Exosomal miR-9 inhibits angiogenesis by targeting MDK and regulating PDK/AKT pathway in nasopharyngeal carcinoma.
Exosomal miR-9 inhibits angiogenesis by targeting MDK and regulating PDK/AKT pathway in nasopharyngeal carcinoma.
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外泌体 miR-9 通过靶向 MDK 并调节 PDK/AKT 通路抑制鼻咽癌血管生成。
DOI：
10.1186/s13046-018-0814-3
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发表时间：
2018-07-13
期刊：
影响因子：
--
通讯作者：
Li XP
中科院分区：
文献类型：
--
作者：
Lu J;Liu QH;Wang F;Tan JJ;Deng YQ;Peng XH;Liu X;Zhang B;Xu X;Li XP
外泌体是一种含有多种功能蛋白质、mRNA和miRNA的小囊泡。来自癌细胞的外泌体miRNA在介导细胞-细胞通讯和肿瘤-微环境串扰中起关键作用，特别是在实现转移和促进血管生成中。我们重点研究了先前被确定为鼻咽癌（NPC）肿瘤发生中的肿瘤抑制因子的miR-9。采用差速离心、透射电子显微镜和纳米颗粒跟踪分析来分离和鉴定外泌体。采用定量PCR和蛋白质印迹法检测miR-9、pri-miR-9、CD 63、TSG 101、MDK、P70 S6 K P-Ser 424和PDK 1 P-Ser 241的表达。使用激光共聚焦显微镜追踪NPC细胞分泌到HUVECs中的外泌体miR-9。分别采用迁移实验、微管形成实验和基质胶塞实验检测exosomal miR-9对HUVECs迁移和微管形成的影响。利用生物信息学分析和荧光素酶报告基因分析证实exosomal miR-9与MDK 3′非翻译区（3′-UTR）的结合，同时利用磷酸化阵列分析鉴定exosomal miR-9处理的HUVECs中的AKT通路。采用免疫组化（IHC）和原位杂交（ISH）方法检测鼻咽癌组织中miR-9、CD 31和MDK的表达。用过表达miR-9的慢病毒转染的NPC细胞在外泌体中释放miR-9。外泌体miR-9直接抑制内皮细胞中其靶基因MDK。机制分析显示，鼻咽癌细胞外泌体miR-9通过靶向MDK和调节PDK/AKT信号通路抑制内皮管形成和迁移。此外，MDK水平在NPC肿瘤样品中上调，并且与微血管密度呈正相关。值得注意的是，外泌体miR-9水平与总生存率正相关，MDK过表达与NPC患者的不良预后正相关，提示外泌体miR-9和MDK的临床相关性和预后价值。总之，我们的数据确定了肿瘤来源的、外泌体相关的miR-9在NPC肿瘤发生中的细胞外抗血管生成作用，并促使进一步研究基于外泌体的癌症治疗疗法。本文的在线版本（10.1186/s13046-018-0814-3）包含补充材料，可供授权用户使用。
Exosomes are small vesicles containing a wide range of functional proteins, mRNA and miRNA. Exosomal miRNAs from cancer cells play crucial roles in mediating cell-cell communication and tumor-microenvironment cross talk, specifically in enabling metastasis and promoting angiogenesis. We focused on miR-9 that was identified as a tumor suppressor previously in nasopharyngeal carcinoma (NPC) tumorigenesis. Differential centrifugation, transmission electron microscopy and nanoparticle tracking analysis were used to isolate and identify exosomes. Quantitative PCR and western blotting analysis were used to detect miR-9, pri-miR-9, CD63, TSG101, MDK, P70S6K P-Ser424 and PDK1 P-Ser241 expression. Laser confocal microscopy was used to trace exosomal miR-9 secreted by NPC cells into HUVECs. The effect of exosomal miR-9 on cell migration and tube formation of HUVECs in vivo and vitro was assessed by using migration assay, tube formation assay and matrigel plug assay, respectively. Bioinformatics analysis and luciferase reporter assay were utilized to confirm the binding of exosomal miR-9 to the 3′untranslated region (3′-UTR) of MDK, while Phosphorylation Array was performed to identify AKT Pathway in HUVECs treated with exosomal miR-9. Furthermore, Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to detected miR-9, CD31 and MDK expression in human NPC tumor samples. NPC cells transfected with miR-9-overexpressing lentivirus, released miR-9 in exosomes. Exosomal miR-9 directly suppressed its target gene - MDK in endothelial cells. Mechanistic analyses revealed that exosomal miR-9 from NPC cells inhibited endothelial tube formation and migration by targeting MDK and regulating PDK/AKT signaling pathway. Additionally, the level of MDK was upregulated in NPC tumor samples and was positively correlated with microvessel density. Notably, the level of exosomal miR-9 was positively correlated with overall survival, and MDK overexpression was positively associated with poor prognosis in NPC patients, suggesting the clinical relevance and prognostic value of exosomal miR-9 and MDK. Taken together, our data identify an extracellular anti-angiogenic role for tumor-derived, exosome-associated miR-9 in NPC tumorigenesis and prompt further investigation into exosome-based therapies for cancer treatment. The online version of this article (10.1186/s13046-018-0814-3) contains supplementary material, which is available to authorized users.
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影响因子：
9.8
作者：
Mineo, Marco;Garfield, Susan H.;Taverna, Simona;Flugy, Anna;De Leo, Giacomo;Alessandro, Riccardo;Kohn, Elise C.
通讯作者：
Kohn, Elise C.
影响因子：
15.9
作者：
Roccaro, Aldo M.;Sacco, Antonio;Ghobrial, Irene M.
通讯作者：
Ghobrial, Irene M.
影响因子：
3.3
作者：
Choi, Yong Won;Kim, Young Hwa;Kim, Jang-Hee
通讯作者：
Kim, Jang-Hee
影响因子：
20.3
作者：
Umezu, Tomohiro;Tadokoro, Hiroko;Ohyashiki, Junko H.
通讯作者：
Ohyashiki, Junko H.
影响因子：
21.3
作者：
Skog, Johan;Wuerdinger, Tom;van Rijn, Sjoerd;Meijer, Dimphna H.;Gainche, Laura;Sena-Esteves, Miguel;Curry, William T., Jr.;Carter, Bob S.;Krichevsky, Anna M.;Breakefield, Xandra O.
通讯作者：
Breakefield, Xandra O.