肿瘤相关巨噬细胞经不均一核糖核蛋白A/B介导上皮-间质转化促进肝癌侵袭转移

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 hepatocellular carcinoma (HCC) cell lines exhibited higher invasive potentials when co-cultured with TAM; this effect may occur through the epithelial-mesenchymal transition (EMT) and up-regulation of hnRNP A/B. In this study, we intend to observe the impact of TAM on promoting the EMT process. The role of hnRNP A/B in TAM-induced HCC invasion and the EMT process will be investigated using short hairpin RNA (shRNA)-mediated down-regulation of hnRNP A/B in HCC cell lines with various metastatic potentials. Furthermore, we will use chromatin immunoprecipitation and gene promotor arrays to screen hnRNP A/B-regulated genes involved in the EMT process in HCC cells. Western blotting and luciferase assays will be respectively used to analyze hnRNP A/B expression and transcription activity in HCC cells co-cultured with TAM. We will then determine whether hnRNP A/B is involved in the transcriptional regulation of these genes in HCC after co-culture with TAM. In addition, the relationship between the expression of these genes and the invasive phenotype of HCC cells will be studied. We will determine whether these genes 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(TAM)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,我们发现TAM能提高肝癌细胞运动侵袭能力，且与上皮-间质转化(EMT)和不均一核糖核蛋白A/B表达上调密切相关。本课题拟观察TAM共培养对肝癌细胞EMT生物标志物表达及亚细胞定位的影响；通过shRNA干预不均一核糖核蛋白A/B表达，观察干预前后TAM对肝癌细胞EMT和侵袭转移的影响的变化，明确不均一核糖核蛋白A/B在TAM诱导肝癌EMT和侵袭转移中的作用；应用启动子芯片筛选不均一核糖核蛋白A/B调控的与EMT发生相关的关键基因；应用免疫印迹、荧光素报告基因分析TAM作用前后肝癌细胞内不均一核糖核蛋白A/B表达和转录活性的改变及其对关键基因转录调控的影响；同时分析关键基因在肝癌组织中的表达及其与肝癌转移复发间的关系，并对其进行体内、外功能分析。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。

肿瘤相关巨噬细胞（tumor associated macrophage，TAM）在肿瘤发生发展中发挥免疫抑制、促血管生成等作用。我们发现肝癌组织中癌内CD206+细胞为TAM，与肝癌患者预后密切相关,可显著提高肝癌细胞侵袭移动能力。EMT主要发生在癌巢边缘,同时,巨噬细胞大量浸润,说明TAM与肝癌发生EMT密切相关。TAM与肝癌细胞共培养后，肝癌细胞运动及侵袭能力均显著增强，共培养上清中IL-8显著增加,可以诱导肝癌细胞EMT,同时促进肝癌细胞运动和侵袭,与JAK2/STAT3/Snail通路活化相关。该结果以第一作者发表在“Int J Oncol”杂志（2015年，IF=3.025）。同时发现，共培养后肝癌细胞内hnRNP AB表达显著上调，提示hnRNP AB在TAM诱导肝癌细胞 EMT和侵袭转移中发挥重要作用。我们发现HepG2细胞中转染hnRNPAB质粒后呈现纺锤形，E-cadherin下调，vimentin表达上调，肝癌细胞运动、侵袭和增殖能力均显著升高；而在HCCLM3中干扰其表达后，则出现相反变化，进一步发现hnRNPAB能转录活化snail而诱导肝癌细胞EMT，与snail表达呈正相关，而与E-cadherin表达呈负相关，hnRNP AB低表达患者总体生存时间长。该结果以第一作者发表于“Cancer Res”杂志(2014年，IF=9.3）。此外，我们还发现Capn4可通过活化FAK-Src通路促进肝癌恶化；CXCL5可活化PI3K/Akt/GSK-3β/Snail通路诱导EMT促进肝癌恶化；该趋化因子还可以招募中性粒细胞，促进胆管癌细胞恶化和转移。这些结果以第一或通讯作者分别发表在“J Pathol”（2014年，IF=7.33）、“Cancer Lett”(2015年，IF=5.621）和“Carcinogenesis”(2013年，IF=5.30）。.以第一作者或通讯作者发表与本课题相关的论文8篇（SCI收录7篇），获得专利授权4项。荣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，明治乳业生命科学奖优秀奖，并入选为科技部“创新人才推进计划重点领域创新团队”核心成员。作为大会秘书长，组织4次大型全国学术会议。在本项目资助下，共培养研究生4人。