| Objective:The aim of this study was to invesgate the effects of cisplatin(DDP)on the expression of extracellular regulated-protein kinase(ERK),phosphorylated extracellular signal regulated kinase(p-ERK)and programmed death ligand 1(PD-L1)in human lung adenocarcinoma A549 cells in vitro and to explore the effect of DDP on the expression of PD-L1 in human lung adenocarcinoma A549 cells and its possible mechanism,and to provide a theoretical basis for DDP combined immunotherapy.Methods:(1)Culture cells: After 3-5 passages,A549 cells in logarithmic growth phase were used for subsequent experiments.(2)A549 cells were intervened by different concentrations of DDP(0 mg/L,0.5 mg/L,1 mg/L,2 mg/L,4 mg/L,8 mg/L)for different time(24h,36 h,48h).The optical density(OD)values of A549 cells in logarithmic growth phase were measured by CCK-8 assay,and the proliferation inhibition rates and 50% inhibitory concentration(IC50)were calculated;(3)Flow cytometry was used to detect the expression of PD-L1.Grouping: to detect the effect of different concentrations of DDP on PD-L1,DDP was divided into different concentration groups(0mg/L,0.5mg/L,1mg/L,2mg/L,4mg/L,8mg/L),In order to explore the effect of ERK pathway inhibitor PD98059 on PD-L1,the cells were divided into the control group,DDP group,PD98059 group and DDP combined with PD98059 group.The control group was added with culture medium,DDP group was added with IC50 concentration of DDP,20umol/L PD98059 was added into PD98059 group,20umol/L PD98059 and IC50 DDP were added into DDP combined with PD98059 group.After intervention for 48 h,the expression of PD-L1 was detected by flow cytometry(4)The expression levels of ERK and p-ERK in each group were detected by Western blot.The cells were divided into control group,DDP group,PD98059 group and DDP combined with PD98059 group.After intervention for 48 hours,the total protein of cells was extracted and the expression levels of ERK and p-ERK in each group were detected by Western blot.Results: 1.Different concentrations of DDP(0mg/L,0.5mg/L,1mg/L,2mg/L,4mg/L,8mg/L)intervened A549 cells.The A549 cells proliferation inhibition rates which were intervened for 24 hours respectively were 0% 、(2.684±0.995)% 、(5.614±1.229)% 、(18.989±2.062)% 、(24.256±2.828)% 、(39.83±2.969)%,the difference was statistically significant(P<0.05),which were intervened for 36 hours respectively were(4.373±1.296)% 、(8.855±2.355)%、(23.88±3.057)%、(39.995±3.365)%、(54.19±2.6)%,which were intervened for 24 hours respectively were 0% 、(5.082±2.793)% 、(10.019±3.77)% 、(30.426±2.533)% 、(56.189±3.165)% 、(75.522±4.216)%,the difference was statistically significant(P<0.05).Except DDP 0mg/L group,there was significant difference in the inhibition rate of proliferation of different concentration groups between different intervention time groups,(P<0.05).After calculation,the IC50 values of DDP intervention for 24 h,36h and 48 h were 11.244mg/L,6.247mg/L and 3.586mg/L,respectively.48 h and 3.586mg/L were selected as the optimal intervention time and IC50.2.A549 cells were intervened for 48 hours by different groups of DDP(0mg/L,0.5mg/L,1mg/L,2mg/L,4mg/L,8mg/L),the expression of PD-L1 was(39.87±1)% 、(42.77±0.85)% 、(51.87±2.05)% 、(62.03±1.31)% 、(70.47±0.21)% 、(75.8±1.85)%,respectively.There was significant difference among the groups(P<0.05).The expression of PD-L1 in control group,DDP group,PD98059 group and DDP combined with PD98059 group was(40.23±2.07)%,(67.07±1.31)%,(38.77±1.69)%,(52.57±1.01)%,respectively.Compared with the control group,the expression of PD-L1 in DDP group and DDP combined with PD98059 group increased(P<0.05).The expression of PD-L1 in DDP combined with PD98059 group was lower than that in DDP group,but higher than that in PD98059 group(P<0.05).There was no statistical significance between PD98059 group and control group(P>0.05).3.The relative protein expression of ERK in the cntrol group,DDP group,PD98059 group and DDP combined with PD98059 group respectively was 1.26±0.13,1.27±0.16,1.27±0.12 and 1.28±0.14,and there was no significant difference among the groups(P>0.05).The relative protein expression of p-ERK in the control group,DDP group,PD98059 group and DDP combined with PD98059 group respectively was 0.29±0.02,0.74±0.02,0.23±0.02 and 0.49±0.05.Compared with the control group,the expression of p-ERK in DDP group and DDP combined with PD98059 group increased,but the expression in PD98059 decreased.The expression of p-ERK in DDP combined with PD98059 group was lower than that in DDP group,but higher than that in PD98059 group(P<0.05).Conclusion: 1.Cisplatin inhibited the proliferation of A549 cells in a dose and time-dependent manner.2.Cisplatin up-regulated the expression of PD-L1 in human lung adenocarcinoma cell line A549 in a concentration dependent manner.3.Cisplatin up-regulated the expression of PD-L1 by activating ERK pathway. |
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