肝癌细胞调控PD-L1表达的表观遗传机制及临床意义

The expression level of PD - L1 on tumor cells is one of the key factors that affect the immune checkpoint blockade curative effect. The progress of hepatocellular carcinoma (HCC) is closely related to the epigenetic changes, our preliminary results show that the IFNγ induced PD-L1 on HCC cells is promoted by EZH2 and DNMT1 siRNA or inhibitors. Therefore, combined with specific epigenetic inhibitors to regulate the exprssion of PD-L1, is expected to improve the therapeutic efficacy of immune checkpoint block therapy. On this basis, this research will combine clinical samples, in vitro models and animal studies to: 1) collect HCC specimens, combined with bioinformatics methods to screen the key epigenetic regulation genes associated with HCC progression; 2) use epigenetic modification inhibitors, RNAi technology in in vitro experiment to reveal the specific epigenetic modification of PD - L1 and its mechanism; 3) explore the potential clinical application of immunotherapy combine with epigenetic modification inhibitors in HCC mouse models and clinical samples. The prospective results will help to reveal the mechanism of epigenetic modification in regulating the PD-L1 epression on HCC, will provide a novel strategy for effective immune therapy.

肿瘤细胞PD-L1的表达水平是影响免疫检查点阻断治疗疗效的关键因素之一。肝癌进展与表观遗传的改变密切相关，我们的前期研结果显示，EZH2、DNMT1的抑制剂或siRNA均能促进IFNγ诱导的肝癌细胞PD-L1的表达。因此，联合特定的表观遗传抑制剂调节PD-L1的表达，有望提高免疫检查点阻断疗法的疗效。以此为基础，本课题拟结合临床样本、体外模型和动物实验研究：1）收集肝癌标本，结合生物信息学手段筛选与肝癌进展相关的关键表观遗传调控基因；2）利用表观修饰抑制剂、RNAi技术等设计体外实验，揭示PD-L1的表观修饰调控方式及其作用机制；3）利用小鼠肝癌模型和临床样本验证，探讨联合表观修饰抑制剂的免疫治疗方案的潜在临床应用。本研究有望揭示肝癌细胞产生PD-L1的表观修饰调控机制，为有效的肝癌免疫治疗提供新思路。

越来越多的研究表明，靶向表观遗传修饰可以提高肿瘤免疫治疗的疗效。然而，这种现象背后的机制在很大程度上仍然未知。在本研究中,我们阐述了表观修饰基因EZH2调控免疫检查点分子PD-L1在肝细胞肝癌（HCC）中表达的机制。我们通过免疫组化和多重免疫荧光染色分析EZH2与PD-L1在HCC组织中的表达及相关性。通过免疫印迹，实时定量PCR，流式细胞术、染色质免疫沉淀和双荧光素酶报告基因实验等揭示EZH2对PD-L1表达的调控机制。体外细胞实验显示EZH2可以负调控IFNγ诱导的肝癌细胞PD-L1的表达。机制研究表明，EZH2可通过促进CD274（编码PD-L1）及PD-L1表达必需的转录因子IRF1基因启动子H3K27me3水平来抑制PD-L1表达，EZH2并该过程并未影响IFNγ/STAT1通路的激活。在HCC临床样本中证实EZH2和PD-L1在免疫活化的HCC组织中（IFNγ信号活跃）表达负相关。多因素Cox分析表明EZH2和PD-L1联合是一个影响HCC患者OS和RFS的独立预后因素。本研究揭示了表观遗传修饰子EZH2可通过直接上调肝癌细胞中CD274和IRF1的启动子H3K27me3水平抑制免疫检查点分子PD-L1的表达，使得EZH2作为免疫活化的HCC的抗PD-L1/PD-1免疫疗法的潜在联合靶点。