PON1介导肝细胞癌外泌microRNAs调控肿瘤MDSC和TAM抑制肝癌侵袭和转移的机制研究

Hepatocellular carcinoma (HCC) is among the most malignant human cancers, with a high mortality rate worldwide. Recently, the tumor microenvironment (TME) in HCC has been reported to play a critical role in tumor development and progression. Using the iTRAQ-based proteomic profiling technique, we previously found paraoxonase 1 (PON1), a member of the calcium-dependent hydrolase protein family, to be significantly correlated with the extent of vascular invasion in over 200 HCC cases. Moreover, downregulation of PON1 expression in tumoral tissue indicated poor prognosis for HCC patients who underwent curative liver resection. Further studies by our group showed that high expression of PON1 in HCCLM3 cell line decreases its tumorigenesis abilities in nude mice model without altering its biological behavior in vitro, indicating a non-cell-autonomous regulation manner of PON1 via modulating the tumor microenvironment. Using nude mice tumorigenesis model, we found that high expression of PON1 in HCCLM3 led to dramatic decreases of the proportions of myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM)，two major components of the tumor microenvironment with potent immune suppressive activities. To further study the mechanism of PON1 regulation, we extracted secreted exosomes from PON1 highly expressed HCCLM3 cell culture medium and then injected into the tumors of the control HCCLM3 nude mice model. Similar decreases of MDSC and TAM proportions were observed. We next extracted exosomal microRNAs from PON1 highly expressed HCCLM3 cells and conducted microRNA array. We finally identified eight differentially expressed exosomal microRNAs regulated by PON1 as potential candidates in MDSC and TAM regulation. In this project, we are aiming to verify the anti-invasive effects of PON1 in HCC, and clarify the underlying mechanism of PON1 regulation on the crosstalk between exosomal microRNAs and the tumor microenvironment. These studies could provide a novel strategy for tumor metastasis prevention and shed new light on the development of more effective tumor immunotherapy.

肝细胞肝癌是恶性程度最高的肿瘤之一，且有较高的死亡率。近期研究表明，肝癌肿瘤微环境对于肿瘤发生和进展起到关键作用。申请者基于蛋白组学的前期研究发现，对氧磷脂酶-1（PON1）与肝癌血管侵犯高度相关，且PON1低表达患者预后较差。PON1是由肝脏合成的钙离子依赖性芳香酯酶，其血清浓度及活性在心血管等疾病中明显下降。预实验中，我们发现过表达PON1的肝癌细胞在体外的增殖及侵袭能力无明显下降，但接种于裸鼠原位后，肿瘤生长明显被抑制，提示PON1通过影响肝癌微环境间接抑制肿瘤进展。在动物实验中，PON1高表达可显著抑制髓系抑制性细胞（MDSC）及肿瘤相关巨噬细胞（TAM）的扩增，而上述两群细胞在肿瘤微环境中起重要的免疫抑制作用，提示此可能为PON1抑制肝癌进展的机制之一。后续实验发现，将纯化后的PON1高表达HCCLM3细胞上清中的外泌体注入裸鼠对照组移植瘤可同样抑制MDSC和TAM细胞群的扩增。

肝细胞肝癌是恶性程度最高的肿瘤之一，且有较高的死亡率。肝癌肿瘤免疫微环境对于肿瘤发生和进展起到关键作用。申请者前期研究发现，对氧磷脂酶-1（PON1）与肝癌血管侵犯高度相关，且PON1低表达患者预后较差。本课题研究发现，在C57BL/6小鼠肝癌移植瘤模型中，相比于对照细胞（Hep1-6-NC），注射PON1过表达的肝癌细胞（Hep1-6-PON1high）的移植瘤生长缓慢，并且局部CD8+T细胞增多，调节性T细胞(Treg)减少。同时我们通过体外共培养实验证明Hep1-6-PON1high细胞可以抑制Treg分化而对CD8+T细胞抗肿瘤效应没有显著影响。近年来研究发现肿瘤细胞可以通过外泌体调控免疫细胞浸润及功能。利用Hep1-6-NC和Hep1-6-PON1high细胞培养上清外泌体抑制Treg分化结果与共培养结果一致。提示PON1可以通过调控外泌体分泌抑制Treg分化，抑制肿瘤进展。申请者通过体内外实验及临床标本研究，深入解析PON1在肝癌免疫微环境的作用，为肝癌免疫治疗提供新思路。