Abstract
AbstractThe discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research, but has left many unanswered questions about how this regulation works and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in blood plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations; and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication^[1-5]^. Such a system would be expected to export specific miRNAs from cells in response to specific biological stimuli. We report here that after serum deprivation several human cell lines tested do export a spectrum of miRNAs into the culture medium. The export response is substantial and prompt. The exported miRNAs are found both within and outside of microvesicles and exosomes. We have identified some candidate protein components of this system outside the cell, and found one exported protein that plays a role in protecting miRNA from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that may involve cell-cell communication.
Highlights
One of the hypotheses advanced to explain the biological significance of the presence of miRNA in the plasma is that they are part of a cell-cell communication system
This biological function would require that the miRNAs convey specific information, and that only certain miRNAs are exported from cells in response to biological stimuli
The Lactate dehydrogenase (LDH) levels in serum free media showed no significant changes during the experimental period, up to 48 hours of serum depletion Finding no detectable evidence of lysis we concluded that the external miRNAs are exported from intact cells, consistent with the observed striking difference between intracellular and extracellular miRNA spectra
Summary
The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research, but has left many unanswered questions about how this regulation works and how it is integrated with other regulatory mechanisms. The LDH levels in serum free media showed no significant changes during the experimental period, up to 48 hours of serum depletion (data not shown.) Finding no detectable evidence of lysis we concluded that the external miRNAs are exported from intact cells, consistent with the observed striking difference between intracellular and extracellular miRNA spectra (see below.). If we compare the changes in levels of certain miRNAs inside and outside the cells, before and after serum deprivation, the rate of export for most of the miRNAs examined increases dramatically during the first 2 hours after SD and declines thereafter (figure 2.) One observation from these data was that some miRNAs exhibited a modest decline in concentration in the medium after increasing strongly in the first few hours (e.g. miR135a, 133b). The elucidation of a novel, and perhaps extensive, biological information transduction system between cells will certainly be of the utmost importance in understanding many biological processes in mammalian systems including development, stress response and tissue renewal
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