胰腺癌外泌体miR-130a通过调控血管生成和血管通透性促进肿瘤进展的机制研究

The main causes of poor prognosis of pancreatic ductal adenocarcinoma (PDAC) include the high malignancy, easy invasion and metastasis. The blood vessels in the tumor microenvironment not only provide nutritional support for tumors, but also a key for blood metastasis. More and more studies showed that exosomes also play an important role in tumor invasion and metastasis for their special roles in intercellular communication. However, it is still unclear whether PDAC regulates the biological behavior of endothelial cells in the tumor microenvironment and then promotes tumor progression and their specific molecular mechanisms. Our previous studies found that exosomes secreted by PDAC cells can promote angiogenesis, and exosomes are rich in microRNA (miRNA), in which the miR-130a is highly expressed. miR-130a can act with HoxA5, a transcriptional factor, to promote angiogenesis in PDAC. We also found that exosomes from PDAC cells can enter into endothelial cells. Therefore, we hypothesized that PDAC regulates the biological behavior of vascular endothelial cells through exosome miR-130a, and further promotes PDAC invasion and metastasis. This project aims to screen out key miRNAs in PDAC exosomes by microarray analysis, explore its specific signaling pathways and molecular mechanisms via RNA pull-down, ChIP and Dual Luciferase Reporter system assays, and then validate the regulation in animal experiments and clinical samples. Tracking and imaging the exosomal inter-cellular communications between PDAC cells and vascular endothelial cells are also included. The research provides a brand-new way and theoretical basis for the treatment of pancreatic cancer by regulating the mechanism of vascular biological behavior to promote invasion and metastasis via exosomes.

胰腺癌是一类恶性程度极高，预后极差的实体肿瘤。肿瘤微环境中血管不但为肿瘤提供营养支持，也是肿瘤血行转移的重要途径，而近几年研究表明外泌体是细胞间“通讯”的重要工具。然而，胰腺癌是否通过外泌体调控微环境中内皮细胞生物学行为进而促进肿瘤侵袭转移及相应机制尚不清楚。我们前期研究发现：胰腺癌分泌的外泌体中富含大量小RNA，其中miR-130a表达量高，且可能调控转录因子HoxA5的表达，进一步与VEGFR-2互作调控血管生成。胰腺癌外泌体亦能作用于内皮细胞。因此我们提出假说：胰腺癌通过分泌外泌体miR-130a调控血管内皮细胞的生物学行为，进一步促进胰腺癌侵袭和转移。本项目拟通过基因芯片、RNA pull-down、ChIP实验、双荧光素酶报告基因实验、动物实验及外泌体共培养模型等技术探索外泌体miRNA调控血管生成的具体信号通路和分子机制及外泌体的通讯过程，为胰腺癌治疗提供新的途径和理论依据。

胰腺癌是世界范围内最恶性、致命的肿瘤之一，易侵袭和转移是胰腺癌预后差的主要原因，控制侵袭转移是治疗胰腺癌的关键。外泌体是一种由包括肿瘤细胞在内的各种细胞分泌的，具有脂质双分子层结构的小体，外泌体内含十分丰富，其内含的生物活性分子在各种生理病理过程中发挥重要作用，本课题的主要任务研究内容即研究胰腺癌外泌体miR-130a的异常表达及其中胰腺癌中发挥的分子作用及其与临床肿瘤特征的相关性。.本课题的重要结果及关键数据：1、胰腺癌细胞系可分泌大量外泌体，外泌体中富含丰富的miR-130a；2、胰腺癌外泌体miR-130a的表达和胰腺癌细胞的恶性程度成明显正相关；3、外泌体miR-130a可以促进胰腺癌细胞的血管生成及血管通透性；4、miR-130a可以负向调控转录因子HoxA5，HoxA5进一步调节VEGFR-2；5、外泌体可以介导胰腺癌细胞及血管内皮细胞之间的信息传递；外泌体miR-130a可以促进体内环境下裸鼠的肿瘤生成；6、外泌体miR-130a可以促进胰腺癌标本的MDV及MVI；7、胰腺癌外泌体miR-130a可能成为胰腺癌的肿瘤诊断或预后的预测标志物之一。.意义：胰腺癌是目前少数我们研究进展缓慢的肿瘤之一，主要在于胰腺癌缺乏早期的肿瘤标志物，对放化疗不敏感，容易复发转移。本课题通过在胰腺癌细胞系中探索外泌体内含的小分子物质，筛选鉴定出了miR-130a，进一步研究发现外泌体miR-130a在胰腺癌中高表达，miR-130a可能在胰腺癌中起重要作用，具体机制为miR-130a负向调控转录因子HoxA5，HoxA5,再进一步调控VEGFR2，通过促进肿瘤进展，内皮细胞血管生成及改变血管通透性从而实现肿瘤的侵袭转移，而这一切都可以由外泌体来介导完成。因此，外泌体miR-130a可能在胰腺癌中发挥重要的肿瘤标志物或预后预测作用。