TIBS 1289 No.

of Pages 10

Review
Extracellular miRNA: A
Collision of Two Paradigms
Andrey Turchinovich,1,* Alexander G. Tonevitsky,2 and
Barbara Burwinkel1
Since their discovery in 2008, extracellular miRNAs (ex-miRNAs) have persisted
Trends
as one of the major themes of molecular and cellular biology. The main reason
Circulating miRNAs have been
for this remarkable interest is the increasing number of research papers report- detected in diverse extracellular uids,
ing that cell-free circulating miRNA mediates both short-range and distant and are either packaged into different
types of membrane vesicles or persist
communication between various cells, and could impact on diverse physiologi- as solely protein-associated entities.
cal and pathological processes. However, there are also multiple conicting
lines of evidence that challenge the biological signicance of circulating ex- miRNAs secreted via membrane vesi-
cles have been reported to elicit various
miRNA, suggesting that they are merely byproducts of cell activity and cell death physiological responses in acceptor
without any particular function. This review aims to summarize these contrast- cells, while conicting evidence sug-
gests that most extracellular miRNAs
ing opinions and to foster further experimental validation of both paradigms.
could be waste byproducts without
any particular biological function.
The Ex-miRNA Story
Recent reports have demonstrated
miRNAs are a subclass of short non-coding RNA molecules expressed in all eukaryotic cells that selective export of specic miRNAs
regulate gene expression at post-transcriptional and, possibly, transcriptional levels [1,2]. Most to the extracellular space, while the
miRNAs are transcribed by RNA polymerase II as primary pri-miRNA transcripts, and undergo mechanisms underlying their secretion,
further processing by Drosha and Dicer nuclease complexes to produce miRNA duplexes 19 delivery, and action in the acceptor
cells remain poorly understood.
24 bp in length. Finally, one of the two strands in the duplex is loaded onto one of the Argonaute
(AGO) proteins, whereas the other stand is thought to be degraded (Box 1). The action of The ultimate question remaining con-
miRNAs is mostly initiated through their binding to the 3-untranslated region (3UTR) of the cerns the proportion of ex-miRNAs (if
target mRNAs, decreasing target stability and translation efciency [[39_TD$IF]13]. However, emerging any) that participate in cell-to-cell com-
munication, and the cell-, tissue-,
data suggest that miRNAs may also participate in transcriptional gene silencing in the nucleus by organ-, and organism-wide impact of
sequence-specic targeting of epigenetic events on DNA [[40_TD$IF]4,5]. More than 2000 different miRNA ex-miRNAs.
species have been discovered in human, and they have been shown to coordinate key cellular
processes including proliferation, DNA repair, differentiation, metabolism, and apoptosis
[1,2,6,7].

In 2008, several research groups reported the presence of nuclease-resistant extracellular

miRNAs (ex-miRNAs) in human blood serum and plasma [811]. Within the next year, the
existence of ex-miRNA was conrmed in all other biological uids [1215]. Circulating ex-
miRNAs were found to be extremely stable and to survive high RNase concentrations, extreme 1
Molecular Biology of Breast Cancer,
variations in pH, boiling, multiple freezethaw cycles, and extended storage [11,16]. It was University Hospital Heidelberg and
Molecular Epidemiology Group,
originally assumed that the stability of ex-miRNAs is due to their encapsulation into membrane German Cancer Research Center,
vesicles (MVs) because these molecules copurify with fractions containing microvesicles (shed- Heidelberg, Germany
2
ding vesicles), exosomes, and apoptotic bodies [1719]. However, later studies have revealed P. Hertsen Moscow Oncology
Research Institute, National Center of
that, at least in blood plasma and in media collected from common human cell lines, 9599% of Medical Radiological Research,
ex-miRNAs are vesicle-free and instead travel within AGO protein-positive ribonucleoprotein Moscow, Russia
(RNP) particles [16,20]. In human, most if not all intracellular mature miRNAs are associated with
one of the four AGO proteins, therefore this outcome appeared fairly intuitive. Consequently, the
*Correspondence:
remarkable stability of AGO proteins in the extracellular milieu logically explained the nuclease a.turchinovich@dkfz.de
resistance of the associated miRNAs [16]. (A. Turchinovich).

Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tibs.2016.08.004 1

2016 Elsevier Ltd. All rights reserved.
 TIBS 1289 No. of Pages 10

According to the current paradigm, supported by multiple reports, ex-miRNAs can be shielded
from RNase degradation by packaging them into MVs including apoptotic bodies, microvesicles
(shedding vesicles), and exosomes, or by complexing with AGO proteins [21]. Some miRNA
species were also found in puried fractions of high-density lipoprotein (HDL) isolated from
human plasma [22]; however, they might constitute only a very minor proportion[41_TD$IF] of the total
circulating miRNAs [23]. It was hypothesized that protein-free (naked) miRNAs could bind to
HDL liposomes, and possibly other membrane vesicles, through divalent cation bridging [22].
However, in mammalian cells miRNAs are produced and persist only in association with AGO
proteins [2,24], while protein-free miRNAs are extremely unstable in the nuclease-rich environ-
ment [16,20]. It is therefore feasible that miRNAs packaged into the MVs are also associated with
the proteins of AGO family. Indeed, several research groups have previously detected AGO2
protein in fractions of puried extracellular vesicles [25,26]. Finally, although synthetic miRNA
was shown to interact with puried nucleophosmin 1 (NPM1) protein in vitro [27], the association
of NPM1 with endogenous miRNAs was not found in vivo [16].

The biological role of ex-miRNAs, as well as of many other extracellular nucleic acid species,
remains a matter of debate. There are a mounting number of reports demonstrating that some
ex-miRNAs could be specically secreted from parental cells, penetrate acceptor cells, and
trigger functional effects. At the same time, substantial experimental data suggest that most, if
not all, ex-miRNAs are non-specically released from cells upon apoptosis, necrosis, or
secretion activity, and do not carry any particular function. In the following sections of this
article we review the experimental evidence in favor of each theory, and argue that further
validation of these concepts is essential.

CellCell Communication Hypothesis

A revolutionary hypothesis, that extracellular miRNA can mediate cellcell signaling via paracrine
or even endocrine routes, emerged after several research groups found that substantial
amounts of miRNA (together with other RNAs) copuried with extracellular MVs including
microvesicles (shedding vesicles) and exosomes [17,18]. This paradigm was substantially
reinforced by multiple follow-up publications demonstrating that miRNAs entrapped within
various MVs can be transferred to recipient cells, alter gene expression and provoke functional
effects [17,2832]. Moreover, several studies reported that miRNA content in exosomes was
signicantly different from that in parental cells, indicating that ex-miRNAs may be selectively
packed into the extracellular vesicles [17,28,31,3335]; however, a comprehensive mechanism
behind this sorting has not been suggested.

Box 1. miRNA Biogenesis and Function

Almost all mammalian miRNAs are transcribed by RNA polymerase II into long (several kb), capped, and polyA-tailed
primary miRNA (pri-miRNA) molecules [[30_TD$IF]82] (Figure I). Importantly, mammalian miRNA genes can exist either as
independent transcription units or as parts of introns and exons of other genes [[31_TD$IF]83[32_TD$IF]85]. In the cell nucleus, pri-miRNAs
undergo further cleavage by the microprocessor complex consisting of Drosha and DGCR8 proteins, and become 60
70 nt stem-loop precursor miRNAs (pre-miRNA) [[32_TD$IF]86]. Next, pre-miRNAs are actively transported into the cytoplasm by
nuclear exportin-5 protein in a complex with the GTPase Ran [[3_TD$IF]87,88], where they are further cut by the endonuclease
Dicer and the associated RNA-binding protein TRBP [[34_TD$IF]89]. The hydrolysis of pre-miRNAs leads to the formation of 22 nt
double-stranded miRNA duplexes. One of the single-stranded (ss)RNA strands of the duplex is loaded onto one of the
AGO proteins and becomes a mature miRNA, while the other RNA strand is rapidly degraded [[35_TD$IF]90,91]. The complex of
mature miRNA and an AGO protein then forms the key component of the miRNA-induced silencing complex (miRISC).
The miRNA strand in the miRISC serves as a guide for recognizing mRNA molecules with fully or partially complementary
sequences, and ultimately results in degradation or/and inhibition of the translation of targeted mRNAs. Importantly,
when the complementarity between the miRNA and the target mRNA is complete, the mRNA can be cut into two parts by
AGO2 protein directly [[36_TD$IF]90,92]. In the case of imperfect complementarity, specic AGO-associated proteins (e.g., GW182)
within the miRISC inhibit translation and facilitate mRNA degradation mainly via deadenylation and decapping [[37_TD$IF]93,94].

2 Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy

TIBS 1289 No. of Pages 10

miRNA gene

s
RNA Pol ll

leu

sm
Nuc

a
opl
Pri-miRNA

Cyt
DGCR8

Drosha
Cap AAA

Pre-miRNA

Dicer
+

AGO 1-4

Mature miRNA
AGO 1-4

+
RNAse aack

Cap AAAAA

Transcripon RNA degradaon

Figure I. [28_TD$IF]Biogenesis and [29_TD$IF]Function of miRNAs.

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Consistent observations of repression of target mRNAs and the accompanying physiological

effects after treatment of cells with exosome fractions remain the strongest support for the
cellcell communication theory of ex-miRNA [2932,3641]. A prominent recent example
includes the destruction of tight junctions in the epithelial cells of blood vessels and the
subsequent promotion of metastasis by exosomal miR-105 secreted by neighboring cancer
cells [38]. Furthermore, overexpression of miR-105 in non-metastatic cancer cells [42_TD$IF]induced
vascular permeability and metastasis[1_TD$IF], whereas inhibition of miR-105 in highly-metastatic
tumors alleviated these effects [38]. Another recent study strongly supports a paradigm of
cellcell communication via ex-miRNA in immune cells. Specically, two prominent miRNAs
regulators of the immune response, the pro-inammatory miR-155 and the immune-sup-
pressive miR-146a, were present in the extracellular exosomes fractions isolated from
dendritic cell cultures and were taken up by the recipient dendritic cells. Both miR-155
and miR-146a were bound to AGO proteins, downregulated their respective mRNA targets,
and reprogrammed the cellular response to endotoxin challenge [41]. Furthermore, immune
cells interchanged the miR-155 and miR-146a in vivo, and injections of miR-146a-containing
exosomes inhibited the inammatory response to endotoxin in mouse models, while
injections of miR-155-containing exosomes promoted the inammation caused by the
same stimulus.

According to currently prevailing opinion, multivesicular bodies (MVBs) act as centers for miRNA
loading and recycling, and are mainly responsible for miRNA export[43_TD$IF] [26,42]. MVBs are mem-
brane compartments (endosomes) that normally mediate sorting of biomolecules to lysosomes
for degradation, to the plasma membrane for secretion, or back to the Golgi apparatus for reuse.
Exosomes are formed within the MVBs, and fusion of MVBs with the plasma membrane results
in the secretion of exosomes into the extracellular space. Primarily, miRNAs, GW182 protein,
and some amount of cytoplasmic AGO2 protein indeed colocalize with MVBs [26,42]. Second,
selective blocking of MVB turnover pathways increased the amount of AGO-loaded miRNAs and
boosted intracellular miRNA silencing [26,42]. It is logical to assume that some miRNAs may be
randomly incorporated into the exosomes for export. However, deep sequencing of total RNA
isolated from a panel of human B cells and their secreted exosomes revealed non-randomly
distributed subsets of miRNA species between B cells and exosomes[4_TD$IF] [44]. Likewise, many other
reports have indicated that exosomes are composed of a set of miRNAs distinct from that of their
parental cells [26,33,43].

The exact mechanisms responsible for the differential sorting of miRNAs into exosomes remain
poorly understood. Some researchers have identied specic sequence motifs (EXOmotifs)
which were over-represented in ex-miRNAs [43]. It was further shown that the nuclear ribonu-
cleoprotein A2B1 (hnRNPA2B1), a ubiquitously expressed RNA-binding protein, binds to a
specic subset of miRNAs through their EXOmotifs and controls their loading into exosomes
[43]. In addition, post-transcriptional modications of miRNAs could also direct their sorting into
MVs because 3-end adenylated miRNAs were found to be enriched in the cells, while 3-end
uridylated miRNAs were over-represented in the extracellular MVs [44]. Finally, the analysis of
RNA proles in macrophages and their exosomes indicated that miRNA sorting into exosomes
could be modulated by changes in the levels of their mRNA targets in the donor cells taking place
upon cell activation. Thus, genetic interference with the expression of several miRNAs or their
target mRNA transcripts promoted the relocation of miRNAs from the cell cytoplasm to the
MVBs (a place of exosome formation) [45]. Although exosomal miRNA can be selected by
proteins, target mRNAs or post-transcriptional modications [4345], the molecular mecha-
nisms underlying the sorting and secretion of miRNA into microvesicles remains unaddressed. In
the literature, furthermore, microvesicles and exosomes are both frequently referred to as
microvesicles, and it is technically challenging to separate these two subclasses of MVs using
physical methods [21].

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Despite the fact that [45_TD$IF]many research groups have reported that ex-miRNA export follows a
specic pattern[46_TD$IF] [17,28,31,3335,46], several lines of evidence argue strongly that secretion of
most ex-miRNA species occurs in a non-selective manner [16]. These data ignited a conicting
hypothesis that most, if not all, extracellular RNAs are released into biological uids passively
after cell death and do not carry any signicant biological function.

The Hypothesis of Cellular Byproducts

The proponents of the trash theory of ex-miRNAs argue that AGO-associated cell-free miRNAs
are apparently non-specic remnants resulting from cellular physiological activity, apoptosis and
necrosis, and cannot fulll a cellcell communication function [[47_TD$IF]16,47]. Intuitively, in addition to a
process of secretion, extracellular vesicles can be released into the extracellular space after cell
death via membrane blebbing. However, the primary evidence supporting this theory is the fact
that miRNAAGO protein complexes can remain stable for prolonged periods in the nuclease-
rich extracellular environment after the parental cells die [[48_TD$IF]16,47]. Indeed, levels of liver, muscle,
and heart tissue-specic miRNAs in the bloodstream increase upon toxicity in those tissues
[[49_TD$IF]48[50_TD$IF]52]. Likewise, the association of AGO protein-bound miRNAs with exosomes, microve-
sicles, and apoptotic bodies could be explained by either the well-known capacity of RNA-
binding proteins to bind to membranes [[51_TD$IF]53,54] or by the random incorporation of cytosolic
content including nucleic acids during the formation of vesicles. Finally, the hypothesis that some
miRNAs are incorporated into extracellular MVs is so far supported only by the fact that MVs and
particular miRNAs copurify by ultracentrifugation and ultraltration. At the same time, large
(>300 kDa) protein aggregates including AGOmiRNA-loaded RISC could copurify with MVs. It
is feasible that all ex-miRNAs detected so far reside outside extracellular vesicles, raising further
dispute about the putative mechanisms of their export and penetration into target cells.

[52_TD$IF]It is plausible that miRNA-loaded RISC complexes become encapsulated randomly into the
newly formed exosomes[53_TD$IF], and are released into the extracellular environment upon fusion of
MVBs with the plasma membrane. The observed differential distribution of miRNAs in exosomal
fractions could be in turn explained by: (i) different decay kinetics of different miRNAs [[54_TD$IF]55,56], (ii)
preferential loss of particular miRNAs during extraction from samples with very low RNA content
(e.g., extracellular uids) [[5_TD$IF]57], or (iii) the afnity of particular miRNAs for the raft-like region in the
outer layers of the MVB membranes [[56_TD$IF]58].

Another strong argument against [57_TD$IF]a physiological role of ex-miRNA came from the calculations of
the copy number of miRNAs in[58_TD$IF] the extracellular space. Proling of miRNAs in human blood
plasma with next-generation sequencing using spiked-in calibrating synthetic oligonucleotides
allowed the calculation of the total and specic concentrations of circulating miRNA [[59_TD$IF]59]. These
data strongly argue against putative hormone-like effects of ex-miRNA on target tissue because
of the extremely low concentration of miRNAs in circulation. The amount of any single miRNA in
plasma would be only a fraction of the measured hundreds of femtomoles of total miRNA[60_TD$IF] per
litre, but even the most active human hormones act at picomolar dilutions and further amplify
their signals by binding to cell receptors. Importantly, the number of intracellular miRNA
molecules required to signicantly inhibit the expression of a target gene must be on average
greater than 1000 copies per cell [[61_TD$IF]60,61]. Taking into account that total miRNA content in the
blood plasma would correspond to only about 0.060.6 molecules per pl (in the range of 100[62_TD$IF]
1000 fM) and given that [63_TD$IF]the average volume of a human HeLa cell is about 3 pl, the number of
total ex-miRNAs per cell volume would correspond to less than one.

Another recent study has further signicantly challenged the cellcell communication hypothe-
sis. By using a novel nanoparticle tracking analysis (NTA) method to quantify the abundance of
extracellular exosomes, and comparing it to the molarity of accompanying miRNAs, the authors
demonstrated that most individual exosomes in standard preparations do not carry a biologically

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relevant number of miRNAs [[64_TD$IF]62]. Thus, in blood plasma, seminal uid, and conditioned cell
media, the ratio of exosomes to miRNA molecules was signicantly greater than one, and on
average 100 exosomes corresponded to only one copy of a given miRNA. The NTA approach
allowed the precise quantication of extracellular exosomes [[65_TD$IF]62,63], unlike the previously used
method of manual counting of microvesicles after visualization by transmission electron micros-
copy [[6_TD$IF]17,32,64]. The fact that most individual exosomes are devoid of miRNA indicates that they
cannot serve as vectors for ex-miRNA-mediated cellcell communication.

Finally, the physiological signicance of the paracrine or endocrine impact of exosomal miRNA in
the body is challenged by observations that the majority of ex-miRNAs could not be within
extracellular vesicles [[67_TD$IF]16,20,47]. Assuming that only vesicle-encapsulated ex-miRNAs are able
to penetrate the cell membrane (via e.g., membrane fusion), the functional population of ex-
miRNAs should be even smaller.

Other RNA Classes in the Extracellular Space

In addition to miRNA, extracellular MVs contain almost all RNA classes including mRNA,
rRNA, tRNA, snRNA, snoRNA, piRNA, scaRNA, Y RNA, vault RNA, and various long non-
coding RNAs [[68_TD$IF]46,65[69_TD$IF]67], and recent deep-sequencing experiments have shed further light
on intracellular and extracellular distribution of different small RNA species [[69_TD$IF]67]. Thus,
intracellular fractions were clearly enriched (>40%) with miRNAs, while RNAs in the extracel-
lular space had an extremely low (<1%) relative abundance of miRNAs, and much higher
(>60%) percentages of rRNA and tRNA fragments [[69_TD$IF]67]. This distribution could be anticipated
because the authors compared the fraction of low molecular weight cellular RNAs versus
degraded total RNA in the extracellular space; indeed, the proportion of miRNAs in the small
RNA fraction is signicantly higher than in the total RNA fraction of a cell. Furthermore, this
observation clearly suggests that the extracellular environment is infested with degradation
products of total RNA from cells. More importantly, subsequent correlation analysis of miRNA
levels between intra- and extracellular fractions showed that miRNA drop-off occurs in a non-
selective manner [[69_TD$IF]67]. It remains to be addressed whether the cleavage and secretion of other
RNA species, including rRNA, tRNA, and snRNA, either within ribonucleoprotein complexes
or MVs, could be non-random and selective. It is also feasible that those RNAs are protected
(completely or partially) from nuclease degradation by associated proteins and remain in the
extracellular space after cell death.

Areas of Reconciliation
Despite [70_TD$IF]all discrepancies between arguments and[2_TD$IF] conicting experimental data supporting the
two opinions regarding a biological function for extracellular miRNA, both theories could in fact
be true. While the majority of ex-miRNAs could still be non-specic remnants without a particular
biological role, some of them could participate in cellcell communication either via vesicle-
association or via alternative modes (Figure 1). For instance, functional transfer of AGOmiRNA
complexes between cells might not be mediated by only MVs. In addition, the observed
functional impact of extracellular RNA could be also explained by the activation of specic
cell-surface receptors.

Primarily, the observation that AGOmiRNA complexes remain stable in the extracellular
environment does not strictly prove that all of them are non-specically secreted, and/or that
they only released after cell death. Indeed, AGO-bound miRNAs have been shown to traverse
between cells via gap junctions [40]. For example, human macrophages in culture efciently
transferred miR-142 and miR-223 to hepatocarcinoma cells using gap junctions formed of
connexin proteins [40]. Furthermore, transfer of these miRNAs inuenced the post-transcrip-
tional regulation of corresponding target mRNAs in the acceptor carcinoma cells, and function-
ally inhibited cell proliferation. Another study has shown that CXCL12 gene-targeting miRNAs

6 Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy

 TIBS 1289 No. of Pages 10

Donor cell

Outward budding
Mulvesicular
body

Shedding
vesicle

Fusion
AGO

Disrupon of cell membrane

(e.g., cell death)

Acvaon

Toll-like receptor

Gap juncon channel

Exosome ?

AGO
Cap AAAA
Fusion

Transcripon RNA degradaon Acceptor cell

Figure 1. [7_TD$IF]Modes of Extracellular MiRNA Export and Its Putative Impact on Acceptor Cells. Argonaute ([10_TD$IF]AGO)
protein-[1_TD$IF]associated miRNA can be exported into the extracellular environment together with exosomes and shedding
vesicles, or be released non-specically upon disruption of the cell membrane (e.g. after cell death)[12_TD$IF]. [13_TD$IF]It [14_TD$IF]is [15_TD$IF]hypothesized [16_TD$IF]that
extracellular miRNA entrapped within exosomes and shedding vesicles can penetrate into the acceptor cells via [17_TD$IF]direct
[18_TD$IF]membrane [19_TD$IF]fusion, while membrane vesicle-free AGO-bound miRNAs could either pass through gap junction channels, or
[20_TD$IF]be [21_TD$IF]transferred by other yet unknown mechanisms (question mark).[2_TD$IF] Inside the acceptor cell, foreign AGO[23_TD$IF]-miRNA
complexes repress target mRNA molecules, [24_TD$IF]while [25_TD$IF]vesicle-free extracellular miRNA can also activate toll-like receptors
([26_TD$IF]TLRs)[27_TD$IF] at the cell surface and induce TLR pathway-related physiological responses.

were transferred from bone marrow stromal cells to the cocultured breast cancer cells via
connexin channels, which both reduced CXCL12 mRNA expression and cell proliferation [[71_TD$IF]68].
Indeed, the size of the average gap junction pores allows penetration by AGO-bound synthetic
siRNAs of 2030 nt in length, which is in the same size range as endogenous miRNAs [[72_TD$IF]69]. It is
not clear whether gap junctions mediate active and specic secretion of AGOmiRNA com-
plexes. It could be hypothesized that these complexes are also secreted and taken up by
recipient cells via other membrane channels or transporters, including double-stranded (ds)
RNA-selective dsRNA-gated channels which have been consistently described in plants, ies,
Caenorhabditis elegans, and even mammals [[73_TD$IF]70].

Second, even though there is, on average, less than one copy of a miRNA per exosome [[74_TD$IF]41,62],
functional transfer of ex-miRNA may occur via the exosomal route at least for selected miRNAs.
Thus, some researchers have hypothesized that a single cell could still produce a sufcient
number of exosomes to deliver miRNA to recipient cells and mediate target knockdown [41].
Others have suggested that this result can be explained by the existence of different subclasses
of exosomes, only some of which are loaded with miRNAs [[73_TD$IF]70]. A third explanation is that both

Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy 7

 TIBS 1289 No. of Pages 10

exosome-associated and free-oating AGO-bound miRNAs puried from uids or cell culture Outstanding Questions
medium do not reect biological miRNA transfer, which might[3_TD$IF] in fact take place between directly Which circulating miRNAs are selec-
adjacent cells via the paracrine route. Indeed, the paracrine mode of cellcell signaling for ex- tively secreted into the extracellular
space, and what is the percentage of
miRNA appears to be more feasible and, unlike the average ex-miRNA levels in a biological uid, non-specically released miRNAs?
the local concentrations of specic ex-miRNAs could sufce to secure the delivery of physiologi-
cally relevant amounts of miRNA from donor to neighboring acceptor cell. Finally, despite the What are the mechanisms underlying
previous estimates assuming that, on average, the number of miRNA molecules required to the selective export of miRNAs into the
extracellular space?
signicantly suppress a gene expression must be greater than 1000 copies per cell [[75_TD$IF]60,61,71],
the inhibitory miRNA level is also determined by the strength and the number of miRNA binding
Are[4_TD$IF] AGO protein-bound ex-miRNAs
sites, as well as by the copy number of mRNA targets [[76_TD$IF]72]. Because cells may contain many able to penetrate through the mem-
different miRNAs targeting the same mRNA, the effective miRNA concentration could be branes of mammalian cells, and, if
signicantly lower [[7_TD$IF]73]. yes, what mechanisms are involved?

Are the concentrations of MV-associ-

Finally, the recent discovery of an interaction between extracellular miRNA and both cell-surface
ated and protein-bound miRNAs in the
and intracellular Toll-like receptors (TLRs) could provide an alternative explanation for the consis- extracellular space above the physio-
tently reported physiological impact of ex-miRNAs on cells [[78_TD$IF]74,75]. Indeed, for a long time specic logical limit to mediate signicant para-
TLRs have been known to bind pathogen-derived nucleic acids and induce various innate or endocrine signaling in vivo?

immune responses [[79_TD$IF]76[80_TD$IF]78], including inhibition of angiogenesis [[81_TD$IF]79,80]. In their work, Fabbri
How many miRNAs in the total extra-
et al. showed that miR-21 and miR-29a secreted by tumor cells are capable of binding to murine
cellular pool participate in cellcell
TLR-7 and human TLR-8 in immune cells, and trigger the secretion of pro-metastatic inamma- signaling?
tory cytokines that may ultimately enhance tumor growth and metastasis [[82_TD$IF]74,81]. The authors
also concluded that ex-miRNAs could function as key regulators of the tumor microenvironment
by acting as paracrine agonists of TLRs [[83_TD$IF]74]. The recent report by Lehmann and colleagues
provided further evidence in favor of this unconventional role of ex-miRNAs [[84_TD$IF]75]. Specically,
intrathecal injection of extracellular let-7b into the cerebrospinal uid of wild-type mice (but not of
TLR-7 knockout mice) resulted in activation of microglia and neurodegeneration. Furthermore,
susceptibility to let-7b-induced toxicity was restored in neurons transfected with TLR-7.

To summarize, despite multiple lines of experimental evidence in favor of both theories, the
concept of cellcell signaling via ex-miRNA in vertebrates remains to be further validated, and
several important questions need to be addressed (see Outstanding Questions). Finally, recent
massively-parallel sequencing experiments have shown that miRNAs represent only a very minor
fraction as compared to other extracellular RNA species (many of which might also carry
regulatory functions), and this raises further questions regarding the biological signicance of
ex-miRNA and opens new exciting horizons for research.

[5_TD$IF]Acknowledgments
We apologize to those whose work was not cited due to space constraints. Our work was supported by the DietmarHopp
Foundation, the Helmholtz Society, the German Cancer Research Center,[6_TD$IF] Bundesministerium fr Bildung und Forschung
(BMBF) grant 01DJ[85_TD$IF]14001, and the Russian Scientic Foundation Grant 14-44-00051.

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