肠道菌群介导的胆汁酸代谢靶向调控HECTD1在HCC中的作用机制研究

Altering gut microbiota could accelerate the development of hepatocellular carcinoma (HCC), however, mechanisms underlying in regulating antitumor immunity remain obscure. Previous data demonstrated that the abundance of commensal gut bacteria was greatly changed in mice induced by diethylnitrosamine, subsequently, we found that altering commensal gut bacteria in mice induced the increase of bile acid (TCA) level by High-Throughput Metabolic Sequencing, and the expression of HECTD1, a novel E3 ubiquitin ligase, was noticeably up-regulated by TCA treatment, which facilitated the progression of inflammation. Thus, we proposed hypothesis that gut microbiota-mediated bile acid metabolism regulates HCC via HECTD1. The subject was started with exploring the vital effects of gut microbiota on HCC development, isolating and identifying the key commensal bacteria by 16S rRNA sequencing. Then the cause of TCA elevation induced by gut microbiota was uncovered. In addition, the CRISPR/Cas9 system, molecular biology techniques combined with pharmacological approaches were utilized to study the detailed molecular mechanisms of HECTD1 in liver injure resulting from TCA and its clinical significance in HCC, thereby providing new targets and novel therapeutic strategies for treating HCC by analyzing from multiple perspectives of microbiota-metabolism-immunity.

肠道菌群的改变可促进肝细胞肝癌（HCC）的发生及进展，然而其对肿瘤免疫的调控作用和机理不明确。我们前期研究发现HCC与正常对照相比肠道菌属丰度显著改变，代谢物高通量测序显示胆汁酸TCA水平明显增高，且TCA显著上调新型E3泛素连接酶HECTD1的表达促进炎症。因此推测肠道菌群通过介导胆汁酸代谢靶向调控HECTD1在HCC中发挥作用。本项目拟从肠道菌群失调为切入点，探讨肠道菌群在HCC发生发展中的重要性，并运用16S rRNA高通量测序等技术分离、鉴定出发挥作用的关键性菌群；其次探究肠道菌群介导胆汁酸代谢紊乱引起TCA显著升高的机制；最后运用CRISPR/Cas9系统、结合分子生物学技术和免疫学手段，研究HECTD1在TCA介导肝损伤中的具体分子机制，并探究其与临床HCC发生发展的相关性。本课题旨在从“微生物-代谢-免疫”多角度分析，为HCC的临床诊疗提供新的靶标及理论依据。

感染（病毒、细菌、寄生虫）与癌症的发生密切相关，且最近研究发现肿瘤内部含有组成特异性的微生物菌群，但其在肝细胞肝癌（hepatocellular carcinoma, HCC）的发生进展，以及免疫调控作用机理仍不明确。我们研究首次发现，在HCC患者肿瘤组织内部细菌α、β多样性显著降低，表现为局部肿瘤微生态失调；进一步分析表明，嗜麦芽糖寡养单胞菌 (Stenotrophomonas maltophilia，S. maltophilia)在肝硬化程度比较高的HCC患者中呈现为高表达，并且从肿瘤组织中成功分离、培养出S. maltophilia，体外实验发现，S. maltophilia可通过促进肝星状细胞(hepatic stellate cells，HSCs)的衰老和活化来促进HCC的发生发展，进一步机制探究表明，S. maltophilia可通过TLR-4-NF-κB信号通路激活NLRP3炎症小体，促进HSCs分泌衰老相关因子（p21, 53BP1, IL-1β, Groα, IL-6 和p16）和纤维化相关蛋白（Collagen Ⅰ, TGF-β和α-SMA）的表达，最终促进HCC的发生发展。本项目成功解决了从HCC肿瘤组织分离、培养及鉴定细菌的关键技术，并建立了细菌与细胞在体外的相互作用模型，首次解析了HCC肿瘤组织内部的菌群特点，发现与HCC发病密切相关的细菌S. maltophilia，并探讨了其致病分子机制，为HCC的诊疗提供新的靶标及理论依据。