Extracellular miRNAs and Cell–Cell Communication: Problems and Prospects

miRNAs mediate post-transcriptional regulation of gene expression and are also present in all biological fluids as extracellular entities in both vesicular and vesicle-free forms. The EV-associated miRNAs are assumed to mediate intercellular communication between cells; however, the contribution of vesicle-free extracellular miRNAs to this process is unknown. Most of the research reports demonstrating the functional transfer of miRNAs between cells provided insufficient evidence that such transfer was mediated by EVs. The theory of cell–cell communication via extracellular miRNAs is challenged by the fact that the described miRNA-sorting mechanisms into EVs do not involve Argonautes, and the latter are usually not detected in EVs. miRNAs are short RNA molecules regulating multiple cellular processes through post-transcriptional gene silencing. Over the past decade, miRNAs have been found in the extracellular space and have been consistently shown to mediate functional communication between cells. While it remains widely accepted that miRNA transfer between cells occurs via extracellular vesicles (EVs), multiple other carriers of cell-free miRNA have been described. In addition, some studies have demonstrated that both miRNAs and their binding partners, Argonaute proteins, remain hardly detectable in common isolates of EVs. In this Opinion article, we summarize the state-of-the-art mechanisms of miRNA sorting and secretion, discuss methodological challenges associated with extracellular miRNA research, and suggest experimental steps to resolve current inconsistencies in the field of miRNA-mediated cell–cell communication. miRNAs are short RNA molecules regulating multiple cellular processes through post-transcriptional gene silencing. Over the past decade, miRNAs have been found in the extracellular space and have been consistently shown to mediate functional communication between cells. While it remains widely accepted that miRNA transfer between cells occurs via extracellular vesicles (EVs), multiple other carriers of cell-free miRNA have been described. In addition, some studies have demonstrated that both miRNAs and their binding partners, Argonaute proteins, remain hardly detectable in common isolates of EVs. In this Opinion article, we summarize the state-of-the-art mechanisms of miRNA sorting and secretion, discuss methodological challenges associated with extracellular miRNA research, and suggest experimental steps to resolve current inconsistencies in the field of miRNA-mediated cell–cell communication. extracellular membrane vesicles 1000–5000 nm in size containing cytoplasm and tightly packed cellular organelles, which are formed during programmed cell death (apoptosis). essential components of the RISC and direct binding partners of miRNAs and siRNAs. series of sequential centrifugations with increasing speed used to separate organelles and other subcellular particles based on their sedimentation rate. a type of cell-to-cell communication in which a cell produces a signal to induce changes in distant cells. recently discovered extracellular, non-membranous nanoparticles ~35 nm in size with distinct protein and lipid contents. a class of EVs 30–150 nm in size released on the fusion of MVBs with the plasma membrane. lipid-membrane-enclosed particles 30–5000 nm in size produced by virtually all cell types. one of the five major groups of lipoproteins that transport fat molecules around the body. HDLs are the smallest and the densest (5–17 nm). HDLs typically comprise 80–100 proteins per particle and can transfer miRNA between cells. membrane vesicles formed by inward budding into the lumen of MVBs; released as exosomes on MVB fusion with the plasma membrane. a type of EV 50–1000 nm in size that is formed by outward budding of the cell membrane. 16–24-bp double-stranded RNA generated on the cleavage of pre-miRNA with RNase Dicer; comprises a guide ‘miRNA’ and a passenger ‘miRNA*’ strand. a specialized type of endosome that contains membrane-enclosed ILVs. The ILVs, which are formed by inward budding of the MVB membrane, can be released into the extracellular space as exosomes on fusion with the plasma membrane or degrade on fusion with lysosomes. a type of cell-to-cell communication in which a cell produces a signal to induce changes in nearby cells. a 60–70-nucleotide hairpin containing RNA that is carved out by the nuclear RNase Drosha from longer pri-miRNA transcripts. In the cytoplasm, pre-miRNA is further cleaved by the Dicer enzyme into 16–24-bp double-stranded miRNA/miRNA* duplexes. 200–300-nucleotide 5′-capped and 3′-polyadenylated RNA polymerase II transcripts containing a 60–70-nucleotide pre-miRNA hairpin. an array of proteins produced by a cell, which are secreted into the extracellular space. either naturally occurring or artificially synthesized double-stranded RNA molecules (typically 18–24 bp long) that elicit the downregulation of a specific gene via the RNAi pathway.