PI3K/AKT通路通过Nrf2途径调控PD-L1在非小细胞肺癌中的表达  

PI3K/AKT regulates the expression of PD-L1 via Nrf2 pathway in non-small cell lung cancer

作　　者：王静[1] 陈洁[1] 胡春 黄诚[1] WANG Jing;CHEN Jie;HU Chun;HUANG Cheng(Department of Oncology,Xiamen Humanity Hospital Affliated to Fujian Medical University,Xiamen 361009,Fujian Province,China)

机构地区：[1]福建医科大学附属厦门弘爱医院肿瘤科,福建厦门361009

出　　处：《中国癌症杂志》2020年第6期419-427,共9页China Oncology

摘　　要：背景与目的:程序性死亡[蛋白]配体-1(programmed death ligand-1,PD-L1)在肿瘤细胞中的表达对肿瘤逃避机体免疫监视具有重要的意义,其表达通常受MAPK、PI3K-AKT或STAT3等多条通路的影响,但这些通路具体经哪个关键环节尚不明确。拟通过PI3K/AKT信号通路对肺癌细胞A549、H460中PD-L1表达的调控具体机制进行探究。方法:使用shRNA技术选择性地沉默A549、H460细胞中的HEB、HTF4、Nrf2及FOXO3a等基因,构建缺陷细胞株;将目的基因PD-L1的3’UTR区域构建至pGL3-basic载体中,通过luciferase双报告基因体系检测PD-L1转录激活情况,并使用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)技术验证细胞内PD-L1基因转录的情况;通过蛋白质印迹法(Western blot)检测细胞内PD-L1的总表达情况;免疫细胞与肿瘤细胞共培养,检测Nrf2基因敲除前后,人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)对A549、H460细胞株的杀伤效果。结果:经10μg/mL的insulin刺激后,A549、H460细胞株中蛋白激酶B(AKT)基因磷酸化水平显著增强,伴随着PD-L1基因的转录水平和表达水平显著增强(P<0.001),在HEB、HTF4及FOXO3a基因敲除的细胞株中,情况相似,而在Nrf2基因敲除的细胞株A549(A549^Nrf2-)和H460(H460^Nrf2-)细胞株中,PD-L1的转录和表达水平则与阴性对照组一样未见明显变化(P>0.05);活性氧(reactive oxygen species,ROS)诱导剂isoproterenol能提高A549、H460细胞株中PD-L1基因的转录和表达水平,而ROS在被NAC解除后,PD-L1的转录和表达水平显著下调,进一步证明Nrf2对PD-L1基因的转录调控作用;wortmannin能逆转insulin刺激引起的AKT磷酸化水平增加,促进PD-L1基因的转录水平和表达水平下降,表明PD-L1的调控与AKT激活程度相关;在insulin和wortmannin的分别干预下,A549、H460细胞株中的Nrf2磷酸化水平能随着AKT磷酸化水平改变而改变,二者相关系数r分别为Background and purpose:Programmed death ligand-1(PD-L1)plays an important role in sheltering tumor cell from surveillance of immune system.While the potential modulation is regarded as being performed at MAPK,PI3K-AKT and STAT3 pathway in most cases,the key control point has never been explicitly reported.This study aimed to probe on this mechanism of lung carcinoma via PI3K/AKT pathway based on the A549 and H460 cell lines.Methods:By using shRNA technology,we created several selectively‘silent’mutations of A 549 and H460 cell lines,involving A549^HEB-,A549HTF4-,A549^Nrf2-A549^FOXO3a-,H460^HEB-,H460^HTF4-,H460^Nrf2-and H460^FOXO3a-.3’UTR region of PD-L1 was constructed into pGL3-basic vector in order to create a dual luciferase reporter gene system which was able to be used for monitoring the transcriptional activation of PD-L1.Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was used to detect the transcriptional level of PD-L1,and the Western blot assay was used for monitoring the total expression of PD-L1;peripheral blood mononuclear cells(PBMCs)co-culturing with A549 and H460 cell lines were utilized to check the function of Nrf2 in tumor immune resistance.Results:Phosphorylation level of AKT and expression level of PD-L1 in A549 and H460 cell lines were simultaneously enhanced after treatment with 10μg/mL insulin(P<0.001),and the same phenomenon was observed in their mutations with defect in HEB,HTF4 and FOXO3a respectively,however,the plot reversal occurred in A549^Nrf2-and H460^Nrf2- cell lines(P>0.001).Isoproterenol could boost both transcriptional activation and expression level in A549 and H460 cell lines,and this auto-action was able to be relieved by NAC.Results above further certified the transcriptional regulation of Nrf2 on PD-L1 gene.Wortmannin could change over the function of insulin to raise the expression of PD-L1,by prohibiting the increasing AKT phosphorylation level.Furthermore,the relationship between the phosphorylation level of Nrf2 and AKT was also analyzed

关 键 词：免疫监视 PD-L1 肺癌 

分 类 号：R734.2[医药卫生—肿瘤]