核受体NR1D1高表达肝癌细胞通过募集并激活巨噬细胞促进侵袭转移的机制及其干预研究

Tumor associated microenvironment plays the key role in the process of hepatocellular carcinoma (HCC) initiation and metastasis. The results of multicenter and large sample analysis showed that profiles of the surrounding nontumoral liver tissue were highly correlated with survival. The dynamic interaction between HCC cells and stromal cells in favor of HCC progression and metastasis step by step, which highlight the initiative transformation role of HCC cells on microenvironment to make it growth and metastasis. However, the mechanism of the process is large unknown. By comparing the gene expression profiles between metastases free and intrahepatic metastases HCC, we established that the metastasis-favoring gene alterations originated from primary tumor. .. One of the is nuclear receptor subfamily 1, group D, member 1 (NR1D1). The following results at mRNA and protein levels showed that , compared with normal liver and cirrhotic liver tissues, NR1D1 expression increased significantly in HCC, especially in extrahepatic metastasis HCCs. Meanwhile, the results of in vitro experiment suggested that down-regulation of NR1D1 in HCC cells resulted in few change in HCC cell proliferation, colony formation, migration and invasion. Much different from in vitro experiment, the results of in vivo study showed that, compared with control group, NR1D1 down-regulation give rise to the significant decrease of tumor burden and lung metastases in MHCC-97HshNR1D1 group. The results of analyzing Affymatrix chip between HCC-LM3, HCC-LM3-shNT and HCC-LM3-shNR1D1suggested that most cytokins and chemokins with obviosly changed were correlated with hypoxia induced factor 1a (HIF-1a). Furthermore, we found the promotion role of NR1D1 on HCC progression is correlated to the macrophage recruitment significantly. Through analyzing the distribution of macrophage in paratumoral liver tissue, we found the density of macrophage in NR1D1 high expression HCC is much higher than those in NR1D1 low expression HCC. The clinical tissue microarray based immunohistochemistry showed the correlation between intratumoral NR1D1 and paratumoral macrophage recruitment. Based on the data, we presented that NR1D1 is the key molecule for HCC to reconstruct microenvironment, which in favor of HCC growth and metastasis. In further study, we will adopted laser confocal fluorescence microscopy, flow cytometry, chromatin immunoprecipitation, co-immunoprecipitation, EMSA-electrophoretic mobility shift assay to screen and verified which molecules play the indispensable role in NR1D1 regulating HCC active reconstruction of microenvironment; exploring the effects of specific inhibitor of chemokine on HCC growth and metastasis.

肝癌细胞通过和间质细胞动态相互作用，主动改造形成有利于自身生长转移的微环境，但机制还不明确。比较伴与不伴转移肝癌间基因表达谱差异，我们发现核受体NR1D1高表达肝癌患者易转移，预后差。沉默NR1D1表达后肝癌细胞体外增殖无明显改变，体内实验却发现生长和肺转移受到抑制，癌旁巨噬细胞（MΦ）密度显著降低。基因芯片提示相应趋化因子的变化均和缺氧诱导因子1α表达下调相关。据此提出“NR1D1通过上调HIF1α改变趋化因子表达谱进而募集并激活MΦ以达到改造肿瘤微环境使之有利于肝癌生长转移”的假说。进一步将在明确NR1D1高表达肝癌细胞对巨噬细胞的募集和激活作用基础上,采用Co-IP、GST亲和层析、ChIP、蛋白芯片等方法明确NR1D1如何调节HIF1α，筛选并验证作用于MΦ膜受体，促进MΦ募集、活化关键趋化因子。探索关键趋化因子抑制剂能否降低NR1D1对MΦ的募集和激活从而抑制肝癌生长转移。

肝癌的转移复发不仅与肿瘤本身有关，而且与肿瘤微环境（Tumor Microenvironment）密切相关。肿瘤微环境是由肿瘤细胞和多种基质细胞、免疫细胞、细胞因子等组成。肿瘤微环境细胞与肿瘤细胞之间的相互作用（The Cross-Talk）已成为肿瘤转移复发研究的热点。本课题组前期研究发现，肝癌微环境在肝癌的转移复发过程中发挥重要作用。NR1D1（nuclear receptor subfamily 1, group D, member 1）是细胞内重要的核受体之一。近期发现，NR1D1可以促进肿瘤发生转移，并且可以通过对炎症因子分泌的调节影响机体炎症微环境。我们题组前期通过基因芯片也发现，NR1D1与肝癌转移密切相关。因此，本项目在结合临床对NR1D1在肝癌中的表达及其对肝癌细胞行为学影响进行研究，发现：1）NR1D1在正常肝组织和癌旁组织中表达低，在肝癌组织中表达高，在伴血管侵犯的肝癌组织中表达最高；HCC中高表达NR1D1提示肿瘤的恶性程度高且患者预后差；2）高转移潜能的肝癌细胞系（MHCCLM3）中NR1D1表达量高；NR1D1对肿瘤增殖的研究发现，体内和体外研究现象相反，NR1D1体外研究发现对肿瘤细胞的增殖能力和克隆形成能力无影响，但是，体内研究发现NR1D1表达增高可以促进肿瘤生长；NR1D1对肿瘤侵袭转移能力研究发现，NR1D1可以促进肿瘤的侵袭能力和迁移能力，同时体内研究发现，NR1D1可以促进肿瘤肺转移；3） NR1D1高表达的肝癌细胞可以募集MΦ，且NR1D1和CD68在HCC患者组织内的表达呈正相关；MΦ与肝肿瘤细胞共培养后，NR1D1高表达的细胞生长、克隆形成能力、侵袭能力和迁移能力明显强于NR1D1低表达的细胞；体内NR1D1高表达的细胞与MΦ联合NR1D1低表达细胞的肿瘤生长能力强于NR1D1低表达的细胞。改研究有助于我们探索关键趋化因子抑制剂能否降低NR1D1对MΦ的募集和激活从而抑制肝癌生长转移。