Exosome传输miRNA调控胃癌细胞与基质细胞交互对话促进肿瘤转移的机制研究

Gastric cancer is one of the most common malignant tumors in China with high incidence of mortality and poor prognosis. Tumor metastasis is a major cause of death in patients with gastric cancer. Epithelial-mesenchymal transition (EMT) and metastatic niche formation are essential to tumor metastasis. It has been proved that exosomes (EXO) play a key role in EMT and regulate the cross-talk between tumor cells and stromal cells to create the metastatic niche. The secreted-miRNA is an important medium of intercellular communication and secreted to the extracellular through EXO. In the preliminary experiment, we found that gastric cancer cell-secreted EXO, rich in a variety of miRNAs, can promote fibroblasts proliferation, migration and transformation into cancer-associated fibroblas (CAF); CAF can promote the secretion of EXO and occurrence of EMT in gastric cancer cells in turn. Thus we speculate that the exosomal transfer of functional microRNAs may play a crucial role in cross-talk between gastric cancer and stromal cellls, and promote tumor metastasis. The project takes gastric cancer cells and CAF as the breakthrough point. We will separate EXO from gastric cancer cells and CAF, and identify the functional microRNAs. Using molecular biology method, gene knockout technique, Confocal microscopy scanning, 3D cell culture technology and etc, we plan to investigate the role and mechanism of secreted-miRNA that involved in the mutual interactions. This study will elucidate the molecular mechanism of secreted-miRNA though regulating intercellular communication, and leading to a favourable metastatic niche from a new perspective, which might provide new biomarkers and targets for the diagnosis and treatment of cancer.

胃癌是我国最常见的恶性肿瘤之一，其发病率高、预后差。肿瘤转移是胃癌患者的主要死因。上皮间质转化（EMT）和肿瘤微环境是肿瘤转移必要条件。Exosome（EXO)参与EMT发生，并调控肿瘤细胞与基质细胞交互对话，营造促癌微环境。分泌miRNA由EXO分泌至胞外，是细胞间交流的重要媒介。本课题组前期研究发现胃癌细胞分泌EXO富含多种miRNA，能促进成纤维细胞增殖和迁移并向肿瘤相关成纤维细胞（CAF）转化；CAF又可促使胃癌细胞分泌EXO并发生EMT。由此推测EXO传输miRNA可能为调控两者交互对话而促进肿瘤转移的元凶。本项目以胃癌细胞和CAF为切入点，分离纯化EXO并鉴定其功能性的miRNA。采用分子生物学、基因敲除、激光共聚焦、3D细胞培养等技术，从整体-细胞-亚细胞三个层次，探讨分泌miRNA调控胃癌细胞-基质细胞对话从而营造促癌转移微环境的分子机制，并为肿瘤诊断治疗提供新的靶位点。

探讨外泌体miRNA在胃癌转移中的作用。胃癌是目前常见的恶性肿瘤之一,死亡率高,早诊率低,放化疗、手术等治疗手段的发展远不能缓解胃癌诊疗的严峻形势。外泌体微小RNA(microRNA,miRNA)是细胞间重要的信息交流介质,由于其在血液中稳定性好,取材方便,且能在胃癌早期释放入血,受到越来越多的关注。外泌体miRNA参与胃癌发生、发展,腹膜转移,耐药性形成以及治疗等各个方面。大量研究提示,外泌体miRNA可作为胃癌诊断的新生标志物及治疗的潜在靶标。利用miRNA芯片分析具有明显侵袭潜力的外泌体的miRNA表达谱，并通过实时荧光定量PCR进行验证。利用miRNA芯片分析具有不同侵袭潜能的外泌体miRNA表达谱，并通过实时定量PCR进行验证。体外和体内实验评估了外泌体miR-196a-1在胃癌转移中的作用。外泌体miR-196a-1的高表达与胃癌患者的生存期显著相关。含miR-196a-1的外泌体是由高侵袭性GC细胞分泌的。异位miR-196a-1表达通过靶向SFRP1促进低侵袭性GC细胞的侵袭。miR-196a-1通过外泌体从高侵袭性胃癌细胞进入低侵袭性胃癌细胞，促进其在体内外向肝脏的转移。体外和体内实验评估了外泌体miR-196a-1在胃癌转移中的作用。外泌体miR-196a-1的高表达与胃癌生存期的低表达显著相关。含miR-196a-1的外泌体由高侵袭性GC细胞分泌。异位miR-196a-1表达通过靶向SFRP1促进低侵袭性GC细胞的侵袭。miR-196a-1通过外泌体从高侵袭性GC转移到低侵袭性GC细胞，促进其在体内外向肝脏转移。 . 实体瘤除了癌细胞外，还包括各种间质细胞。癌相关成纤维细胞(CAFs)是肿瘤基质的重要组成部分，在包括胃癌在内的多种恶性肿瘤的肿瘤进展和转移中起着重要作用。然而，CAFs对胃癌细胞的进展和转移的影响尚未得到很好的研究。我们的研究表明，由CAFs分泌的外泌体中的基质金属蛋白酶11 (MMP11)可以被传胃癌细胞通过外泌体MMP11部分迁移。此外，MMP11在从胃癌患者血浆和肿瘤组织中纯化的外泌体中过表达，并与胃癌患者的总体生存相关。我们还发现在胃癌的CAFs中，外泌体miR-139负调控MMP11。体外miR-139通过降低体内外MMP11的表达来抑制胃癌细胞的肿瘤生长和转移。因此，我们认为来自胃癌的外体miR-139可