肿瘤相关巨噬细胞上调肝癌细胞中Nanog信号的作用及其机制研究

Stemness-related genes have been found to be abnomally overexpressed in many tumors, and demonstrated to be involved in the regulation of tumor stemness and malignant progression. But little is known about the cause of their upregulation. Our previous work revealed the functions of Nanog in promoting stemness properties and abilities of invasion and metastasis of hepatocellular carcinoma (HCC) by inducing epithelial-mesenchymal transition (EMT) through activating Nodal/smad3 signaling pathway, and also showed that Nanog expression was enhanced mainly in tumor cells at tumor-stroma interface. Based on the well-known concepts that inflammatory tumor microenvironments regulate tumor stemness and contribute to tumor evolution toward metastasis, we speculate that the main inflammatory cell population, tumor-associated macrophage(TAM), within tumor stroma may be the culprit that stimulate Nanog expression, which was supported by the fact indicated in preliminary experiment that TAM could increase Nanog mRNA level. This project aims at firstly testing the hypothesis that TAM can promote stem cell-like phenotypes and malignant invasion of HCC cells through upregulating Nanog signal, and secondly clarifying the underlying signaling pathway of how TAM upregulates Nanog. This project will provide mechanistic information of TAM-Nanog inducing HCC metastasis from tumor-microenvironment interactions view, and contribute to development of novel targeted therapy for HCC.

研究表明，干性相关基因在多种肿瘤中呈现异常表达，它们参与调控肿瘤的干性和恶性进展，但是关于它们表达异常上调的机制尚缺乏深入研究。我们前期研究证实Nanog通过激活Nodal/smad3通路启动肝癌细胞发生上皮间质转化，促进肝癌的干性和侵袭转移，并发现Nanog在临床肝癌组织中的高表达多位于肿瘤-间质交界处。基于炎性微环境在肿瘤干性和转移中的重要作用，我们推测主要间质炎症细胞-肿瘤相关巨噬细胞（TAM）可能是上调Nanog的“元凶”，预实验结果显示TAM能够上调Nanog的mRNA水平。本项目拟利用共培养系统，分析TAM上调肝癌细胞中Nanog信号的作用及其在调节肝癌干性中的地位；并采用抗体芯片等技术，通过联合分析TAM分泌因子和肝癌细胞膜表面受体等变化解析其信号调控机制。本项目立足肿瘤微环境、干性和转移之间的调控关系，分析Nanog表达上调原因和探讨肝癌转移机制，为靶向生物治疗提供新依据。

干性相关基因在多种肿瘤中呈现异常表达，并被证实参与调控肿瘤的恶性表型，但关于它们表达异常的机制尚无定论。我们前期研究已证实Nanog对肝癌细胞侵袭转移的重要作用，本项目则是为了寻找Nanog上调的原因和机制。首先通过诱导THP-1获得M2型巨噬细胞（TAM2），将其和肝癌细胞（MHCC97H，简称97H）共培养，发现可以显著上调Nanog等干性基因表达，并且可以促进肝癌细胞的增殖和侵袭运动能力。然后采用质谱和抗体芯片技术比较分析共培养和单独培养的分泌蛋白组差异，共鉴定318个细胞因子表达差异，其中共培养组比两组单独培养都上调有179个，主要富集在NF-κB信号通路、Toll样受体信号通路上；其中有58个因子在单独培养的TAM2中的表达显著高于97H，主要参与凋亡生物过程调节，提示TAM2可能主要通过调节凋亡途径作用肝癌细胞。进一步我们选择了在共培养组中上调且TAM2表达高于97H的PD-ECGF，分析它对Nanog上调的作用和机制。结果表明在培养基中加入PD-ECGF因子，可以显著上调肝癌细胞中Nanog的表达；而且TAM2对Nanog的上调作用可以被PD-ECGF中和抗体明显抑制，暗示TAM2通过分泌PD-ECGF上调Nanog信号；对信号通路分子表达分析结果显示加入PD-ECGF后Wnt通路分子beta-catenin发生了上调，而且也引起了EMT相关分子的表达变化，提示PD-ECGF通过激活wnt/beta-catenin通路上调Nanog信号，进而诱导EMT。综上所述，我们的研究结果不仅证实了TAM2对Nanog信号的上调作用，而且解析了TAM2通过PD-ECGF-wnt/beta-catenin通路上调Nanog信号的机制，该结论解决了本项目拟解答的关键科学问题，为今后着眼于肿瘤炎性微环境和肿瘤干性的抗癌靶向生物治疗提供了新的依据。