17β-雌二醇联合苯并芘异常激活人肺腺癌A549细胞中AHR/PD-L1轴

17β-Estradiol combined with benzopyrene abnormally activates AHR/PD-L1 axis in human lung adenocarcinoma A549 cells

目的:探讨17β-雌二醇(17β-estradiol,E2)和苯并芘(benzopyrene,BaP)对人肺腺癌A549细胞程序性死亡配体1(programmed death ligand 1,PD-L1)表达的影响及其潜在分子机制。方法:分别选用浓度梯度为1~1000 nmol/L的E2和浓度梯度为0.008~5μmol/L的BaP处理A549细胞,筛选出最适浓度;实验分为对照组、E2组、BaP和E2+BaP组,每组实验重复3次。RT-qPCR检测PD-L1的mRNA水平;Western blot检测PD-L1、芳香烃受体(aryl hydrocarbon receptor,AHR)和缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)表达水平,以及蛋白激酶B(protein kinase B,PKB/AKT)和细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)的磷酸化水平。分别加入AKT特异性抑制剂LY294002和ERK1/2特异性抑制剂PD98059进行反向验证,观察PD-L1表达的变化。结果:与E2组和BaP组相比,E2+BaP组A549细胞中PD-L1的mRNA和蛋白表达水平均显著上调(P<0.05);AHR、HIF-1α、p-AKT和p-ERK1/2蛋白水平均显著高于其他各组(P<0.05);LY294002和PD98059能够逆转E2+BaP对A549细胞PD-L1表达的上调作用(P<0.05)。结论:E2联合BaP可能通过异常激活AHR-AKT-ERK1/2信号通路诱导人肺腺癌A549细胞PD-L1表达升高。

AIM:To investigate the effects of 17β-estradiol(E2)and benzopyrene(BaP)on programmed death ligand 1(PD-L1)expression in human lung adenocarcinoma A549 cells and their potential molecular mechanisms.METHODS:The A549 cells were treated with 1~1000 nmol/L E2 and 0.008~5μmol/L BaP,and the optimal concentrations were identified.The cells were divided into control group,E2 group,BaP group,and E2+BaP group.Each set of experiments was repeated 3 times.RT-qPCR was used to determine the mRNA level of PD-L1.Western blot was used to detect the expression levels of PD-L1,aryl hydrocarbon receptor(AHR)and hypoxia-inducible factor-1α(HIF-1α),as well as the phosphorylation levels of protein kinase B(PKB/AKT)and extracellular signal-regulated kinase 1/2(ERK1/2).The AKT-specific inhibitor LY294002 and ERK1/2-specific inhibitor PD98059 were used for reverse verification,and the changes in PD-L1 expression were observed.RESULTS:Compared with E2 group and BaP group,the mRNA and protein levels of PD-L1 in the A549 cells of E2+BaP group were significantly up-regulated(P<0.05).The protein levels of AHR,HIF-1α,p-AKT and p-ERK1/2 in E2+BaP group were significantly higher than those in other groups(P<0.05).LY294002 and PD98059 reversed the up-regulation of PD-L1 induced by E2+BaP(P<0.05).CONCLUSION:E2 combined with BaP up-regulates PD-L1 expression in human lung adenocarcinoma A549 cells through abnormal activation of the AHR-AKT-ERK1/2 signaling pathway.