Endogenous and exogenous extracellular microRNA transfer

Abstract

MicroRNAs are small non-coding RNA molecules that post-transcriptionally regulate gene expression by repressing specific target mRNAs. Although most miRNAs regulate gene expression intracellularly, extracellular miRNAs have been observed in many body fluids. As a result many researchers have focused on understanding the origin and function of extracellular miRNAs. Many studies have reported that extracellular RNAs are transferred between cells and can exert regulatory function, however the biological contexts and mechanisms of miRNA transfer are not well understood. We set out to investigate miRNA transfer in the context of the bone marrow microenvironment and our initial results indicated that there was transfer of miRNAs from monocytic to stromal cells. After further investigation, we determined that these results were artifacts of unbound fluorophore uptake and contamination of cell populations due to inaccurate sorting during flow cytometry-based sorting. These findings indicate that investigators carrying out miRNA transfer studies should exercise caution when relying on fluorescence to track miRNAs, as well as when using flow cytometry-based sorting to separate recipient and donor cells to determine miRNA transfer after co-culture. Until recently, miRNA transfer studies have focused on endogenous transfer within an organism, but a report suggested exogenous miRNAs are acquired from dietary consumption. This was the first report to indicate that there may be cross-kingdom regulation from dietary-derived miRNAs and sparked intense specialist and lay interest. Research groups conducted follow-up studies to verify these results, however most of these reports have conflicting results to the initial study. None of these studies have been definitive due to limitations in the study design or not being done in humans. We designed the first controlled study in humans, where individuals were fed a specific plant-based meal and collected serial blood draws to determine whether dietary-derived miRNAs in the human bloodstream are acquired orally. Through this study we determined that plant miRNAs, ath-miR-156a and hvu-miR-168a, are not detected at any timepoint in the majority of participant serum samples. The maximum serum concentration of plant miRNA observed in any participant is low with only 22,071 copies per uL of serum. However, even in participants with detectable levels above the limit of quantification, absolute concentrations are low and miRNA levels did not replicate in independent analyses. We concluded that dietary plant miRNA absorption is unlikely to occur in healthy individuals. At best plant miRNA absorption into the human bloodstream is highly inefficient and absolute concentrations detected in this study are so low that they are unlikely to regulate endogenous gene expression.