Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, 226001, China.
Department of Neurosurgery, Affiliated Hospital of Nantong University, Nantong, China.
Biochem Biophys Res Commun. 2019 Oct 15;518(2):325-330. doi: 10.1016/j.bbrc.2019.08.057. Epub 2019 Aug 14.
Exosomes are a type of extracellular vesicles derived from cells and mediators of intercellular communication. Different cell types have their own unique exosomes for exchanging information. We previously found that SASH1, a tumor suppressor, was lowly expressed or absent in glioma tissues and glioma C6 cells, but the structure and function of the corresponding exosomes had been unclear. Hence, we aimed to investigate whether exosomes generated from normal glial cells and glioma cells form different protein patterns and whether those derived from normal glial cells affect SASH1 expression in glioma cells. We collected exosomes from astrocytes and C6 cells and identified their exosomal proteins through mass spectrometry. We also performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses, whose results showed that both the total and unique exosomal proteins from each cell type were similar. Moreover, the KEGG analysis revealed different clusters of unique exosomal proteins in glial cells and glioma cells. In the normal glial cells, the top clusters were mainly involved in processes with RNA transcripts and proteins, whereas in glioma cells the clusters were attributed to PI3K-Akt signaling, cell adhesion, and cancer-related pathways. Western blot analysis showed that HMGB1 exists in exosomes derived from cultured astrocytes, although its expression was higher in glioma C6 cells. Furthermore, we found that exosomes extracted from astrocytes could increase SASH1 expression in C6 cells (P = 0.040), whereas those derived from HMGB1-depleted astrocytes could not (P = 0.6133). The expression levels of SASH1 decreased after the addition of extracellular recombinant HMGB1 protein, whereas that of TLR4 increased. Our study is the first to demonstrate that HMGB1 plays different roles depending on its form: as an extracellular protein, HMGB1 decreases SASH1 expression, but as an exosomal protein, HMGB1 increases SASH1 expression. Nevertheless, the mechanism, which partly depends on the TLR4 pathway, behind these opposing effects requires further study. Our novel findings on the structure-dependent roles of the cytokine HMGB1 in promoting or inhibiting cancer provide a fresh insight into the interactions of cancer cells with the microenvironment.
外泌体是一种源自细胞的细胞外囊泡，是细胞间通讯的介质。不同的细胞类型有自己独特的外泌体来交换信息。我们之前发现，肿瘤抑制因子 SASH1 在神经胶质瘤组织和神经胶质瘤 C6 细胞中低表达或缺失，但相应外泌体的结构和功能尚不清楚。因此，我们旨在研究正常神经胶质细胞和神经胶质瘤细胞产生的外泌体是否形成不同的蛋白质模式，以及源自正常神经胶质细胞的外泌体是否影响神经胶质瘤细胞中的 SASH1 表达。我们从星形胶质细胞和 C6 细胞中收集外泌体，并通过质谱法鉴定其外泌体蛋白。我们还进行了基因本体论（GO）和京都基因与基因组百科全书（KEGG）分析，结果表明，每种细胞类型的总外泌体蛋白和独特外泌体蛋白都相似。此外，KEGG 分析显示，神经胶质细胞和神经胶质瘤细胞的独特外泌体蛋白簇不同。在正常神经胶质细胞中，主要簇主要涉及 RNA 转录物和蛋白质过程，而在神经胶质瘤细胞中，簇与 PI3K-Akt 信号转导、细胞黏附和癌症相关途径有关。Western blot 分析表明，HMGB1 存在于培养的星形胶质细胞衍生的外泌体中，尽管其在神经胶质瘤 C6 细胞中的表达水平更高。此外，我们发现星形胶质细胞提取的外泌体可以增加 C6 细胞中 SASH1 的表达（P=0.040），而源自 HMGB1 耗尽的星形胶质细胞的外泌体则不能（P=0.6133）。添加细胞外重组 HMGB1 蛋白后 SASH1 的表达水平下降，而 TLR4 的表达水平增加。我们的研究首次证明，HMGB1 因其形式而异发挥不同的作用：作为细胞外蛋白，HMGB1 降低 SASH1 的表达，但作为外泌体蛋白，HMGB1 增加 SASH1 的表达。然而，这些相反作用背后的机制部分依赖于 TLR4 途径，需要进一步研究。我们关于细胞因子 HMGB1 在促进或抑制癌症方面的结构依赖性作用的新发现为深入了解癌细胞与微环境的相互作用提供了新的视角。
