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

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0.86和0.93,为强正相关;Nrf2敲除后,A549、H460细胞株与PBMC共培养后凋亡数量显著增加。结论:PI3K/AKT通路对肺癌细胞A549、H460中PD-L1表达具有关键调控作用,该调控是通过磷酸化激活Nrf2而实现的。

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 by using Wortmannin and insulin,and the results confirmed that the two parties correlated to each other positively,for the correlation coefficient r was 0.86 and 0.93 in A549 and H460 cell lines respectively.At last,A549^Nrf2- and h460^Nrf2- cell lines became more sensitive to the attack of PBMCs than that of their wild type owing to Nrf2-deficiency,for increased apoptosis was observed in co-culture experiment.Conclusion:PI3K/AKT pathway plays a vital role in regulating PD-L1 expression in A549 and H460 cell lines,and this action is potentially realized via phosphorylating Nrf2.