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.
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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
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Acknowledgements
We thank the technology platform support staff at the GIGA Research Center. We thank Marie-Alice Meuwis for her support in designing and interpreting the mass spectrometry data. We thank Nicolas Bovy and Marc Thiry for the generation of the electronic microscopy data. This study was supported by the University of Liège (ULiege), the Fonds National de la Recherche Scientifique (FNRS), Télévie and the Fonds Léon Frédéricq. The authors declare that they have no competing interests.
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JPB designed, supervised, conducted and performed experiments and statistical analyses, interpreted the data, and wrote the manuscript. AB performed the experiments with HMVECs, immunofluorescence, fractionation experiments, and exosome characterization. ML performed experiments and provided technical backup. FD participated in data analysis and provided scientific suggestions. IS conceived and designed the study, coordinated the experiments, and wrote the manuscript. All authors read and approved the final manuscript.
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18_2019_3425_MOESM2_ESM.tif
Fig S1: Design of miR-503-biotin and cel-miR-67-biotin duplexes. In black is shown the natural miR-503 and cel-miR-67 sequences and the modifications are indicated in colors: orange for the biotin addition, blue for the 3' tailing, light purple for the phosphorylations and in bold for the mismatch included to de-stabilize the carrier (reverse) strand (TIF 746 kb)
18_2019_3425_MOESM3_ESM.tif
Fig S2: Biotinylation of miR-503 does not prevent the exosomal export of miR-503 triggered by Epirubicin and transfection validations. (a) Cells were transfected with miR-503-biotin. After 24h, cells were treated with Epirubicin and the export of miR-503 was assessed by RT-qPCR in exosomes from treated (green) and untreated (grey) cells. (b) The efficiency of the transfection of the synthetic RNAs was assessed by RT-qPCR in mock and (b) miR-503-bio or (c) cel-miR-67-bio transfected cells. (d) Cells were transfected with cel-miR-67. After 24h, cells were treated with Epirubicin and the exosomal and cellular abundance of cell-miR-67 was assessed by RT-qPCR in treated (green) and untreated (grey) conditions. Data shows mean and SEM of three independent experiments (TIF 821 kb)
18_2019_3425_MOESM4_ESM.tif
Fig S3: cel-miR-67 is not immunoprecipitated with ANXA1 and hnRNPA2B1. HUVECs were transfected with cel-miR-67 (10 nM). 48h later, immunoprecipitation assays were performed using the indicated antibodies or an IgG control (IgG). Then the levels of cel-miR-67 were evaluated by qPCR. Plots show (a) fold change of cel-miR-67 in the immunoprecipitated (IP) vs input fractions (IN) in non-treated conditions and (b) fold change of immunoprecipitated cel-miR-67 in Epirubicin-treated (EPI) vs non-treated cells (NT). Plots represent mean and SEM from three independent experiments,* p<0.05; ** p<0.01, ***p<0.001. IN= 1% of the cellular lysate before immunoprecipitation (TIF 14120 kb)
18_2019_3425_MOESM5_ESM.tif
Fig S4: ANXA2, Thrombspondin-1 (TSP-1) and Vimentin (VIM ) do not re-localize into the nucleus after Epirubicin treatment. (a) Confocal images of ANXA2 (top panels), TSP1 (middle panel) and VIM (lower panels) in green and DAPI in blue of HUVECs treated with Epirubicin for 24h. Pictures taken at the indicated times after starting the treatment. (b) Western blotting for GAPDH (cytoplasm marker) and Histone H3 (nucleus marker) in different subcellular fractions (TIF 17473 kb)
18_2019_3425_MOESM6_ESM.tif
Fig S5: Validation of the knock down of the components of ExoMiR complex. Cells were transfected with siRNAs against the indicated genes or control siRNA (siScr). The levels of (a) mRNA were assessed at 24, 48 and 72h, and the levels of (b) protein were assessed with a 24h delay by Western Blotting. (* p<0.05; ** p<0.01, ***p<0.001) (TIF 14393 kb)
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Pérez-Boza, J., Boeckx, A., Lion, M. et al. hnRNPA2B1 inhibits the exosomal export of miR-503 in endothelial cells. Cell. Mol. Life Sci. 77, 4413–4428 (2020). https://doi.org/10.1007/s00018-019-03425-6
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DOI: https://doi.org/10.1007/s00018-019-03425-6