IL-35调控髓系抑制性细胞介导食管癌免疫逃逸机制研究

Interleukin-35(IL-35) is a novel member of IL-12 family. Recent studies have demonstrated that IL-35 exerted immunological function in autoimmune diseases, and infections. However, the role of IL-35 in tumor immune escape is unclear. Our preliminary study showed that IL-35 was highly expressed in serum and tumor tissue of esophageal cancer, and IL-35 level was positively correlated with CD14+HLA-DR-MDSC. IL-35 treatment could effectively induce the migration of this population. These results suggested that IL-35 and MDSC may have internal link in the immune escape mechanism of esophageal cancer. To further characterize the molecular mechanism by which IL-35 regulate MDSC expansion and function, we propose here to (1) detect the frequency, number, phenotype of MDSC, and the level of IL-35 in peripheral blood and tumor tissue, as well as the correlation between them and their clinical parameter; (2) investigate the effect of IL-35 on MDSC expansion, migration, survival and function; (3) characterize the molecular mechanism by which IL-35 affect the MDSC’s quantity and function by using the combination of RNA array, bioinformatics and RNAi techniques. Through this study we aim to elucidate the role of IL-35 on the regulation of MDSC, with the implication of the novel immune-therapeutic avenue for the esophageal cancer, even other tumors.

IL-35是一类具有免疫调节作用的细胞因子，在多种自身免疫性疾病中发挥重要的调节作用。但迄今为止其在肿瘤免疫微环境中的作用尚不清楚。本课题前期研究结果发现食管癌患者的血浆和癌组织中IL-35水平显著升高，且与肿瘤组织中髓系抑制性细胞（MDSC）聚集相关；体外实验研究发现IL-35能有效诱导MDSC迁移。为此，我们提出假说：IL-35可能通过调节MDSC参与食管癌的免疫逃逸。为了验证这一假设，本课题拟进一步收集食管癌患者外周血及肿瘤组织标本，分析MDSC的表型、频率及数目，检测IL-35表达水平，结合临床指标，分析其临床意义；利用体外及体内实验探讨IL-35在调控MDSC扩增、迁移、存活和免疫抑制功能中的作用；利用基因芯片技术、生物信息学分析手段和功能验证实验，明确IL-35调节MDSC的信号传导通路。本研究将有助于揭示食管癌免疫逃逸的新机制，并为肿瘤免疫干预提供新靶点。

食管癌的发生发展是一个多因素、多环节、多阶段的过程，涉及肿瘤细胞自身及肿瘤微环境的影响等。本项目通过GEO数据库和TCGA数据库分析发现IL-35是食管癌患者早诊和预后的关键基因；临床样本分析发现IL-35的两个亚基EBI3和p35在食管癌患者癌组织及食管癌细胞株中均高表达。但从细胞水平和动物水平两方面验证均未发现IL-35与食管癌增殖、迁移、克隆形成等功能的相关性。综上可知，IL-35可能对食管癌的发生、发展无显著影响。.近年来，表观遗传学在肿瘤发生及耐药中的作用逐渐被揭示，因此，本项目从表观遗传学角度系统研究了食管癌发生及耐药的驱动因素及机制。首先利用改进的Nano-hmC-Seal技术对150例食管癌患者和177例健康志愿者血浆游离DNA（cfDNA）中微量5hmC修饰进行全基因组测序，描绘了食管癌患者的cfDNA 5hmC图谱;利用机器学习方法建立了食管癌诊断模型，预测的灵敏度和特异性分别达到93.75%和85.7%，表明cfDNA 5hmC测序可能成为食管癌早期检测、诊疗与复发监测的有力工具。其次在非编码RNA方面，发现circ-FIG4/miR-99a/ABHD4 可通过EMT信号通路调控食管癌恶性表型；沉默circ-FIG4能够显著抑制食管癌细胞EMT表型，而抑制miR-99a或者过表达ABHD4可部分逆转此现象。因此，circ-FIG4可通过吸附miR-99a促进ABHD4的表达形成ceRNA调控网络，circ-FIG4/miR-99a/ABHD4轴可通过影响EMT通路调控食管癌的生物学行为。与此同时，本研究解析了组蛋白甲基转移酶NSD2可诱导MACC1-AS1介导ESCC对顺铂耐药，而干预MACC1-AS1表达可逆转顺铂耐药。综上，本研究从表观遗传学视角证实了cfDNA 5hmc可作为食管癌发生的标志物，阐明了circ-FIG4/miR-99a/ABHD4轴对食管癌发生发展的调控作用及机制，解析了NSD2诱导MACC1-AS1介导食管癌对顺铂耐药的作用及机制，为食管癌的发生发展研究开辟新方向，为食管癌的临床治疗提供新的理论依据和药物靶点。