分泌型Syntenin/MDA9在胶质瘤中的生物学功能和临床应用研究

Syntenin, also called MDA9, is an intracellular PDZ domain containing adaptor protein that is ubiquously expressed among various animal species. Syntenin is a multifunctional protein due to its intercalation with various proteins. By in silico analyzing the the Cancer Genome Atlas (TCGA) and REMBRANDT database, we found SDCBP (encoding Syntein protein in humans) is significagtly upregulated in gliomas，especially in the most malignant type--glioblastoma multiforme (GBMs), and its expression is inversely correlated with the survival rate of GBM patients. Our preliminary data showed that Syntenin is indeed overexpressed at both mRNA and protein levels in a collection of GBM specimens and cell lines, and knockdown of Syntein using RNA interference approach dramatically inhibited cell proliferation, while over expression of Syntenin increased GBM cell growth. Furthermore, it was found that Syntenin is able to significantly secreted in multiple glioma cell lines mainly through exosome pathway. Functional experiments showed that the isolated exosomes from Syntenin overexpressing GBM cells is able to stimulate glioma cell proliferation and invasion, and enhance tube formation of the human brain microvascular endothelial cells (HBMEC) in vitro. All together, these results suggested that Syntenin might be a novel diagnosis/prognosis marker and therapeutic target gene in glioblastoma. This project will further investigate the biological function and molecular mechnisms of Syntenin especially its secreted protein in the gliomagenesis and development, and explore its possibility of being a novel therapeutic target and/or a novel marker for glioblastoma diagnosis and prognosis.

Syntenin（又称MDA9）是广泛存在于原核及真核生物中的胞内衔接蛋白(adaptor protein)。胶质母细胞瘤(GBM)是一种最常见的颅内恶性肿瘤。我们的初步实验表明，Syntenin在胶质瘤病人样本和细胞系中异常高表达，且其表达水平与GBM病人的生存率成负相关。RNAi干扰敲除Synteinin可抑制胶质瘤细胞增殖，而Syntenin的过表达可促进细胞生长。我们还发现，在多个胶质瘤细胞系中Syntenin蛋白可通过外泌体（exosome）途径大量分泌到胞外，且从高表达Syntenin的GBM细胞培养基中分离纯化的外泌体可明显促进GBM细胞的增殖和侵袭，并可促进脑血管内皮细胞的血管新生。本项目将在此基础上进一步研究Syntenin特别是其分泌蛋白在GBM发生发展中的生物学功能及分子机制，探讨其作为一个新的胶质瘤生物治疗靶点以及作为临床诊断和预后评价的新指标的可行性。

胶质母细胞瘤（glioblastoma, GBM）是最常见的原发性中枢神经系统恶性肿瘤，具有异质性、浸润生长、易复发、预后差等特点。MDA9参与调控多种肿瘤细胞转移，且在胶质瘤特别是GBM 中异常高表达。我们发现MDA9可在多个胶质瘤细胞系中通过外泌体(exosome) 途径分泌到细胞外，提示分泌型MDA9可能是一个新发现的参与胶质瘤发生、发展和侵袭的靶标蛋白，有望作为临床诊断和治疗的新靶点。本课题重点研究了高表达MDA9后，外泌体分泌水平、生物学功能、蛋白组分的变化，并探索了相关的分子机制。主要结果包括：a.MDA9在胶质瘤细胞系中表达升高 ：MDA9基因的过表达和GBM 病人的生存率成负相关关系。而且MDA9蛋白在胶质瘤病人样本和胶质瘤细胞系中高表达。b.高表达MDA9促进外泌体分泌增多：发现外泌体CD63表达水平与细胞中MDA9表达水平呈正相关。NTA，透射电镜等检测发现高表达MDA9细胞分泌的外泌体直径和数量都有所增加。c.高表达MDA9的外泌体促进胶质瘤细胞的增殖、侵袭及血管发生：高表达MDA9后细胞生长能力显著增强；而高表达MDA9的外泌体较对照外泌体显著促进了野生型细胞的增殖、侵袭及血管形态发生；d.高表达MDA9的外泌体更易于被野生型细胞吞噬 ：通过共聚焦观察及定量检测，发现高表达MDA9的外泌体更易于被野生型细胞吞噬。e.高表达MDA9的外泌体通过Stat3通路发挥生物学功能：高表达MDA9的细胞较对照细胞中p-Stat3表达水平显著升高，且MDA9高表达的外泌体更能促进受体细胞Stat3磷酸化。stat3抑制剂WP1066可以明显抑制MDA9细胞的增殖，以及抑制MDA9-exo引起的受体细胞的侵袭及血管发生。f.相对定量蛋白组学分析：高表达MDA9外泌体及对照外泌体送样质谱检测及蛋白组学分析、GO功能富集及KEGG通路富集等，以期发现外泌体MDA9高表达后发生改变的生物学过程，分子功能，细胞组分及信号通路，以及关键蛋白。通过本项目，我们期望分泌型MDA9可以作为临床诊断胶质母细胞瘤恶性程度的一个指标或治疗靶点。