活化肝星状细胞通过NR4A2/OPN轴促进肝内胆管癌上皮间质转化

Activated hepatic stellate cells (aHSCs) is associated with the malignant behavior of ICC, the underlying mechanism is yet to be elucidated. In our preliminary experiments, we established the co-culture system of activated HSCs and ICC cell lines (HUCCT1, HCCC9810, RBE), which indicated that aHSCs enhance epithelial-mesenchymal transition (EMT) in ICC; moreover, aHSCs upregulated nuclear receptor subfamily 4, group A, member 2 (NR4A2) and osteopontin (OPN) in ICC, which are involved in proliferation and invasion of malignant tumor. Up-regulation of NR4A2 in ICC cell lines can promote the proliferation, invasion and EMT in ICC. In addition, NR4A2 mediated the effect that aHSCs promote EMT in ICC. OPN, as target gene of NR4A2, is a key regulator of EMT. We hypothesized that NR4A2/OPN axis plays a core role in the effect that aHSCs promote EMT in ICC. In this proposal, we will confirm this hypothesis and illuminate the underling molecular mechanism, the main pathway which mediate the pro-tumor role of NR4A2-OPN axis will be studied by protein array, CHIP assay and etc. The study may provide new clues for the development of new ICC drug therapy strategy.

活化肝星状细胞(aHSCs)与肝内胆管癌(ICC)恶性生物学行为密切相关。前期研究发现ICC以间质丰富及纤维沉积为特征，纤维间质组织学成熟度及成纤维细胞数量均与预后相关。已知aHSCs是ICC肿瘤微环境中纤维间质及成纤维细胞主要来源，我们利用aHSCs与ICC细胞直接共培养体系发现aHSCs可促进ICC增殖、侵袭及上皮间质转化；基因表达谱芯片筛选发现与aHSCs细胞共培养的ICC细胞显著高表达增殖侵袭相关分子NR4A2、OPN，生信分析发现OPN为NR4A2靶基因，两者均与ICC不良预后相关；进一步研究发现NR4A2可介导aHSCs促进ICC上皮间质转化作用，且诱导上皮间质转化相关分子OPN高表达。我们推测：aHSCs经由NR4A2-OPN促进ICC上皮间质转化。本研究拟通过蛋白芯片及CHIP等技术，验证该假说，寻求aHSCs促进ICC恶性生物学行为的潜在可干预靶点，为药物治疗提供新思路。

肝内胆管癌（Intrahepatic cholangiocarcinoma，ICC）是一种高度致命的恶性肿瘤，其特征表现为大量的肿瘤内成纤维细胞浸润。这些成纤维细胞可能参与维持ICC的高侵袭性，但相关的机制仍不明确。在这里，通过建立ICC细胞和肝星状细胞（hepatic stellate cells，HSCs）的共培养模型，我们确定HSCs触发了ICC细胞中一种转录因子核受体家族4亚群A成员2（NR4A2）的表达。在功能上，NR4A2促进肿瘤增殖、转移，是ICC患者总体生存的独立预后指标。机制上，NR4A2通过转录激活上调骨桥蛋白（osteopontin，OPN）表达，从而增强Wnt/β-catenin的活性。在共培养模型中，血管内皮生长因子（VEGF）不仅增强了NR4A2的表达，而且抑制NR4A2-OPN轴降低VEGF水平。综上，本研究表明NR4A2/OPN/Wnt/β-catenin轴是HSC诱导ICC进展的关键信号通路，该通路在和VEGF的正反馈可能进一步促进ICC进展。