肝癌治疗新靶标VASN蛋白的自分泌和旁分泌双重靶向效应的作用机制研究

Cancer cells are able to release angiogenic factors to promote tumor angiogenesis and neo-vasculature growth towards tumor mass. Therefore, blocking the crosstalk between cancer cells and endothelial cells is an important strategy in post-surgery treatment. Our lab has discovered that vasorin (VASN) released by the hepatoma cells might function as an important angiogenic factor. It is reported that VASN plays a role in developmental angiogenesis and involved in the response to vascular injury stress, while it might also contribute to cancer cell metastasis by other studies. Using high throughput screening, we have succeeded in identifying VASN as a serum biomarker of hepatoma. We further revealed that VASN is overexpressed in liver cancer cells, and demonstrated that VASN exhibits a dual-targeting function. VASN secreted by liver cancer cells promotes the proliferation and migration of cancer cells via the autocrine signaling pathway. VASN can also be internalized by cultured human umbilical vein endothelial cells (HUVECs) to promote migration of HUVECs via the paracrine signaling pathway. We also identified that CD71, a membrane receptor of liver cancer cells, interacts with VASN. This study aims to elucidate the dual-targeting mechanism of VASN protein through autocrine and paracrine signaling pathways, and to identify its receptor and downstream pathways, which lays the foundation for targeting VASN as a liver cancer treatment.

肿瘤细胞释放血管生成因子，促进肿瘤血管生成和新生血管向肿瘤的生长。因此这类因子是抗肿瘤新生血管形成的热点药物靶点。我们发现VASN（vasorin）蛋白可能是肝癌细胞分泌的血管活性分子。研究认为，VASN是诱导血管形成的发育相关蛋白，参与机体对血管损伤应激的应答。近年来发现VASN参与肿瘤侵袭过程。我们首次从肝癌血清中高通量筛选获得VASN标志物分子。进一步研究发现，VASN在肝癌细胞中高表达，肝癌细胞分泌的VASN可以靶向两种效应细胞：不仅促进肝癌细胞的增殖与迁移（自分泌作用），肝癌细胞外泌体内的VASN还能够被培养的脐静脉内皮细胞内化并促进内皮细胞的迁移（旁分泌作用）。我们钓取了VASN蛋白在肝癌细胞膜上的结合分子，鉴定为CD71分子。本研究旨在阐明VASN蛋白的双重靶向效应细胞的自分泌旁分泌作用机制，鉴定其作用受体与下游关键信号分子，解析VASN蛋白的功能，奠定其生物治疗靶标的地位。

肿瘤产生的血管生成因子是抗肿瘤药物的热点靶点。我们发现VASN（vasorin）蛋白不仅被肝癌细胞中高表达，促进肝癌细胞的增殖与迁移（自分泌作用），肝癌细胞外泌体内的VASN还能够被培养的脐静脉内皮细胞内化并促进细胞内皮的迁移（旁分泌作用）。.本项目对VASN的自分泌和旁分泌机制进行了深入探讨。鉴定了VASN胞外区（sVASN）内皮细胞表面的靶分子，并进行了验证与阻断实验 。结果显示，真核表达的rhsVASN蛋白、HepG2细胞培养上清中天然形式的sVASN均可以促进内皮细胞HUVEC的迁移与增殖；sVASN与HUVEC细胞表面CD71相互作用，CD71介导sVASN内化至内皮细胞内，转铁蛋白封闭CD71后可减弱sVASN的内化。首次阐述了sVASN的非TGF-β依赖的、CD71信号通路介导的对内皮细胞的旁分泌生物学功能。.在上述基础上，我们利用噬菌体肽库筛选了VASN自分泌方式靶向肝癌细胞的活性模拟肽。首先制备了VASN的单抗并在细胞上筛选获得了一株具有阻断VASN功能的单抗V21，利用噬菌体肽库技术获得了该单抗的数条模拟表位，其中两条表位肽V21P1和V21P2与VASN蛋白的Cys432-Cys441区段高度相似，位于蛋白EGF样功能域内，合成的肽段能够抑制功能抗体的活性，针对该肽段的抗体能够抑制重组和细胞分泌的VASN靶向肝癌细胞。.此外，我们还采用基因编辑技术CRISPR/Cas9构建了VASN基因稳定敲除的 HepG2细胞株，为详尽阐明VASN的自分泌、旁分泌作用的研究奠定了基础，也为以后靶向分子的生物学作用验证提供了细胞模型。