针对程序性细胞死亡蛋白1 (PD-1) 和程序性死亡配体1 (PD-L1) 之间相互作用的免疫治疗是非小细胞肺癌 (NSCLC) 患者的一种治疗选择。 PD-L1 的表达决定了治疗的有效性，但 PD-L1 DNA 甲基化和表达之间的关系尚未明确描述。我们在NSCLC细胞系和肿瘤活检标本中调查了PD-L1 DNA 甲基化、mRNA表达和蛋白表达。我们使用聚合在一起的定期间隔短回文重复序列相关蛋白9 (CRISPR-Cas9) 来修改PD-L1基因环境和内切酶缺陷的Cas9 (dCas9) 与十一转位甲基胞嘧啶二氧化酶1 (TET1) 和DNA (胞嘧啶-5)-甲基转移酶3A (DNMT3A) 融合来操作PD-L1 DNA甲基化。在NSCLC细胞系中，我们发现特定的PD-L1 CpG位点甲基化水平与PD-L1 mRNA表达呈负相关。然而，使用γ干扰素诱导PD-L1 mRNA表达并没有降低这些CpG位点的甲基化水平，并且使用CRISPR-Cas9，我们发现CpG位点并没有直接造成负调节。dCas9-TET1和dCas9-DNMT3A分别可以诱导PD-L1的低甲基化和高甲基化，后者导致表达下降，显示甲基化的功能影响。在NSCLC活检中，PD-L1甲基化和表达之间的负相关性较弱。我们得出结论，PD-L1 DNA甲基化与表达之间存在调节性联系。然而，由于这些措施之间的联系较弱，这项研究强调了在PD-1/PD-L1免疫治疗中，PD-L1 DNA甲基化可以作为生物标志物和药物靶点，需要进一步研究来提高有效性的措施。

Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.