MiR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles.

Andrew Chin,Nadia Zaffaroni,Sungyong You,Blandine Victor,Javier Mariscal,Edwin Posadas,Muller Fabbri,Michael R Freeman,Elisabetta Broseghini,Keith Syson Chan,Minhyung Kim,Jlenia Guarnerio,Chen Qian,Giorgia Guerra,Dolores Di Vizio,Paolo Gandellini,Shivani Sharma

doi:10.3390/cancers13225850

Abstract

Simple SummaryExtracellular vesicles (EVs) are heterogenous in size, cargo, and mechanism of biogenesis. While the mechanism of formation of small EVs, such as exosomes, has been widely investigated, little is known about the pathobiology of large EVs. We identify here a microRNA that alters cellular vesicologenesis increasing shedding of large EVs and slightly reducing shedding of small EVs. We also demonstrate that this microRNA, miR1227, targets SEC23A to promote this phenotype. Importantly, large EVs are released by cells undergoing a mesenchymal-amoeboid transition that functionally translates into a more metastatic phenotype.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.

Highlights

Extracellular vesicles (EV) exert potent physiologic effects and mediate the intercellular transfer of biomolecules, such as protein and RNA
In addition to the differences in the cells from which they are shed, small EVs (S-EV) and larger (1–10 μm) EVs (L-EV) differ in their biogenesis, and S-EV are generated through multiple mechanisms, including the inward budding of endosomal membranes to generate multi-vesicular bodies, followed by fusion of multi-vesicular bodies to the plasma membrane to release exosomes and direct budding from the plasma membrane to release small ectosomes
Plasma EVs isolated from prostate cancer patients by differential centrifugation contained significantly more miR-1227 than plasma EVs isolated from pooled healthy donors (Figure 1B), indicating an association between the expression of miR-1227 and prostate cancer

Summary

Introduction

Extracellular vesicles (EV) exert potent physiologic effects and mediate the intercellular transfer of biomolecules, such as protein and RNA. The transfer of cancer-associated micro-RNAs (miRNAs) in EVs has attracted great attention, due to the potential for alterations in protein translation in distant cells. Most studies on the role of EVs in cancer have focused on small (~100 nm diameter) EVs, such as exosomes; cells shed a wide range of EV populations, differing in size and cargo, and the functional role of distinct EV populations in cancer is poorly understood [3]. L- and S-EV originate from different molecular pathways and carry distinct cargo [5], suggesting that L- and S-EV should be differentially regulated in diseases, such as cancer. We performed orthogonal studies to identify the direct targets of miR-1227 and demonstrated that miR-1227’s differential alteration on the shedding of L- and S-EV occurs by directly targeting the COPII vesicle protein SEC23A. Our data suggests that this may be, in part, due to alterations in EV shedding

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