肝癌外泌体携载胞浆型磷脂酶cPLA2α参与调控肝癌血管生成的作用及分子机制研究

Exosomes may be involved in tumor angiogenesis, but the mechanism remains to be elucidated. Our previous studies have shown that cPLA2α can regulate the invasion and metastasis of various malignant tumors, and exosomes are important carriers for the release of cPLA2α. Regulation of the expression of cPLA2α in hepatocellular carcinoma (HCC) cells can significantly affect its content in exosomes. Overexpression of cPLA2α can promote angiogenesis of HCC, and its secreted exosomes can significantly promote the proliferation, motility and angiogenesis of endothelial cells. Immunohistochemistry of HCC confirmes that the expression of cPLA2α is positively correlated with the expression of vascular marker CD31, both of which are independent risk factors affecting the prognosis of HCC. Therefore, we speculate that HCC exosomes can remodel the microenvironment of HCC and promote the progression and metastasis of HCC by carrying cPLA2α to mediate tumor angiogenesis. This project aims to explore the regulation of cPLA2α in exosomes on tumor angiogenesis by means of enzyme-linked immunoassay, immunoblotting, co-culture of exosomes and cells, and in vivo experiments in mice; to further clarify the mechanisms through whole transcriptome sequencing, pathway blockade and rescue experiments; to further explore the clinical value of anti-angiogenesis therapy of cPLA2α inhibitor in HCC through 3D culture technique of tumor tissue in vitro and PDX model.

外泌体可参与介导肿瘤血管生成，但其机制仍亟待阐明。申请人前期研究表明磷脂酶cPLA2α可参与调控多种恶性肿瘤的侵袭转移，外泌体是其释放出胞的重要载体，调控肝癌细胞cPLA2α的表达可显著影响其在外泌体中的含量；过表达cPLA2α可促进肝癌的血管生成，其分泌的外泌体可显著促进内皮细胞的增殖、运动和成管能力；免疫组化证实cPLA2α与血管标记分子CD31的表达显著正相关，且二者均为影响肝癌预后的独立危险因素。因此我们推测，肝癌外泌体通过携载cPLA2α介导肿瘤血管生成重塑肝癌微环境，促进肝癌的进展和转移。本项目拟通过酶联免疫检测、免疫印迹、外泌体-细胞共培养、动物实验等方法探究外泌体中cPLA2α含量的改变对肿瘤血管生成的调控作用；通过全转录组测序、通路阻断及拯救实验明确这一调控的具体机制；应用肿瘤组织体外三维培养技术和PDX模型进一步探究cPLA2α抑制剂抗肝癌血管生成治疗的临床应用价值。

血管生成是影响肝癌治疗效果、造成患者预后不佳的重要原因。由新生血管导致肿瘤微环境的重塑有利于肿瘤的转移和治疗抵抗，促进了肿瘤的恶性进展。研究发现外泌体在介导肿瘤血管生成的过程中发挥重要作用。我们前期研究表明磷脂酶cPLA2α可参与调控多种恶性肿瘤的侵袭转移，肝癌组织cPLA2α的表达与肿瘤内部的微血管密度显著正相关，且二者均为影响肝癌预后总生存期和无病生存期的独立危险因素；外泌体是cPLA2α释放出胞的重要载体，调控肝癌细胞cPLA2α的表达可显著影响其在外泌体中的含量；体外细胞功能学实验结果提示过表达cPLA2α可促进肝癌的血管生成，其分泌的外泌体可显著促进内皮细胞的增殖、运动和成管能力；小鼠体内实验结果表明，在接种后的相同时间内，过表达cPLA2α的肝癌细胞所成肿瘤体积总是大于对照组，HE染色和免疫组织证实cPLA2α过表达的肝癌细胞所成的肿瘤中具有丰富的新生血管；我们将cPLA2α不同表达水平的肝癌细胞分泌的外泌体与血管内皮细胞共培养后进行全转录组测序发现，cPLA2α低表达组与对照组细胞间差异基因主要富集在Wnt/β-catenin信号通路中，提示肝癌可能通过肿瘤细胞外泌体携载cPLA2α的分泌激活Wnt/β-catenin信号通路进而调控微环境中的血管生成。总之，我们的结果提示cPLA2α有望发展成为评价肝癌血管生成及预后的标志物、治疗肝癌的药物靶标，以及肝癌个体化治疗的靶点。