MLN4924通过抑制类泛素化通路上调人肺腺癌A549细胞中PD-L1的表达

MLN4924 enhances PD-L1 expression in human lung adenocarcinoma A549 cells by inhibiting the Neddylation pathway

目的:探究MLN4924对肺腺癌A549细胞中程序性死亡-配体1(PD-L1)表达的影响及其可能的内在机制。方法:体外培养A549细胞,按(0、0.25、0.5、0.75、1、5、10μmol/L)MLN4924分组分别处理24、48、72 h后,CCK-8法检测细胞增殖抑制率,作图并计算各组半数抑制浓度(IC_(50)),流式细胞术检测PD-L1的表达,选择最适时间72 h及其IC_(50)用于后续实验。后续实验按(0、0.469μmol/L)MLN4924分组培养72 h,Western blot法检测FBXW2蛋白、肌段同源盒2(MSX2)蛋白、Y-染色体性别决定基因盒2(SOX2)蛋白、PD-L1蛋白表达,qPCR法测定PD-L1 mRNA表达。结果:MLN4924在24、48、72 h的IC_(50)值分别为(1.976、0.647、0.469)μmol/L;与对照组相比,实验组A549细胞PD-L1表达均上调,呈时间-浓度依赖性。与对照组相比,实验组A549细胞FBXW2、SOX2蛋白表达降低,MSX2蛋白、PD-L1 mRNA及蛋白表达均增加。结论:MLN4924通过抑制类泛素化通路上调人肺腺癌A549细胞中PD-L1表达。

Objective:To investigate the effect of MLN4924 on the expression of programmed death-ligand 1(PD-L1)in lung adenocarcinoma A549 cells and its possible underlying mechanism.Methods:Human lung adenocarcinoma A549 cells were cultured in vitro and treated with different doses of MLN4924(0,0.25,0.5,0.75,1,5,10μmol/L)for 24,48 and 72 h.The inhibition rate of cell proliferation was detected by CCK-8 method.The median inhibitory concentration(IC_(50))of each time group was calculated.At the same time,the expression of PD-L1 was detected by flow cytometry.Meanwhile,the obvious up-regulation time of PD-L1 and its IC_(50)were selected for subsequent experiments.Follow-up experiments were divided into two groups:Blank control group and IC_(50)MLN4924 treatment group.After 72 hours of culture,the expression of FBXW2 protein,muscle segment homeobox2(MSX2)protein,sex-determining region Y-box containing gene 2(SOX2)and PD-L1 protein was detected by Western blotting method,and the expression of PD-L1 mRNA was detected by qPCR method.Results:The IC_(50)values at 24,48 and 72 h were 1.976,0.647,0.469μmol/L.Compared with the control group,the expression of PD-L1 in A549 cells treated with MLN4924 was up-regulated in a time and concentration dependent manner.Compared with the control group,the expressions of FBXW2 and SOX2 proteins in MLN4924 treatment group were decreased,while the expressions of MSX2 protein,and the expressions of the mRNA and protein of PD-L1 were increased.Conclusion:MLN4924 enhances the expression of PD-L1 in A549 cells by inhibiting Neddylation pathway.