巨噬细胞内HSP27表达调控其亚型转化并影响肝癌侵袭转移的作用机制

Tumor-associated macrophages (TAM) are a major component of the cancer-related microenvironment and play a central role in tumor metastasis. In our previous study, we found that the specific marker CD206 was found to identify TAMs in hepatocellular carcinoma (HCC) tissues. Furthermore, it was found that overexpression of HSP27 in macrophages could promote invasive ability of HCC cell lines in vitro; this effect may be associated with macrophage subsets transformation and the epithelial-mesenchymal transition (EMT) of HCC cells. This was a novel finding of the impact of HSP27 in macrophages on cancer progression. This study will establish in vivo and in vitro model of human HCC immune microenvironment.Using the model, we will investigate whether HSP27 is involved in regulation of macrophage suvival, migration and phenotype switch. In addition, through short hairpin RNA (shRNA) interference or up-regulation of HSP27 expression in macrophages, we will determine the role of HSP27 in macrophage-induced HCC invasion and the EMT process. Furthermore, we will use antibody arrays to screen HSP27-regulated key proteins involved in macrophage subsets transformation and HCC progression. Western blotting and co-immunoprecipitation will be used to analyze the interaction between HSP27 and the key proteins, and investigate the role of HSP27 expression on regulation of proteins. In addition, the relationship between the expression of these proteins and the invasive phenotype of HCC cells will be studied. We will determine whether these proteins could be important independent factors in determining clinical outcomes of HCC. Using specific RNA interference or up-regulation of these genes in HCC cells, we will assess their roles in tumor invasion and in vivo metastasis. This study will provide a biological foundation for the discovery of new potential therapeutic targets for HCC.

肿瘤相关巨噬细胞(TAMs)是肿瘤免疫微环境中的重要成分，在肿瘤转移中发挥重要作用。前期工作中,我们在发现肝癌组织中TAMs特异性标志物的基础上，率先揭示巨噬细胞内HSP27过表达可显著增加肝癌细胞运动侵袭能力,且与巨噬细胞向M2亚型转化和肝癌细胞上皮-间质转化（EMT）密切相关。本课题模拟人体肝癌免疫微环境建立体内和体外模型，通过干预巨噬细胞中HSP27表达，观察巨噬细胞生长凋亡、运动和亚型转化的变化，并明确HSP27在巨噬细胞诱导肝癌EMT和侵袭转移中的作用；应用抗体芯片筛选巨噬细胞中HSP27表达调控的与其亚型转化和肝癌发展相关的关键分子；应用共转染、免疫印迹和免疫共沉淀分析HSP27对关键分子表达的影响及二者相互作用关系，同时分析它们在肝癌组织中的表达及其与肝癌转移复发的关系，并进行体内、外功能分析，明确其在肝癌转移中的作用。通过上述研究，为探讨肝癌治疗中新的干预靶点提供实验依据。

我们发现肝癌组织中癌内CD206+细胞为肿瘤相关巨噬细胞(TAM)，与肝癌患者预后密切相关。HSP27在CD206+巨噬细胞中表达显著升高，HSP27过表达质粒转染后，巨噬细胞分泌高水平M2型抗炎因子(IL-10和TGF-β)和低水平M1型促炎因子(TNF-α和IL-12)，与肝癌细胞共培养后，肝癌细胞内E-钙粘蛋白下调，N-钙粘蛋白上调，运动侵袭能力显著增强。通过信号通路芯片筛选及免疫共沉淀发现，NFkB与HSP27有相互作用，与转染NFkB过表达质粒的巨噬细胞共培养后，肝癌细胞运动侵袭、体内成瘤和肺转移均明显增强，同时发生上皮间质转化(EMT)改变。提示NFkB可能在HSP27促进巨噬细胞亚型转化和肝癌细胞恶化中发挥重要作用。我们还发现肝癌中中性粒细胞高丰度分泌CCL2招募巨噬细胞，同时分泌CCL17介导Treg浸润，促进肿瘤生长、侵袭转移，导致了索拉菲尼耐药。该成果以通讯作者，发表在Gastroenterology(2016,IF=20.77)。我们还发现，巨噬细胞和miR-28-5p-IL-34构成的反馈环可以调控肝癌转移，而巨噬细胞分泌细胞因子TGF-β1形成反馈回路参与miR-28-5p对IL-34的调控网络，该成果发表在Hepatology(2016,IF=14.08)。我们发现巨噬细胞与肝癌细胞共培养后，可以诱导肝癌细胞EMT,与JAK2/STAT3/Snail通路活化相关，以第一作者发表在Int J Oncol(2015,IF=3.33)。此外，我们还发现miR-29a通过拮抗TET酶家族抑制SOCS1-Stat3-MMP9信号通路促进肝癌恶化；SEMA3A过表达和PDEF表达下调均可促进肝癌细胞体内外生长和转移，以第一或通讯作者分别发表在Cell Death Dis(2017,IF=5.64)、Oncotarget(2017,IF=5.17)和Expert Rev Gastroenterol Hepatol(2018,IF=2.96)等杂志。 .以第一或通讯作者发表本课题相关论文10篇（SCI收录8篇）,获专利授权1项,获教育部自然科学奖一等奖,第二十七届上海市优秀发明选拔赛优秀发明金奖,汤钊猷-上海建行肝癌诊疗研究奖，入选科技部“创新人才推进计划重点领域创新团队”核心成员,中山医院十佳优秀青年导师。作为大会秘书长,组织6次全国学次全国学术会议。