Effect And Mechanism Of DNMT1 On PD-L1 Expression In Non-small Cell Lung Cancer

Objective:To detect the expression and correlation of DNA methyltransferase 1(DNMT1)and programmed death ligand-1(PD-L1)in human non-small cell lung cancer(NSCLC)and explore the effect and mechanism of DNMT1 in regulating PD-L1 expression.Methods:(1)Human lung adenocarcinoma A549 cells were treated with 5-Aza-2’-deoxycytidine(DAC),a DNA methyltransferase 1 inhibitor,and the drug was set up in five concentration gradients of 0.5μmol/L,1.5μmol/L,2.5μmol/L,5μmol/L,and 10μmol/L in experimental groups.And the drug cell proliferation-toxicity assay was performed by Cell Counting Kit-8cell counting reagent(CCK-8)at 6h,24 h,48h and 72 h to observe the proliferation of A549 cells.(2)Downloading tumor transcriptome data(TCGA-LUAD)and lung adenocarcinoma methylation data using the UCSC Xena database.The gene expression data of PD-L1 in the lung adenocarcinoma gene transcriptome data from The cancer genome atlas(TCGA)database and the methylation data of five Cp G sites in the PD-L1 promoter region in lung adenocarcinoma(cg15837913,cg02823866,cg14305799,cg13474877,and cg19724470)methylation data,the methylation box plot of the 5 Cp G sites in the PD-L1 promoter region and the correlation scatter plot were performed using the Sanger Box3.0 database for data visualization to analyze the methylation level of each Cp G site and the correlation with PD-L1 expression.(3)According to TCGA database,methylation and unmethylation primers were designed for Cp G sites cg19724470 and cg15837913 in PD-L1 promoter region.Methylation of the above sites in PD-L1 promoter region and methylation of two sites in lung adenocarcinoma A549 cells treated with DAC were detected by methylation specific PCR(methylation-specific PCR,MS-PCR).(4)Real-time fluorescence quantitative PCR(q PCR)experiments were performed to detect the changes of DNMT1 and PD-L1 m RNA expression in A549 cells treated with DAC at 2.5μmol/L and 5μmol/L concentrations.Results:(1)After applying DAC at concentrations of 0.5μmol/L,1.5μmol/L,2.5μmol/L,5μmol/L,and 10μmol/L to human lung adenocarcinoma A549 cells for 6h,24 h,48h,and 72 h,it was found that DAC significantly inhibited the growth of A549 cells,and the inhibitory effects of the above concentration gradients of DAC on cells were dose-dependent at 24h(P<0.05).(2)Analyzing the data of 448 lung adenocarcinoma patients in the dataset,among the five Cp G loci associated with the PD-L1 promoter in lung adenocarcinoma(cg15837913,cg02823866,cg14305799,cg13474877,cg19724470),the cg15837913 locus and cg19724470 locus showed a large range of methylation(P<0.001),and the methylation levels of both loci were significantly and negatively correlated with PD-L1 expression.(3)As verified by methylation-specific PCR assay,the PD-L1 promoter region Cp G sites cg19724470 and cg15837913 were significantly methylated in human lung adenocarcinoma A549 cells,and the two sites showed a demethylation status after adding DAC.(4)When human lung adenocarcinoma A549 cells were treated with DAC at concentrations of 2.5μmol/L and 5μmol/L,DNMT1 expression was decreased(P<0.05)and PD-L1 expression was increased(P<0.05)by real-time fluorescence quantitative PCR.Conclusions:cg19724470 and cg15837913 may be two key Cp G methylation sites of PD-L1 promoter in human lung adenocarcinoma A549 cells,and inhibition of DNMT1 can lead to demethylation of these 2 sites along with a significantly higher expression of PD-L1,indicating that DNMT1 may regulate the PD-L1 expression in non-small cell lung cancer cells through such sites.