补体激活在结肠炎恶性转化过程中发挥关键作用的网络调控研究

The incidence of colorectal cancer (CRC) increased fastest among all types of cancer in China. It is highly correlated with inflammation; however, the underlying mechanisms are not fully understood, and it requires the effective therapeutics. Using AOM/DSS-induced inflammation-mediated malignant transformation (MT) mouse model, we collected mouse colon samples of different stages of MT, which were further evaluated in omics-level. The results were used to build up 80 kinds of dynamic changes by bioinformatics method. Meanwhile, we induced MT with AOM/DSS in knockout mice of 8 complement genes, and found that the knockout of C5 gene and especially C5aR gene near-completely impeded the process of MT, in which the underlying mechanisms were primarily illustrated. Moreover, we still interestingly observed that there were consistently a tiny minority of treated mice free from MT. These mouse feces were subsequently harvested for bacterial sequencing in comparison to the mouse feces suffering from MT, and the difference between them was revealed. Therefore, in this proposal we will further establish the regulatory network for colitis MT, and elucidate the detailed molecular mechanisms for C5- and C5aR-KO mice free from colitis-induced MT. Then we will discover the key molecules in the network and their action mechanisms in combined with the C5a/C5aR signaling. In addition, the effect of C5a/C5aR signaling blockade and complement inhibition on colitis-induced MT will also be investigated. Finally, we will further investigate the effect and the underlying mechanisms of colorectal bacteria on colitis-induced MT in the levels of genus and species. These findings will be verified in clinical specimen, and we hope that they will benefit prevention, diagnosis and treatment on CRC.

大肠癌发病率增长最快，与炎症密切相关，但发病机制不完全清楚，也缺乏有效治疗手段。我们前期以AOM/DSS诱导小鼠结肠炎癌转化为模型，在野生型小鼠中收集了炎癌转化动态标本，通过生物信息学手段分析多种组学数据，初步建立了炎癌转化过程的mRNA/蛋白动态变化规律；另外还发现诱导过程中总有极少数小鼠不产生恶性转化，通过测序揭示了发生与不发生炎癌转化的小鼠肠道菌群差异。最重要的是，在8种补体基因敲除小鼠中发现，C5尤其是C5aR基因敲除后几乎完全阻止结肠炎癌转化，并初步揭示了细胞与分子机制。本课题在上述突破性进展基础上，将构建结肠炎癌转化的调控网络，并与C5a/C5aR信号调控密切结合，揭示关键节点分子并诠释其功能，阻断C5a/C5aR信号或抑制补体激活对炎癌转化的影响；同时，还将在属和种的水平研究菌群对炎癌转化的影响及其机制。研究结果还将在临床样本中验证，并最终为结肠癌预防、诊断和治疗提供新思路。

直结肠癌（Colorectal Cancer, CRC）的发病率与死亡率均位居所有组织类型肿瘤的第三位，是威胁性很大的肿瘤类型，但其发病机制还不清楚。炎症与肿瘤关系密切，它不但与炎症性肠病（inflammatory bowel disease）和肠炎相关性肿瘤（colitis-associated cancer, CAC）有关，与其他类型的CRC也密切相关。另外，补体系统是固有免疫的重要组成部分，也是沟通固有免疫与获得性免疫的桥梁，在维持免疫自稳与免疫监视方面发挥关键作用。我们利用AOM（azoxymethane，氧化偶氮甲烷）/DSS（dextran sodium sulfate, 右旋糖酐硫酸酯钠）诱导制备小鼠结肠炎癌转化模型，发现补体C5，尤其是C5AR1基因，而非C3或C5AR2基因敲除后，几乎完全阻断炎癌转化的发生。细胞水平上的机制研究发现，炎症局部补体激活后产生的C5a通过与C5aR1结合招募MDSCs（Myeloid-Derived Suppressor Cells，髓性来源的免疫抑制细胞）至炎症局部，抑制CD8+T细胞增殖与功能，在炎症局部形成一个强烈的免疫抑制微环境，最终诱发炎癌转化，该结果进一步通过骨髓移植实验所证实。分子水平上的机制发现，首先，C5a/C5aR1与PDE4（Phosphodiesterase 4，磷酸二酯酶4）以及TLRs相互作用，调控众多细胞因子、趋化因子和炎症介质（如COX2）的水平，进而促进炎癌转化进程；其次，C5a/C5aR1与E3泛素化酶结合，增加β-catenin的K63多聚泛素化，降低K48多聚泛素化，从而增加β-catenin的稳定性，最终也促进炎癌转化进程。另外，我们发现C5aR1拮抗剂几乎可完全阻断，而PDE4抑制剂可显著抑制小鼠的结肠炎癌转化。上述参与炎癌转化的重要分子也在临床样本中得到证实。因此，我们的研究揭示了C5aR1为结肠炎癌转化的核心分子，具有重要开发价值的潜在预防药物靶点，其拮抗剂具有很好的开发前景。