Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion
The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying th...
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doaj-ae8e38fcc6d84a959bd9d3253b00dd472021-07-01T00:34:52ZengMDPI AGCells2073-44092021-06-01101543154310.3390/cells10061543Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular SecretionHelena Sork0Mariana Conceicao1Giulia Corso2Joel Nordin3Yi Xin Fiona Lee4Kaarel Krjutskov5Jakub Orzechowski Westholm6Pieter Vader7Marie Pauwels8Roosmarijn E. Vandenbroucke9Matthew JA Wood10Samir EL Andaloussi11Imre Mäger12Department of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, SwedenDepartment of Paediatrics, University of Oxford, Oxford OX3 9DU, UKDepartment of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, SwedenDepartment of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, SwedenDepartment of Paediatrics, University of Oxford, Oxford OX3 9DU, UKCompetence Centre on Health Technologies, 50 411 Tartu, EstoniaScience for Life Laboratory, Department of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Stockholm University, Solna, Box 1031, SE-171 21 Stockholm, SwedenDepartment of Clinical Chemistry and Haematology, University Medical Center Utrecht, 3584 CX Utrecht, The NetherlandsBarriers in Inflammation Lab, VIB Center for Inflammation Research, VIB, 9052 Ghent, BelgiumBarriers in Inflammation Lab, VIB Center for Inflammation Research, VIB, 9052 Ghent, BelgiumDepartment of Paediatrics, University of Oxford, Oxford OX3 9DU, UKDepartment of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, SwedenInstitute of Technology, University of Tartu, 50 411 Tartu, EstoniaThe extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R<sup>2</sup> = 0.69–0.87), quantitatively an outstanding 96.2–99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.https://www.mdpi.com/2073-4409/10/6/1543extracellular vesiclessmall RNASECextracellular RNAmiRNA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Helena Sork Mariana Conceicao Giulia Corso Joel Nordin Yi Xin Fiona Lee Kaarel Krjutskov Jakub Orzechowski Westholm Pieter Vader Marie Pauwels Roosmarijn E. Vandenbroucke Matthew JA Wood Samir EL Andaloussi Imre Mäger |
spellingShingle |
Helena Sork Mariana Conceicao Giulia Corso Joel Nordin Yi Xin Fiona Lee Kaarel Krjutskov Jakub Orzechowski Westholm Pieter Vader Marie Pauwels Roosmarijn E. Vandenbroucke Matthew JA Wood Samir EL Andaloussi Imre Mäger Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion Cells extracellular vesicles small RNA SEC extracellular RNA miRNA |
author_facet |
Helena Sork Mariana Conceicao Giulia Corso Joel Nordin Yi Xin Fiona Lee Kaarel Krjutskov Jakub Orzechowski Westholm Pieter Vader Marie Pauwels Roosmarijn E. Vandenbroucke Matthew JA Wood Samir EL Andaloussi Imre Mäger |
author_sort |
Helena Sork |
title |
Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion |
title_short |
Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion |
title_full |
Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion |
title_fullStr |
Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion |
title_full_unstemmed |
Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion |
title_sort |
profiling of extracellular small rnas highlights a strong bias towards non-vesicular secretion |
publisher |
MDPI AG |
series |
Cells |
issn |
2073-4409 |
publishDate |
2021-06-01 |
description |
The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R<sup>2</sup> = 0.69–0.87), quantitatively an outstanding 96.2–99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future. |
topic |
extracellular vesicles small RNA SEC extracellular RNA miRNA |
url |
https://www.mdpi.com/2073-4409/10/6/1543 |
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