炎症小体NLRP6调节肿瘤相关巨噬细胞与肝细胞癌恶性转化的相关机制研究

Inflammatory responses play decisive roles at different stages of tumor development including initiation, promotion, malignant conversion, invasion, and metastasis. Inflammasome complexes generally have three main components: a cytosolic pattern-recognition receptor, the enzyme caspase 1 and an adaptor protein. The receptor is either a member of the NOD-like receptors (NLR) family of proteins or a member of the pyrin and HIN domain containing (PYHIN) family of proteins. Infections with hepatitis B (HBV) or C (HCV) viruses increase the risk of hepatocellular carcinoma (HCC). The inflammatory response triggered by infection precedes tumor development and is a part of normal host defense, whose goal is pathogen. Inflammation also affects immune surveillance and response to therapy..In our early researches, we found out that the expression of inflammasome NLRP6 in peripheral blood mononuclear cells (PBMCs) of HCC patients was associated with the progression of disease. Nlrp6 exhibits a tissue and cell-type specific pattern of expression, with high level in intestine，colon，liver，monocytes and neutrophils. NLRP6 as a negative regulator of inflammatory signal in antibacterial immune responses has recently been documented. Yet absence of NLRP6 accelerated colitis associated tumor growth in mice. It is suggested that NLRP6 may play an opposite role in the regulation of tumor development in immune cells and tissues. We analyzed the lymphocyte subpopulation of HCC patients and healthy population, and it showed that NLRP6 was increased mononuclear cells in HCC patients. When the tumor occurs, monocytes accumulate into the liver tissue and differentiate into mature macrophages. Therefore, it is significant to study the effect of NLRP6 on the immune function in mononuclear macrophages for the revelation of its mechanism on tumor occurrence, recurrence and metastasis..In this project, we will investigate the molecular mechanism of NLRP6 on the immune function of macrophages based on regulating the nlrp6 expression in THP-1 cell line. Next，we will co-culture the macrophages and hepatoma cells to revealed the mechanism of the interaction between NLRP6 imbalanced macrophages and hepatoma cells. Third, the diethylnitrosamine (DEN)-HCC model of nlrp6 deficiency mice will be used to testify the effect and mechanism of NLRP6 on the occurrence and development of HCC.This project focus on the molecular mechanism of HCC generation and malignant transformation, in which NLRP6 imbalanced in the tumor microenvironment. It will provide a new strategy for clinical prevention or treatment of new drug targets for HCC.

研究表明炎症小体家族成员NLRP6在癌症的发生、复发与转移过程中起着重要的作用，在免疫细胞与肿瘤细胞中分别发挥着不同的功能，关于NLRP6在肝癌中的研究尚属空白。我们前期研究发现肝细胞癌（HCC）患者外周血单核淋巴细胞（PBMCs）中单核巨噬细胞的NLRP6表达增高，并与HCC患者病情进展正相关，提示：单核巨噬细胞中炎症小体NLRP6的表达活化可能参与调控HCC的发生、发展。本项目拟调节NLRP6在单核巨噬细胞中的表达水平，揭示NLRP6影响单核巨噬细胞免疫功能的相关分子机制；进一步基于体外巨噬细胞-肝癌细胞共培养实验解释NLRP6失调巨噬细胞对肿瘤细胞增殖、迁徙等功能的影响机制；最终结合nlrp6敲除小鼠模型，研究NLRP6失调对肿瘤微环境、炎癌转化的作用与分子机制，深入揭示炎症小体NLRP6与肝细胞癌恶性转化的互动调控分子机制，为寻找新药物靶点和肝癌的临床预防与治疗提供新策略。

炎症小体家族成员NLRP6在癌症发生、复发与转移过程中起着重要的作用。同时在免疫细胞与肿瘤细胞中分别发挥着不同的功能。我们前期研究发现肝细胞癌患者外周血单核巨噬细胞的NLRP6表达较健康人显著增加，并且与肝细胞癌发展呈正相关。进一步地，在本项目支持下，我们发现NLRP6在肝炎、肝硬化、肝癌患者外周血单核细胞中的表达呈递增趋势。另通过对肝癌病人外周血单核细胞进行流式分析并实时定量PCR验证，发现NLRP6在CD14+细胞中呈高表达。本项目进而选择巨噬细胞与肝细胞作为研究对象，分别对其NLRP6表达水平进行基因编辑，通过实时定量PCR、蛋白质免疫印迹、激光共聚焦、流式等手段观察分析NLRP6对各细胞功能的影响。结果表明，对于肝细胞，NLRP6可促进细胞的生长及粘附功能，而对于巨噬细胞，本项目并未观察到NLRP6对巨噬细胞的增殖功能的影响，但NLRP6敲低可显著抑制单核细胞的凋亡，并通过IRF5/NF-kB通路调节巨噬细胞的吞噬功能，促进巨噬细胞CD86表达并向M1型极化，同时促进细胞因子IL18及IL1b分泌。另外我们对NLRP6敲除小鼠进行DEN肝癌造模，结果表明，NLRP6敲低后显著降低了肿瘤发生。同时，免疫组织化学染色结果表明CD86+M1型巨噬细胞浸润明显增加，但其深层分子机制还有待进行后续深入探索。