EZH2-STAT4-IFN-γ通路介导神经胶质瘤免疫逃逸的机制研究

Glioma is the most common brain tumor with poor clinical prognosis. Glioma cells evade the immune system through promoting the skew of glioma associated microglia/macrophage (GAM) from activated M1 to M2 phenotype, with a mechanism unelaborated. Our previous work demonstrates that oncogenic EZH2 plays a vital role in glioma. Further studies show that genes negatively related with EZH2 are found to be mainly implicated in immune response, and moreover, inhibition of EZH2 in glioma cells is able to promote M1 activation of GAM cells. Our preliminary work shows that up-regulation of STAT4 and IFN-γresults from inhibition of EZH2 in glioma cells, while IFN-γ is capable of inducing M1 phenotype transformation of GAM cells as well. The analysis of genome locus using UCSC genome browser shows that EZH2 is likely to suppress STAT4 expression through methylation of H3K27 histone within the promoter of STAT4. Collectively, we come up with the hypothesis that the EZH2-STAT4-IFN-γsignaling pathway mediates the activation of the M2 phenotype of GAM cells in glioma, eventually resulting in the immune evasion of glioma, whereas inhibition of this pathway will remodel the immune effect of GAM cells against glioma. The expected outcome of this project will help to unravel the molecular mechanism involved in the immune evasion of glioma, and suggest potential intervention targets of immunal therapies against glioma.

神经胶质瘤是发生率最高的颅内肿瘤，临床预后差。胶质瘤细胞可促使胶质瘤相关小胶质细胞（GAM）由抗癌的M1型向促癌的M2型转化，导致胶质瘤免疫逃逸，机制仍不明确。申请人前期已发表文章发现，EZH2表达调控在胶质瘤中发挥重要作用。进一步研究发现，胶质瘤中与EZH2表达呈负相关的基因主要参与免疫应答，而且抑制EZH2可促进GAM细胞M1型转化。初步研究结果发现，抑制胶质瘤细胞EZH2表达可上调STAT4和IFN-γ表达；而IFN-γ亦可诱导GAM细胞M1型转化。生物信息学分析显示，EZH2可能通过调控STAT4启动子区H3K27的甲基化而抑制其表达。综上，我们提出假设，胶质瘤中存在EZH2-STAT4-IFN-γ通路促进GAM细胞M2型活化，诱导胶质瘤免疫逃逸；抑制该通路则重塑GAM细胞对胶质瘤的免疫效应。本项目的研究结果有助于揭示胶质瘤免疫逃逸的分子机制，为胶质瘤免疫治疗提供潜在的干预靶点。

研究背景：胶质母细胞瘤是成人恶性程度最高的胶质瘤，临床预后极差。这主要归因于其发生发展的机制仍不明确，发病机制异质性很大。肿瘤免疫应答参与肿瘤发生发展受到关注。癌基因EZH2参与胶质瘤增殖、凋亡、细胞周期、分化及干细胞干性维持等进程，但其是否参与肿瘤免疫尚不明确。主要研究内容：阐明EZH2对胶质瘤细胞炎症因子表达影响；2.阐明共培养条件下，抑制胶质瘤细胞EZH2表达对小胶质细胞吞噬功能与极性转化影响。重要结果和关键数据：1. 采用siRNA和DZNep敲低和抑制人和小鼠胶质瘤细胞中EZH2表达与功能，转化生长因子TGFβ1-3表达下调，抗炎因子IL1β和IL-6表达上调，iNOS、TNFα与IFNγ未检测到。该结果表明，EZH2参与胶质瘤免疫应答。2. 采用小鼠胶质瘤细胞GL261与原代小胶质细胞共培养，小胶质细胞细胞因子表达发生显著改变，IL1β和IL-6表达抑制，而IL-10、TGFβ1-2、iNOS显著上调，TNFα与TGFβ3无显著改变。抑制EZH2表达与功能，小胶质细胞M1型标志物IL1β、IL-6与CD11c未发生改变，TNFα和iNOS显著上调；许多M2型标志物表达显著下调。采用人胶质瘤细胞U87与人外周血单核细胞（PBMC）来源的巨噬细胞共培养，得到类似结果。此结果表明，胶质瘤细胞与巨噬细胞共培养，能够诱导巨噬细胞向M2型转化。3.沉默GL261细胞 EZH2的表达，与小胶质细胞共培养，后者吞噬功能显著增强；采用INOS抑制剂1400W和siEZH2/DZNep共同处理GL261细胞，再与与原代小胶质细胞共培养，小胶质细胞吞噬功能则显著下降。小胶质细胞吞噬功能增强，而该吞噬功能可能通过INOS实现。科学意义：本研究首次发现EZH2参与胶质瘤细胞免疫应答，通过调控iNOS表达，在胶质瘤与小胶质细胞相互作用中发挥重要功能。本研究有助于揭示肿瘤免疫抑制分子机制，对胶质瘤靶向治疗提供潜在的靶点与理论基础。