MLN4924 Up-regulates Pd-L1 Expression In A549 Human Lung Adenocarcinoma Cells Via Inhibiting The Neddylation Pathway

Objective: In this study,we designed experiments to explore the effect of MLN4924 on the expression of programmed death-ligand 1(PD-L1)in human lung adenocarcinoma A549 cells,and preliminarily explore its possible molecular mechanism.Methods: 1.The optical density(OD)values of A549 cells treated with different concentrations of MLN4924(0,0.25,0.5,0.75,1.0,5.0,10.0μM)for different times(24h,48 h,72h)were detected by CCK8 method.The proliferation inhibition rate and 50% inhibiting concentration(IC50)of each time group were calculated.2.The expression of PD-L1 in A549 cells treated with different concentrations of MLN4924(0,0.25,0.5,0.75,1.0,5.0,10.0μM)for different times(24 h,48 h,72h)was detected by Flow cytometry.3.The 72 hours with the most obvious up-regulation of PD-L1 expression and its IC50 were selected for subsequent experiments.The control group was added with culture medium,and the experimental group was added with 72 h IC50concentration of MLN4924.The expression of FBXW2,muscle segment homeobox 2(MSX2),Sex-determining region Y-box-containing2(SOX2)protein,and PD-L1 protein was detected by Western blot.4.The control group was added with culture medium,and the experimental group was added with72 h IC50 concentration of MLN4924 to intervene for 72 h,respectively.The PD-L1 m RNA level of each group was detected by q PCR.Results:(1)After A549 cells were treated with different concentrations of MLN4924(0,0.25,0.5,0.75,1.0,5.0,10.0μM)for 24 h,48h,and 72 h,CCK8assay showed that OD value increased with the prolongation of treatment concentration or time,suggesting that cell proliferation inhibition rate gradually increased.The IC50 values of each time group(24h,48 h,72h)were 1.976,0.647,0.469μM,respectively.The expression levels of PD-L1 in A549 cells treated with different concentrations of MLN4924(0,0.25,0.5,0.75,1.0,5.0,10.0μM)for different times(24h,48 h,72h)were detected by flow cytometry,and the expression of PD-L1 in A549 cells gradually increased.There was a time-concentration dependence(P<0.05).3.Western blot showed that:Compared with the control group,the expression of FBXW2 and SOX2 proteins in the experimental group was down-regulated,while the expression of MSX2 protein and PD-L1 protein was up-regulated,and the differences were statistically significant(P<0.05).(4)The results of q PCR showed that the PD-L1 m RNA in the experimental group was up-regulated compared with the control group,and the difference was statistically significant(P<0.05).Conclusion: 1.MLN4924 inhibited the proliferation of A549 cells in a time-concentration dependent manner.2.MLN4924 could up-regulate the expression of PD-L1 in A549 cells in a time-concentration dependent manner.3.The molecular mechanism by which MLN4924 up-regulates the expression of PD-L1 in A549 cells may be related to the down-regulation of SOX2 expression.