Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion
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
Affiliations
Helena Sork
Department of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, Sweden
Mariana Conceicao
Department of Paediatrics, University of Oxford, Oxford OX3 9DU, UK
Giulia Corso
Department of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, Sweden
Joel Nordin
Department of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, Sweden
Yi Xin Fiona Lee
Department of Paediatrics, University of Oxford, Oxford OX3 9DU, UK
Kaarel Krjutskov
Competence Centre on Health Technologies, 50 411 Tartu, Estonia
Jakub Orzechowski Westholm
Science for Life Laboratory, Department of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Stockholm University, Solna, Box 1031, SE-171 21 Stockholm, Sweden
Pieter Vader
Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands
Marie Pauwels
Barriers in Inflammation Lab, VIB Center for Inflammation Research, VIB, 9052 Ghent, Belgium
Roosmarijn E. Vandenbroucke
Barriers in Inflammation Lab, VIB Center for Inflammation Research, VIB, 9052 Ghent, Belgium
Matthew JA Wood
Department of Paediatrics, University of Oxford, Oxford OX3 9DU, UK
Samir EL Andaloussi
Department of Laboratory Medicine, Karolinska Institutet, SE-141 52 Huddinge, Sweden
Imre Mäger
Institute of Technology, University of Tartu, 50 411 Tartu, Estonia
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 (R2 = 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.
