MicroRNA-1246介导肝癌细胞与活化的肝星状细胞之间的相互对话

Tumor microenvironment is associated with the characteristic of HCC. The study of the characteristic of HCC could help to develop new therapy strategy. Previously, we found that peri-tumoral hepatic stellate cells (HSCs) is associated with tumor invasion and metastasis, in vitro study showed that quiescent HSCs can be activated by HCC cell lines. Thus indicating cross-talk between these two cells. In our preliminary experiments, activated HSCs up-regulated the expression level of microRNA-1246 (miR1246), which promote proliferation and invasion of HCC cell lines through down-regulation of RORα; moreover, up-regulation of miR1246 in HCC cell lines can promote the accumulation and activation of quiescent HSCs. We hypothesized that there is a cross-talk between hepatoma cells and activated HSCs, and miR1246 plays a core role in this cross-talk. In this proposal, we will confirm this hypothesis and illuminate the underling molecular mechanism, the key transcription factor which regulate the expression of miR1246 and the main target gene which mediate the pro-tumor role of miR1246 will be studied by protein array, CHIP assay and etc. The study may provide new clues for the development of new HCC drug therapy strategy.

肿瘤微环境与肝癌恶性生物学行为密切相关。我们前期研究发现：癌周肝星状细胞与肝癌侵袭转移相关，肝癌细胞体外刺激肝星状细胞活化，提示肝癌细胞与肝星状细胞间可能存在相互联系。预实验中：活化的肝星状细胞上调肝癌细胞MicroRNA-1246（miR-1246）表达，后者通过RORα促进肝癌细胞增殖及侵袭；上调miR-1246可促进肝癌细胞对肝星状细胞的募集与活化，两者间形成正反馈循环。我们推测：miR-1246在两种细胞间的相互对话中处于核心地位。我们拟通过蛋白芯片及CHIP等技术，验证该假说并研究相互对话的机制，寻找调控miR-1246表达的关键转录因子及受miR-1246调控的下游关键靶基因，寻求打破相互对话的潜在可干预靶点，为肝癌治疗提供新的策略。

肝星状细胞（Hepatic stellate cells，HSC）在肝细胞癌（hepatocellular carcinoma， HCC）的发生发展中起着重要作用。然而，目前仍缺乏关于肝癌细胞与HSC直接互动机制的研究。在这项研究中，我们建立了HCC细胞系和HSC的直接共培养模型肝癌细胞。我们发现HSC触发了一组miRNA在HCC中的高表达，尤其是miR-1246。通过转录组测序和数据库预测，我们确认了RORα为miR-1246的靶基因。在体内和体外试验中，我们验证了miR-1246的过度表达或RORα的敲除通过Wnt/β-catenin途径激活增强肝癌在体内外的增殖、侵袭和转移能力并促进上皮间质转化（EMT）。此外，临床数据提示miR-1246的上调和RORα的抑制均是HCC患者生存的独立危险因素。目前共发现RORα的四个不同转录本，我们发现RORα-1在四个转录本（RORα-1/-2/-3/-4）中对HCC的起主要影响，RORα-1的过度表达显著抑制MHCC97H细胞增殖、迁移和入侵的能力。我们分析了RORα-1过表达和对照组的MHCC97H细胞中的差异表达功能富集分析显示，肿瘤微环境ECM与RORα-1的抗肿瘤作用有关。趋化因子CXCL5被RORα-1过度表达显著下调。RORα-1的过度表达显著抑制肝癌细胞增殖、迁移、侵入以及β-catenin、c-Myc、Cyclin D1和N-cadherin（EMT相关蛋白）的水平。此外，CXCL5的过度表达显著减弱了细胞凋亡的抑制作用MHCC97H中RORα-1过度表达诱导的增殖、迁移和侵袭，表明RORα-1通过下调CXCL5发挥抗肿瘤作用。总之，我们证明了MiR1246-RORα-Wnt/β-catenin轴是HSC促进HCC进展的重要途，RORα-1通过调节趋化因子CXCL5在MHCC97H细胞中发挥作用。