Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma
Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma
复制标题
血管生成素-2 通过外泌体诱导人肝细胞癌中的血管生成
DOI：
10.1186/s12964-020-00535-8
复制
发表时间：
2020-03-17
影响因子：
8.4
通讯作者：
Min, Jun
中科院分区：
文献类型：
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作者：
Xie, Ji-yan;Wei, Jin-xing;Min, Jun
背景肝细胞癌（Hepatocellular carcinoma，HCC）是最常见的原发性肝癌，是一种高度血管化的实体瘤.血管生成素-2（ANGPT 2）已被描述为抗血管生成治疗的有吸引力的靶点。外泌体是由大多数细胞类型分泌的小的细胞外囊泡，并且通过将功能性货物递送至受体细胞而有助于细胞间通讯。方法采用免疫印迹、酶联免疫吸附试验和免疫金标记法检测肝癌细胞来源exosomes中ANGPT 2的表达，并采用激光共聚焦显微镜、免疫共沉淀和免疫印迹法观察exosomes中ANGPT 2的内化和回收。我们使用了两种肝癌细胞系（Hep 3B和MHCC 97 H）通过慢病毒感染过量表达ANGPT 2或通过CRISPR/Cas系统敲低ANGPT 2，然后分离exosomes与人脐静脉内皮细胞（HUVECs）共培养，通过Matrigel微管形成试验、transwell迁移试验、伤口愈合试验、细胞计数kit-8试验观察血管生成，结果我们发现HCC来源的外泌体携带ANGPT 2并通过外泌体内吞作用将其递送到HUVECs中，这种递送通过Tie 2非依赖性途径导致血管生成的显著增加。同时，我们观察到HCC细胞分泌的外泌体ANGPT 2被受体HUVEC回收，并可能被重复使用。此外，敲低ANGPT 2的CRISPR-Cas系统显著抑制了HCC细胞分泌的exosomal ANGPT 2诱导的血管生成，并明显抑制了HCC中上皮-间质转化活化。这条通路可能成为抗血管生成治疗的潜在靶点。视频摘要
BackgroundHepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown.MethodsWe detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay.ResultsWe found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC.ConclusionsTaken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy.Video Abstract