Chin Andrew, Mariscal Javier, Kim Minhyung, Guerra Giorgia, Victor Blandine, Qian Chen, Broseghini Elisabetta, Posadas Edwin, Freeman Michael R, Sharma Shivani, Gandellini Paolo, Zaffaroni Nadia, You Sungyong, Chan Keith Syson, Guarnerio Jlenia, Fabbri Muller, Di Vizio Dolores
Department of Surgery, Division of Cancer Biology and Therapeutics, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
Cancer Biology Program, University of Hawai'i Cancer Center, Honolulu, HI 96813, USA.
Cancers (Basel). 2021 Nov 22;13(22):5850. doi: 10.3390/cancers13225850.
Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV.
癌细胞会释放出细胞外囊泡（EVs）的异质混合物，其大小和组成各不相同，可能以不同方式影响生理过程。然而，细胞如何差异地控制这些不同类型的细胞外囊泡的释放，目前还知之甚少。在这里，我们发现，与起源细胞相比，富含于前列腺癌细胞外囊泡中的miR-1227，但不存在于源自前列腺良性上皮细胞的细胞外囊泡中，它能诱导大型细胞外囊泡（如大型肿瘤小体）的释放，同时抑制小型细胞外囊泡（如外泌体）的释放。我们使用了从稳定表达miR-1227的细胞中进行RNA测序、一种经过改良的RISCTRAP检测方法（该方法可稳定并纯化用于RNA测序的mRNA-miR-1227复合物）以及计算机靶标预测工具，来鉴定可能介导细胞外囊泡释放这种改变的miR-1227靶标。COPII囊泡蛋白SEC23A被发现，并通过qPCR、蛋白免疫印迹法和荧光素酶测定法验证为miR-1227直接靶标。抑制SEC23A足以诱导大型细胞外囊泡的释放。这些结果确定了一种新的细胞外囊泡释放机制，即miR-1227对SEC23A的抑制会导致细胞外囊泡释放的转变，并有利于大型细胞外囊泡而非小型细胞外囊泡的释放。
