hnRNPA2B1 inhibits the exosomal export of miR-503 in endothelial cells

Abstract

The chemotherapeutic drug epirubicin increases the exosomal export of miR-503 in endothelial cells. To understand the mechanisms behind this process, we transfected endothelial cells with miR-503 carrying a biotin tag. Then, we pulled-down the proteins interacting with miR-503 and studied their role in microRNA exosomal export. A total of four different binding partners were identified by mass spectrometry and validated by western blotting and negative controls, among them ANXA2 and hnRNPA2B1. Using knock-down systems combined with pull-down analysis, we determined that epirubicin mediates the export of miR-503 by disrupting the interaction between hnRNPA2B1 and miR-503. Then, both ANXA2 and miR-503 are sorted into exosomes while hnRNPA2B1 is relocated into the nucleus. The combination of these processes culminates in the increased export of miR-503. These results suggest, for the first time, that RNA-binding proteins can negatively regulate the exosomal sorting of microRNAs. Keywords Exosomes • EVs • MicroRNAs • Exosomal export • RNA-binding proteins Abbreviations Epi Epirubicin EVs Extracellular vesicles HUVECs Human umbilical vein endothelial cells HMVECs Human microvascular endothelial cells ANXA2 Annexin A2 hnRNPA2B1 Heteronuclear ribonucleoprotein A2/B1 TSP1/thbs1 Thrombospondin 1 SYN Syntenin VIM Vimentin Vinculin Vinculin MEC MicroRNA exporting complex Cellular and Molecular Life Sciences Jennifer Pérez-Boza and Amandine Boeckx contributed equally to the work.

Sorting Mechanisms for MicroRNAs into Extracellular Vesicles and Their Associated Diseases

“…It becomes sumoylated in a similar fashion to hnRNPA2B1, suggesting a potential role of sorting miRNA into EVs [12]. While these collective findings have demonstrated a positive interaction between hnRNPs and miRNA loading into exosomes, other reports have specifically shown a negative interaction between hnRNPA2B1 and miR-503 [13]. Knockdown of hnRNPA2B1 correlated with increased miR-503 levels within exosomes.…”

RNA packaging into extracellular vesicles: An orchestra of RNA‐binding proteins?

“…Among others, N-methyl-N-[3-(3-methyl[1,2,4] formation induced an O-GlcNacylation in hnRNPA2B1, essential for the sorting of AGG and UAG motifs-containing miR-17 and -93 into MVs, guiding hnRNPA2B1 towards MVs rather than exosomes. On the other hand, hnRNPA2B1 was recently identified as an inhibitor of the exosomal sorting of miR-503 in endothelial cells (Pérez-Boza et al, 2020). This group determined that epirubicin increases the exosomal export of miR-503 in endothelial cells by disrupting the interaction between hnRNPA2B1 and the miRNA, probably mediated by ANXA2 (below described).…”

“…The authors demonstrated that, in endothelial cells, the chemotherapeutic drug epirubicin induced the increased exosomal export of miR‐503 by disrupting the interaction between hnRNPA2B1 and miR‐503. They also observed that, upon treatment, hnRNPA2B1 relocates in the nucleus while a fraction of the initial MicroRNA exporting complex (MEC), composed by ANXA2 and miR‐503, is remarkably sorted into exosomes despite the modest variation at intracellular level (Pérez‐Boza et al., 2020).…”

“…On the other hand, hnRNPA2B1 was recently identified as an inhibitor of the exosomal sorting of miR‐503 in endothelial cells (Pérez‐Boza et al., 2020). This group determined that epirubicin increases the exosomal export of miR‐503 in endothelial cells by disrupting the interaction between hnRNPA2B1 and the miRNA, probably mediated by ANXA2 (below described).…”

“…Another study in 2020 reported the interplay between ANXA2 and hnRNPA2B1 as key mediators of the exosomal export of miR‐503 (Pérez‐Boza et al., 2020). The authors demonstrated that, in endothelial cells, the chemotherapeutic drug epirubicin induced the increased exosomal export of miR‐503 by disrupting the interaction between hnRNPA2B1 and miR‐503.…”