EGFR抑制剂通过c-myc途径影响PD-L1在肺癌细胞中的表达

EGFR inhibitor modulates the PD-L1 expression via c-myc pathway in lung carcinoma cell

目的:以EGFR抑制剂为介质,研究EGFR/KRAS通路对人非小细胞肺癌A549和H1299细胞中免疫检查点抑制剂PD-L1表达的影响,探讨靶向治疗与免疫治疗联用在相关肿瘤临床治疗中的潜在价值。方法:Western blotting检测A549和H1299细胞经Erlotinib处理后细胞中c-myc的表达量变化;RT-PCR法检测Erlotinib对A549和H1299细胞中c-myc下游基因分化抗原簇47(cluster of differentiation 47,CD47)及PD-L1转录水平的调控,同时平行检测在c-myc基因沉默后,Erlotinib对CD47及PD-L1转录水平的调控;Western blotting检测Erlotinib用量与PD-L1表达的相关性;通过与肿瘤浸润T细胞(tumor infiltrating T cell,TIL-T)共培养,检测Erlotinib影响前后A549和H1299细胞对TIL-T细胞治疗的敏感度。结果:Erlotinib能显著下调c-myc在A549和H1299细胞内的表达量( P <0.01);c-myc蛋白表达量受Erlotinib影响下调后,PD-L1的转录水平也受到了明显的影响( P <0.01),但Erlotinib对于c-myc沉默的A549和H1299细胞株中CD47及PD-L1的转录水平影响很小( P >0.05);Erlotinib剂量对A549和H1299细胞中PD-L1的表达量呈现出显著的正相关性,但对c-myc沉默的细胞株影响不大( P >0.05),结果与转录水平一致;Erlotinib处理后,A549和H1299细胞对TIL-T细胞的抵抗能力显著降低,细胞凋亡信号caspase-3激活更加明显。 结论:Erlotinib能通过EGFR通路调控c-myc在细胞中的表达水平,进而介导PD-L1表达水平降低,增强TIL-T细胞对A549和H1299细胞的杀伤能力。

Objective:To probe on a mechanism that whether the EGFR inhibitor is able to modulate the expression of programmed death-ligand-1(PD-L1),an immune checkpoint inhibitors,via EGFR/KRAS pathway in A549 and H1299 cell lines,then explore the potential application of combination therapy involves immunotherapy and targeted means on clinical tumor treatment. Methods:Western blotting assay was utilized here to detect the c-myc expression change,which induced by Erlotinib in A549 and H1299 cell lines.Transcriptional level of cluster of differentiation 47(CD47) and PD-L1,which belong to downstream gene of c-myc were monitored by RT-PCR technique.In addition,gene silencing of c-myc was required here as a control to assess the importance of c-myc on PD-L1.Except for the detection at the level of gene,protein expression about PD-L1 which modulated via Erlotinib was also detected by Western blotting assay.Ultimately,we also had evaluated that whether Erlotinib could provide assistant effect for tumor infiltrating T cell(TIL-T) to kill tumor cells by conducting co-culture experiments. Results:Erlotinib was able to suppress the PD-L1 transcription via downregulate the expression of c-myc( P <0.01) in both A549 and H1299 cell lines.However such phenomenon had not yet been discovered in c-myc silencing cell line( P >0.05).In keeping with the detection at transcriptional level,Erlotinib could also restrain the expression of PD-L1,but fail to affect the others whose c-myc gene was silenced( P >0.05).Furthremore,the result of cell co-culture experiment also showed that Erlotinib had an aptitude for enhancing the killing ability on A549 and H1299 cell lines,due to the activiation of caspase-3 in these groups were more significant than non-Erlotinib groups. Conclusion:Erlotinib can decrease the expression of PD-L1 via EGFR/c-myc in A549 and H1299 cell lines,then empowers TIL-T cell more ability to kill them.