肝内胆管癌细胞诱导肝癌细胞上皮细胞-间充质转化在混合型肝癌转移中的机制及意义

Simultaneous containing hepatocellular carcinoma cells and cholangiocarinoma cells components, and metastatic feature are important characteristics of combined hepatocellular-cholangiocarcinomas. In the initial research period, we verified that high-level epithelial-mesenchymal transition (EMT) had arose in the combined hepatocellular-cholangiocarcinomas. Further study confirmed that cholangiocarinoma cells were able to induce hepatocellular carcinoma cells to arise EMT and the phenomenon might depend on Ln-332 and TGF-β1 which were secreted by cholangiocarinoma cells. On the basis of initial research, we will study the influence of Ln-332 and TGF-β1 to growth, migration, invasion and resistance of hepatocellular carcinoma cells, and clear the mechanism of EMT in hepatocellular carcinoma cells induced by cholangiocarinoma cells. We will also use a experimental lung metastasis model to determine the relation between the EMT in hepatocellular carcinoma cells induced by cholangiocarinoma cells and metastatic feature of combined hepatocellular-cholangiocarcinomas. Our research tries to explain the reason combined hepatocellular-cholangiocarcinomas has a dismal prognosis. It will offer a potential therapeutic approach for treatment of combined hepatocellular-cholangiocarcinomas.

肝癌细胞和肝内胆管癌细胞并存、高转移率导致预后极差是混合型肝癌的两大特点。前期研究中我们发现，混合型肝癌中存在较高水平的肿瘤细胞上皮细胞-间充质转化（EMT）。进一步研究表明，肝内胆管癌细胞可诱导肝癌细胞发生EMT，并可能与肝内胆管癌细胞分泌Ln-332和TGF-β1相关。本研究拟在前期结果的基础上，深入研究肝内胆管癌细胞分泌Ln-332和TGF-β1对肝癌细胞增殖、迁移、侵袭及耐药的影响，明确肝内胆管癌细胞诱导肝癌细胞发生EMT的作用机制；利用混合型肝癌肺转移动物模型，证明肝内胆管癌细胞诱导肝癌细胞EMT与混合型肝癌易转移之间的联系。本研究是从新的角度研究混合型肝癌预后不良的原因，结果将为临床治疗混合型肝癌提供新的思路和可能的靶点。

本项目研究结果主要包括两方面。首先，本研究通过分析肝细胞癌细胞和肝内胆管癌细胞的互作关系，分析造成混合型肝癌易转移和预后极差的潜在原因。我们发现肝内胆管癌细胞培养上清能够促性肝细胞癌细胞发生上皮细胞-间充质转化（EMT），并能够促进肝细胞癌细胞迁移和侵袭，但对肝细胞癌细胞增殖影响不明显。不仅如此，肝内胆管癌细胞培养上清能够增强肝细胞癌细胞的化疗耐受能力。通过培养上清Western blot，我们发现LAMC2在肝内胆管癌细胞培养上清中有表达，而在肝细胞癌细胞培养上清没有表达。LAMC2敲除的肝内胆管癌细胞培养上清失去诱导肝细胞癌细胞发生EMT的能力，并失去促进肝细胞癌细胞迁移的作用，提示肝内胆管癌细胞分泌LAMC2是产生该现象的关键因素。其次，我们发现解偶联蛋白（UCP2）介导的能量代谢异常对胆管癌细胞EMT有重要影响。我们发现UCP2在胆管癌组织中高表达，且与淋巴转移和预后不良有关。细胞实验发现，敲减UCP2表达有效抑制胆管癌细胞增殖和迁移。UCP2敲减的胆管癌细胞能量代谢发生改变：需氧糖酵解受到抑制，而线粒体氧自由基和AMP/ATP比值明显升高，继而导致AMPK信号通路被激活，最终抑制胆管癌细胞EMT。应用抑制剂genipin抑制UCP2活性，可增强胆管癌细胞对化疗药物的敏感性，提示UCP2介导的需氧糖酵解调节可能是治疗胆管癌的新靶点。我们的研究为混合型恶性肝癌研究提供一个新的参考，为胆管癌的治疗提供一个新的思路。