Prospects & Overviews

Recently in press

Beyond nutrients: Food-derived

microRNAs provide cross-kingdom
regulation
Mengxi Jiang1), Xiaolin Sang2) and Zhi Hong2)

Food turns out to be not only the nutrient supplier for Introduction
our body but also a carrier of regulatory information.
Interestingly, a recent study made the discovery that The ecosystem in which we are living is inter-connected:
individual species are not isolated, but are associated with
some plant/food-derived microRNAs (miRNAs) accumu-
each other. There is constant communication between all
late in the serum of humans or plant-feeding animals, and living organisms, as well as between the organisms and their
regulate mammalian gene expression in a sequence- habitat. Cells communicate with each other using common
specific manner. The authors provided striking evidence signaling molecules such as hormones, cytokines, growth
that miRNAs could function as active signaling molecules factors, and second messengers. Recently, secreted
to transport information across distinct species or even microRNAs (miRNAs) have emerged as novel signaling mol-
ecules to mediate intercellular communication [1–3]. miRNAs
kingdoms. Although the mechanism of how miRNAs are
are a class of 19–24-base-long nucleotides derived from the
shuttled between different organisms is still not well primary miRNA hairpin portion of transcripts. They were first
characterized, initial results point to the involvement of characterized as non-coding RNAs from the nematode
microvesicles and specific RNA-transporter-like proteins. Caenorhabditis elegans in 1993 [4, 5]. They are known to
These findings raise both speculation about the potential function in a sequence-specific manner to silence specific
protein-coding genes at the post-transcriptional level by tar-
impact that plants may have on animal physiology at the
geting the 30 untranslated region (30 UTR) of mRNA [6]. Where
molecular level, and an appealing possibility that food- there is extensive complementary base pairing, miRNAs can
derived miRNAs may offer us another means to deliver direct mRNA cleavage; often, where there is only partial
necessary nutrients or therapeutics to our bodies. homology, miRNAs can lead to translational repression and

.
Keywords:
cross-kingdom regulation; microRNA; microvesicle; RNA
transporter
mRNA destabilization [6]. Using the recently developed deep-
sequencing technology, miRNAs have been found in a wide
spectrum of eukaryotes from plants to mammals, including
humans, algae, and viruses [7]. So far, over 15,000 miRNA
gene loci distributed over 140 species have been annotated in
the miRBase database [8]. With a rapid increase in knowledge
accumulated over the last decade, miRNAs are now recognized
as a crucial regulatory element that can manipulate various
DOI 10.1002/bies.201100181 biological processes including cell growth and differentiation,
development, apoptosis, and metabolism [9].
1)
Department of Microbiology and Immunology, University of Michigan In mammals, miRNAs have recently been found in serum,
Medical School, Ann Arbor, MI, USA
2) plasma, urine, saliva, and other body fluids [10–14].
State Key Laboratory of Pharmaceutical Biotechnology, School of Life
Sciences, Nanjing University, Nanjing, China Surprisingly, contrary to traditional beliefs on extracellular
*Corresponding author: RNA stability, biochemical analyses indicate that, in mam-
Zhi Hong mals, these circulating miRNAs are highly stable and resistant
E-mail: [email protected] to RNase activity, as well as extreme pH and temperature [15, 16].
Abbreviations: More importantly, it has been reported that the profiles of
Ago2, argonaute-2; GI, gastrointestinal; LDL, low density lipoprotein;
LDLRAP1, low-density lipoprotein receptor adapter protein 1; MV,
circulating miRNAs, particularly serum miRNAs, are tightly
microvesicle; RISC, RNA-induced silencing complex. correlated with a variety of diseases, including cancer and

280 www.bioessays-journal.com Bioessays 34: 280–284,ß 2012 WILEY Periodicals, Inc.

.... Prospects & Overviews M. Jiang et al.

diabetes, and with tissue injury. This suggests that miRNAs Escherichia coli expressing target gene dsRNA can effectively
could be used as biomarkers to diagnose and monitor human initiate RNA interference (RNAi) on the target mRNA, indicat-
diseases [15–23]. In this review, we focus on the findings of ing that bacteria-derived dsRNA can regulate gene expression

Recently in press
Zhang et al. [24] that secreted miRNAs can function as signal- in a higher organism [35]. In addition to C. elegans, many other
ing molecules in a cross-kingdom manner. species, such as planaria or certain parasitic nematodes, have
the ability to take up environmental dsRNA into their cells [36, 37].
Similarly, feeding insects plants that express dsRNA may also
trigger RNAi [38]. For example, when cotton bollworm larvae
miRNAs are selectively packaged into are fed on plant material expressing dsRNA targeting
microvesicles to transport information CYP6AE14, whose gene product helps the insect to counteract
from cell to cell the deleterious cotton metabolite gossypol, the transcript level
of this gene is decreased and causes larval growth retardation
Despite numerous reports of the detection of secreted miRNAs, [39]. In mammals, miRNAs have been identified in both cow
the exact mechanisms of how these miRNAs are transported milk and human breast milk [13, 14], suggesting a potential
and act as signaling molecules are not clear. They have been route for inter-individual communication via miRNAs.
implicated in stem cell function, hematopoiesis, and immune Another interesting example of miRNA cross-kingdom regu-
regulation [3, 25–28]. Recently, several lines of evidence have lation comes from the discovery that engineered E. coli
suggested that miRNAs are selectively packaged into micro- expressing short hairpin RNA (shRNA) against a mammalian
vesicle (MV) compartments to function efficiently in mamma- gene can induce cross-kingdom RNAi both in vivo and in vitro
lian cells. MVs are membrane-covered vesicles and can be [40]. These findings may provide the groundwork for future
released by various kinds of cells. Based on the mechanism design of RNAi-based therapies.
of formation and the intracellular origin, MVs may be divided Cross-kingdom regulation via miRNA/dsRNA also comes
into shedding vesicles (SVs), which directly bud from the cell from the virus-host interplay. In plants and insects, host
surface, exosomes, which are derived from endosomal mem- cells can process viral dsRNA into small interfering RNAs
branes [29, 30], and apoptotic bodies in response to apoptotic (siRNAs), which can be maintained and amplified later to
stimuli [31]. It is believed that being packed in MVs can help target parasitic RNA [41]. Certain animal cells can encode
miRNAs escape RNase digestion since treatment with deter- their own miRNAs as a defense mechanism to fight off foreign
gents, which destroy the lipid structure of vesicles, can lead to viral genomes [42]. On the other hand, many viruses are
the miRNA degradation [3]. This indicates that the vesicle known to encode suppressor proteins that can counter this
structure is critical for miRNA stability. Some miRNAs are defense with RNA silencing [41]. For example, Lecellier et al.
present at a relatively higher level in MVs than in the parental showed that a cellular miRNA, miR-32, is able to effectively
cells [28, 32]. Furthermore, MVs have been shown to possess inhibit the accumulation of retrovirus primate foamy virus
specific types and levels of miRNAs compared to those found type 1 (PFV-1) in human cells. Tas, a protein encoded by
overall in cells under various conditions, supporting the hy- PFV-1, however, can suppress miR-32-mediated translation
pothesis that the miRNA packaging process is a selective inhibition [43].
rather than a random event. Recently, it was demonstrated
that miRNAs contained in exosomes are released through a
ceramide-dependent secretory machinery, and that these Plant-derived miRNA may regulate
secreted miRNAs can function in recipient cells. For example, mammalian gene expression
a tumor-suppressive miRNA can be transported between cells
via this secretory pathway and can produce cell growth inhi- In a recent publication by Zhang et al. [24], the authors
bition in recipient cells [1]. explored the function of plant-derived miRNAs as active sig-
In addition to MVs, argonaute-2 (Ago2), a protein involved naling molecules to mediate intercellular communication.
in the RNA-induced silencing complex (RISC) [33], and high- They demonstrated that mature single-stranded plant
density lipoprotein (HDL) [34] have recently been proposed to miRNAs are present in both serum and tissues of mammals
act as carriers of miRNAs. These proteins may provide alterna- that use plants such as rice as their food sources. Using state-
tive, non-vesicular means of transporting miRNAs between of-the-art Solexa sequencing and traditional quantitative RT-
cells. PCR, with or without oxidation of small RNAs with periodate,
they were able to verify that the miRNAs detected are bona fide
plant miRNAs [24]. In addition, in a murine model, the authors
tested the hypothesis that the plant miRNAs accumulate in the
miRNA/double-stranded RNA regulates
sera and tissue as a result of food intake. They showed that the
gene expression in a cross-kingdom exogenous mature plant miRNAs in food can pass through
manner mouse gastrointestinal (GI) tract to reach the sera and organs.
Moreover, the authors identified the low-density lipoprotein
Cross-kingdom regulation through miRNA/double-stranded receptor adapter protein 1 (LDLRAP1) as a target for MIR168a, a
RNA (dsRNA) has been observed in many organisms and plant miRNA that was present at a relatively high level in
engineered systems. The uptake of exogenous miRNA/ human sera. The presence of exogenous pre-MIR168a or ma-
dsRNA often leads to an alteration of gene expression in ture MIR168a miRNA can significantly reduce LDLRAP1
the recipient cells. For example, feeding nematode larvae with protein level in culture. Furthermore, feeding mice with rice

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Recently in press M. Jiang et al. Prospects & Overviews ....

Figure 1. A schematic view for the uptake of

food-derived miRNAs into human cells. Food
enters the gastrointestinal (GI) tract, where it is
digested, releasing small pieces of debris and
molecules that human cells can absorb, includ-
ing amino acids, fatty acids, and miRNAs.
Exogenous miRNAs may be taken up into the
epithelial cells lining intestine by SID-like trans-
porters and selectively packaged into MVs.
miRNAs are secreted into the circulatory system
enclosed in MVs, together with components of
the RISC. On reaching the final recipient cells in
organs such as liver, miRNAs are released and
regulate the target gene in a sequence-specific
manner. MV, microvesicle; MVB, multivesicular
body; SID, systemic RNAi deficient; ncRNA,
non-coding RNA; RISC, RNA-induced silencing
complex.

that produces MIR168a reduced the amount of LDLRAP1 transmembrane protein, SID-1 (systemic RNAi deficient-1), is
protein in liver, which in turn resulted in an elevation of believed to form a channel that could allow passive diffusion
the LDL level in mouse plasma. Both the decrease of of dsRNA [45]. The mammalian SID-1 homolog is able to
LDLRAP1 and the increase of LDL in plasma, however, could enhance siRNA uptake [45, 46]. SID-2 is another recently
be blocked by the addition of an anti-MIR168a antisense identified transmembrane protein in C. elegans that is import-
oligonucleotide [24]. These elegantly executed experiments ant for environmental RNAi [47]. In contrast to the ubiquitous
not only confirm the role of circulating miRNAs in intercellular expression of SID-1, SID-2 is expressed in the intestine and is
communication, but also suggest that miRNAs can transport localized to the luminal membrane; it is thought to mediate
and function in a cross-kingdom manner. A schematic sum- the endocytosis of dsRNA from the lumen [47]. The presence of
mary of how food-derived miRNAs can regulate mammalian an RNA transporter protein on the cell surface may also
gene expression is shown in Fig. 1. provide a means for the plant miRNAs to be transported across
For the plant-derived miRNAs to be taken up by the animal the intestinal lumen in mammals. However, there are several
body from food sources and eventually reach the end organs, gaps that need to be filled when trying to identify the potential
they must overcome numerous challenges. First, once in the transporter proteins in mammalian cells for plant miRNAs.
mammalian GI tract, the exogenous miRNAs face many Although SID-1/SID-2 proteins are capable of transporting pre-
extreme conditions such as various enzymes, phagocytosis, miRNAs and single-strand RNA with hairpin structures [47, 48],
and a low pH environment. These unfavorable surroundings whether SID-like proteins are capable of delivering natural
require miRNAs to have stable structures to protect themselves plant pre-miRNAs or mature miRNAs still needs to be eval-
from degradation prior to being taken up into the recipient uated. It is worth noting that there is no direct evidence that
cells. Plant miRNAs are methylated at the 20 -hydroxyl group of mammalian Dicer and Ago2, the two key enzymes for mature
the 30 -terminal nucleotide, which inhibits 30 -end uridylation miRNA biogenesis, can recognize natural plant pre-miRNAs
and subsequent 30 -to-50 exonuclease digestion [44]. Consistent and liberate single-stranded mature miRNAs. However, at
with this, plant miRNAs have a slower degradation rate com- least some of the plant miRNAs are known to be taken up
pared to their synthetic forms [24], suggesting that methyl- by mammalian cells in their mature single-stranded form [24],
ation indeed may contribute to the stability of plant miRNAs raising the possibility that a receptor or transporter gene
in vivo. specific for single-stranded RNA is present on mammalian
Second, how does the plant miRNA pass through the GI cell surface.
tract? One possible mechanism may be that the intestinal The final challenge is how the plant miRNAs get shuttled
epithelial cells can somehow take up plant miRNAs from food, from the initial cells that take up these molecules to the end
albeit the exact trafficking pathway of the plants miRNA from organs such as liver in mammals. Using fluorescently labeled
the gut to these cells is not known. In C. elegans, a multispan MVs and miRNAs, it was demonstrated that miRNAs packaged

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.... Prospects & Overviews M. Jiang et al.

in MVs can be delivered to downstream recipient cells and years. Some researchers are dedicated to isolating the
regulate target genes in these cells [3, 24]. Zhang et al. [24] also active/functional components from herbs; however, these
hypothesized that plant miRNAs in food could be transported components continue to mystify and elude us. The miRNAs

Recently in press
through a similar mechanism: intestinal epithelial cells take that are now being discovered may provide additional clues
up miRNAs, which are then packaged into MVs, to be released for evaluating the active therapeutic components of herbs.
into the circulatory system (Fig. 1). Consistent with this, more The fact that oral ingestion of plant-derived miRNA/sRNA
than half of the MIR168a in the serum was in a MV-packaged can regulate human metabolism and gene expression also
form [24]. raises concerns on transgenic crops. For decades there have
On reaching the mammalian target cells, the plant miRNAs been debates on the safety of transgenic food with regards to
also develop several strategies to guarantee their functions human health and the environment. This profound discovery
within the cells. It turns out that MVs package not only by Zhang et al. [24] should make decision takers more cautious
miRNAs, but possibly also protein components of the RISC when considering the issues that may arise from the consump-
to ensure that packaged miRNAs are active [3, 24]. MIR168a tion of transgenic crops.
was found to be associated with Ago2 in the MVs from human Lastly, if plant miRNAs could act in animal cells, is the
intestinal epithelial cells [24]. In addition, after being deliv- reverse scenario possible? That is, could mammalian miRNA
ered to the target liver cells, MIR168a is still found to be regulate gene expression in a plant cell? Preliminary studies
associated with Ago2 along with its target LDLRAP1 mRNA have shown animal-specific miRNAs can be detected in plant
[24]. These data also suggest that plant miRNAs are able to cells cultured on the medium supplemented with dairy milk
utilize mammalian Ago2 to execute their function. (Prof. Jianqun Chen, personal communication). These pio-
How do plant-derived miRNAs regulate their target mRNAs neering studies may initiate a new level of understanding
in mammalian cells? Whereas plant miRNAs tend to be per- of the co-evolutionary processes existing between the king-
fectly or nearly perfectly complementary to their target doms of life.
sequence and induce mRNA cleavage, mammalian miRNAs
only possess partial homology to targets and often cause
translational repression [49]. The fact that MIR168a decreases Conclusions
the LDLRAP1 protein level, but does not affect the mRNA level,
indicates that it behaves like a mammalian miRNA in liver By studying circulating miRNAs we are realising that these
cells. It would be interesting to analyze other plant miRNA molecules represent a novel class of signaling molecules for
regulation patterns in mammalian cells. intercellular communication. The discovery that plant-specific
miRNAs accumulate in the sera and regulate the gene expres-
sion of human or plant-feeding animals extends the function
Speculations raised by miRNA-mediated of miRNAs. These could represent a cross-kingdom regulatory
cross-kingdom regulation signal. Food intake, possibly via the intestinal epithelia of the
GI tract, may represent a general pathway for ingestion of
With the discovery that the exogenous miRNAs or shRNAs food-derived or food-associated miRNAs. Food-derived
derived from plants or bacteria can regulate gene expression of exogenous miRNAs may also be qualified as a novel nutrient
mammalian cells [24, 40], many issues (some related to our component, like vitamins and minerals, and may provide us
daily lives) are raised. First, what is the scope and degree of with a novel system to deliver therapeutic small RNAs into
food-derived miRNAs on regulation of mammalian genes? animals. However, the exact processes of miRNA transport
Solexa sequencing has revealed that there are approximately through organisms and the mechanism of cross-kingdom gene
30 plant miRNAs in sera from Chinese individuals [24]. What regulation are still unclear. Future studies examining the
are the mammalian targets (other than LDLRAP1) of these trafficking pathway, the miRNA cross-kingdom recognition
miRNAs? Also, what are the potential effects of these food- and regulatory mechanism, and the extent of cross-kingdom
derived miRNAs on our metabolism and health? Initially, due regulation are warranted.
to the complexity of the physiology involved, a panel of organs
and potential targets need to be further examined in animal
models. It would also be interesting to extract the contribution Acknowledgments
of individual miRNAs in regulating mammalian genes, for We would like to thank the members of Hong Laboratory for
example, using engineered bacteria expressing individual helpful discussion. We thank Ross Bohler for critical reading
plant miRNAs. of the manuscript. This work was supported in part by a
Second, this study implies that while we are consuming grant from National Natural Science Foundation of China
food, we not only absorb its nutrients, but also inherit signal- (31070246) to Z.H.
ing and regulatory information from these food sources. This
may have an impact on the future design of genetically engin-
eered plants or identification of active ingredients in certain
plants that have therapeutic effects. In fact, the success of
engineered bacteria expressing an shRNA specifically silenc- References
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