外泌体circ-SMAD2激活FOXM1/Snail通路促进胰腺癌侵袭转移的机制研究

As the most aggressive human malignancies, pancreatic cancer is characterized by its high risk of metastasis. Exosomes secreted by cancer cells contain a wide range of functional contents, promoting tumor progression through communication between the tumor and surrounding stromal tissue. However, the role of exosomes on pancreatic cancer is still elusive. In the previous study, we found that exosomes derived from hypoxic pancreatic cancer cells increased the migration of cancer cells. By RNA sequencing of normoxic and hypoxic cancer cells-derived exosomes, circ-SMAD2 stood out as one of the most significantly upregulated circRNAs under hypoxic conditions. Furthermore, down-regulation of circ-SMAD2 inhibited the cell migration and epithelial-mesenchymal transition (EMT). By RNA-pull down and mass spectrometry, we found that circ-SMAD2/FOXM1/Snail signaling pathways may enhance EMT and lead to metastasis under hypoxia condition. Therefore, in this study we will aim at illustrating the molecular mechanisms of exosomal circ-SMAD2 promotes cancer cell metastasis under hypoxia condition. Our studies will advance our understanding of the mechanisms in the metastasis of pancreatic cancer, provide a novel orientation, and dig out potential targets for treatment.

胰腺癌是一类极易转移的消化道恶性肿瘤。外泌体是细胞分泌的囊泡状小体，在肿瘤转移中发挥重要作用。项目申请人前期研究发现，胰腺癌细胞在缺氧环境下分泌的外泌体显著促进其转移；高通量测序进一步揭示外泌体中circ-SMAD2高表达可诱导EMT发生；RNA pull down及质谱分析证实转录因子FOXM1为circ-SMAD2的作用靶点，可激活EMT相关重要转录因子Snail。在此基础上，我们提出假说：缺氧环境通过激活癌细胞外泌体circ-SMAD2/FOXM1/Snail信号通路促进转移。然而，上述信号分子间的具体关系和分子机制有待深入研究。据此本项目旨在首先明确外泌体circ-SMAD2在胰腺癌细胞转移过程中的具体作用，继而深度挖掘circ-SMAD2/FOXM1/Snail信号轴异常的功能效应及分子机制，将从外泌体层面揭示胰腺癌侵袭转移的分子机制，为诊治胰腺癌提供新思路新靶点。

胰腺癌是恶性程度极高的消化系统肿瘤，预后极差，其主要原因是胰腺癌较早发生局部侵袭和远处转移。胰腺癌发生和发展过程中肿瘤内部明显乏血供处于缺氧环境，缺氧诱导上皮-间质转化（Epithelial-Mesenchymal Transition, EMT）改变而有利于转移。研究表明，癌细胞主动分泌的外泌体携带的蛋白质、mRNA以及非编码RNA(ncRNA)在胰腺癌的转移过程中扮演了重要角色。前期实验中，我们发现与正常氧浓度下的外泌体相比，缺氧环境下胰腺癌细胞分泌的外泌体能够明显促进癌细胞的转移能力。通过高通量测序技术发现缺氧环境下细胞分泌的外泌体中存在大量环状RNA(circRNA)，并且多种circRNA表达水平较对照外泌体差异性明显，提示外泌体携带的circRNA在胰腺癌细胞转移中可能扮演重要角色。这其中circ-SMAD2在缺氧外泌体中表达升高最明显，而且在细胞中下调其表达时显著抑制细胞转移能力，同时EMT通路中Snail和N-Cadherin的表达水平受到下调，相对应地促进了E-Cadherin的表达。目前研究认为circRNA能够通过直接结合蛋白质分子调控肿瘤细胞增殖、侵袭等，我们通过RNA-pull down结合质谱分析发现circ-SMAD2可特异性与转录因子FOXM1结合，circ-SMAD2的敲低能抑制FOXM1的表达水平。FOXM1是EMT发生发展过程中的重要因子，可以通过调节转录因子Snail而影响EMT通路中 E-Cadherin的表达水平。基于这些前期研究，本课题进一步证实缺氧环境下胰腺癌细胞分泌的外泌体中携带的物质发生改变；circ-SMAD2通过外泌体在细胞间进行传递，激活FOXM1/Snail通路诱导EMT进展，最终促进了癌细胞的转移。本项目明确了外泌体中circ-SMAD2与FOXM1和Snail的作用关系，并初步揭示了 circ-SMAD2/FOXM1/Snail信号轴在转移中的具体机制，从外泌体层面揭示胰腺癌侵袭转移的分子机制，为诊治胰腺癌提供新思路新靶点。