell was interfered by PBMC,the survival rate of HGC-27 cell was not affected(P>0.05),GCMSC induced HGC-27 escaped from immune circumstance.In miR-140 mimics GCMSC/HGC-27 and HGC-27 cells,the survival rate was decreased once interfered with PBMC(P<0.01;P<0.01),Up-regulating miR-140 in GCMSC reversed HGC-27 immune escape and increased cytokines secretion by PBMC.5.MiR-140 mimics GCMSC down-regulated PD-L1 m RNA,PD-L1 protein and PD-1 protein in HGC-27 cells and reversed HGC-27 immune escape.6.Luciferase report showed that PD-L1 was the direct target of miR-140.Conclusion1.In gastric cancer tissues,miR-140 is down-regulated and PD-L1 is up-regulated,they correlate with tumor diameter and TNM stage.2.Up-regulating miR-140 in gastric cancer mesenchymal stem cells mayinhibit gastric cancer cell proliferation and promote apoptosis.3.Up-regulating miR-140 in gastric cancer mesenchymal stem cells may reverse gastric cancer cell immune escape.4.MiR-140 reverses GCMSC induced gastric cancer cell immune escape by target inhibiting PD-L1.