EZH2在肺癌细胞PD-L1表达调控中的作用及机制研究

英 文 摘 要.High expression of PD-L1 was an important factor to promote lung cancer cells immune escape and may contribute to chemotherapy resistance and poor prognosis. However, the regulation mechanism of PD-L1 expression in lung cancer is not clear. We found a significantly positive correlation between the expression of EZH2 and PD-L1 in lung cancer by analyzing the results of gene chip from GEO DataSets, and our previous research found that knockdown of EZH2 in LLC could inhibit the expression of PD-L1 significantly, suggesting that EZH2 could regulate PD-L1 expression and promote lung cancer immune escape. In the present project, we aim to investigate the impact of EZH2 on the expression of PD-L1 in vitro and in vivo, and to elucidate the potential mechanisms by which EZH2 regulating PD-L1 expression and promote lung cancer immune escape using STAT3/NF-κB inhibition and ChIP-Seq. We would also illuminate the efficacy of EZH2 inhibitor combined with adoptive immunotherapy. This study will help to illustrate a new underlying mechanism of PD-L1 regulation, and provide new strategy for the lung cancer immunotherapy.

过表达PD-L1是肺癌细胞发生免疫逃逸的重要原因,并与肺癌的治疗抵抗及不良预后密切相关,但目前PD-L1在肺癌中的表达调控机制尚不明确。申请者前期统计分析GEO DataSets数据库中基因芯片结果后发现,肺癌中Zeste增强子同源物2(EZH2)的表达与PD-L1的表达呈正相关,且前期实验发现,敲低小鼠肺癌细胞LLC中EZH2的表达可显著下调PD-L1的表达,提示EZH2可调控肺癌细胞PD-L1的表达,进而影响肺癌细胞的免疫逃逸。本项目拟使用sh-EZH2慢病毒、EZH2抑制剂处理肺癌细胞,体内外验证EZH2对PD-L1的调控;采用STAT3/NF-κB信号通路抑制剂及ChIP-Seq技术阐明EZH2调控PD-L1的分子机制,并探讨使用EZH2抑制剂下调PD-L1表达后对肺癌过继免疫治疗疗效的影响。本研究不但从全新角度阐释PD-L1的调控机制,也为肺癌的免疫治疗发掘新的治疗靶点。

过表达PD-L1是肺癌细胞发生免疫逃逸的重要原因,并与肺癌的治疗抵抗及不良预后密切相关,但目前PD-L1在肺癌中的表达调控机制尚不明确。申请者前期统计分析GEO DataSets数据库中基因芯片结果后发现,肺癌中Zeste增强子同源物2(EZH2)的表达与PD-L1的表达呈正相关,且前期实验发现,敲低小鼠肺癌细胞LLC中EZH2的表达可显著下调PD-L1的表达,提示EZH2可调控肺癌细胞PD-L1的表达,进而影响肺癌细胞的免疫逃逸。进一步的研究发现采用sh-EZH2慢病毒或EZH2抑制剂GSK343敲降/抑制肺癌细胞中EZH2的表达可显著下调PD-L1的mRNA及蛋白水平。体内实验证明敲降EZH2可增强机体的抗瘤免疫应答，延缓肿瘤生长。同时，通过缺氧培养发现缺氧可诱导Hif-1和PD-L1的表达上调。采用Hif-1脯氨酸羟化酶-2抑制剂可下调PD-L1的mRNA水平，说明EZH2是通过Hif-1a调控PD-L1的表达。本项目明确了EZH2在肺癌组织中高表达，且与肺癌患者的不良预后密切相关。通过EZH2敲降，低氧诱导等实验证明了EZH2通过影响Hif-1a调控PD-L1的表达。通过体内实验验证了敲降肺癌细胞EZH2的表达可提高机体的抗瘤免疫应答，延缓肿瘤生长。本课题为发掘肺癌联合免疫治疗新策略提供了新方向。