肿瘤血管内皮细胞CD109低表达诱导内皮-间质转化促进肝癌转移的机制研究

Tumor angiogenesis plays an important role in hepatocellular carcinoma (HCC) progression, however anti-angiogenic therapies have poor efficacy and opposite effect to promote tumor progression. In addition to involved in tumor angiogenesis, tumor-associated endothelial cells (TEC) have been demonstrated to play more important active roles in tumor progression. Studies have shown that endothelial-mesenchymal transition (EndMT) of TEC promote tumor progression, but its role in HCC progression is unclear. Our previous work revealed that CD109 was one of differentiated expressed genes that determined TEC heterogeneity. The further study demonstrated that TEC with low CD109 expression were correlated with poor prognosis in HCC patients and CD109 knockdown TEC promoted HCC progression by paracrine interleukin-8 (IL-8) in vitro and in vivo. We observed that CD109 knockdown changed HUVEC shape from the rounded cobblestone-like morphology to an elongated spindle-like appearance, suggesting that CD109 knockdown might induce the EndMT of HUVEC. We aimed at verifying the effect of CD109 downregulation inducing the EndMT in HCC and unveiling its underlying mechanism through in vitro and in vivo experiments, further demonstrating the role of EndMT in HCC progression. The potential of inhibiting the EndMT to prevent the HCC metastasis in animal experiments may suggest the therapeutic targets for HCC.

肿瘤血管生成与肝癌进展密切相关，但抗血管生成疗效不佳且有促肿瘤的反作用。除参与肿瘤血管生成外，肿瘤血管内皮细胞的主动促肿瘤作用更值得关注。研究表明肿瘤血管内皮细胞通过内皮间质转化（EndMT）促进了肿瘤进展，但在肝癌中作用不清。我们前期研究发现CD109在肝癌血管内皮细胞与正常血管内皮细胞存在差异表达，且肝癌血管内皮细胞CD109低表达与患者不良预后相关，我们证实肿瘤血管内皮细胞CD109低表达通过旁分泌IL-8促进了肝癌进展；同时我们也发现CD109低表达的血管内皮细胞外形从圆形铺路石样向长梭形转变，提示CD109低表达诱导了EndMT。本研究拟深入研究肝癌血管内皮细胞CD109表达下调诱导了EndMT，揭示CD109调控EndMT的关键机制，阐明EndMT在肝癌进展中的作用，并通过动物实验明确阻断CD109表达下调诱导EndMT对肝癌转移的作用，以期为肝癌转移的防治提供新的靶点。

肿瘤血管生成与肝癌进展密切相关，以肿瘤血管内皮细胞为主要靶点的抗血管生成药物疗效不佳且具有促肿瘤的反作用。研究表明肿瘤血管内皮细胞通过内皮间质转化（EndMT）促进了肿瘤进展，但在肝癌中作用不清。本研究在体外通过下调血管内皮细胞CD109的表达以及免疫荧光检测人肝癌标本中EndMT标志物，我们发现肝癌血管内皮细胞CD109低表达诱导了EndMT。通过全基因组芯片筛选CD109下调后的差异表达基因，进行KEGG通路分析，我们发现变化最明显的10条通路中包括了与EndMT相关的TGF-β信号通路以及PI3K/Akt信号通路。通过利用通路阻断剂检测通路相关基因和蛋白的变化，证实了血管内皮细胞CD109表达下调后激活了TGF-β信号通路以及下游的PI3K/Akt信号通路诱导了EndMT。进一步研究表明，CD109表达下调是通过上调了TGF-β受体以及配体的表达激活了血管内皮细胞自分泌的TGF-β信号通路来诱导EndMT。我们通过体外肝癌细胞与血管内皮细胞粘附实验以及肝癌细胞跨血管内皮细胞迁移实验，发现血管内皮细胞CD109表达下调后促进了肝癌细胞与血管内皮细胞的粘附并减弱了血管内皮细胞的屏障功能，在体外促进了肝癌的转移。TGF-β信号通路抑制剂在体外可以逆转CD109表达下调促肝癌的转移作用。通过慢病毒转染的方法获得了CD109稳定低表达的血管内皮细胞株，并构建了血管内皮细胞与人肝癌细胞HCCLM3混合接种裸鼠原位瘤模型，我们发现血管内皮细胞CD109表达下调体内促进肝癌的进展，TGF-β信号通路抑制剂在体内可以抑制肝癌的转移。阻断CD109表达下调诱导EndMT可以抑制肝癌的转移，具有潜在的临床应用价值。