In multicellular organisms, the cell-to-cell communication is of particular importance for any physiological process, and the proper organization of the entire organism. Numerous studies over the past years suggest a horizontal transfer of cellular secreted microRNAs between cells, tissues and organs. Hence extracellular RNAs (primarily small non-coding RNAs) represent a novel form of inter-cellular communication by transferring genetic information from a donor cell to a recipient cell. This points to an important new role for small RNAs in inter-cellular communication on the paracrine- and endocrine-level.
Recent papers report a cell-to-cell microRNA exchange and communication via gap junctions.

Small RNAs (primary microRNAs, piRNAs and other small RNA families) can be exported out of the donor cells and transported as free circulating DNA or RNA or by various carriers, e.g. membrane-derived vesicles (exosomes, microvesicles, ectosomes, apoptotic bodies, and more), microRNA-binding protein complexes (RBP), or high density lipoproteins (HDL). Secreted microRNAs can be uptaken and delivered into recipient cells where they function as endogenous microRNAs, simultaneously regulating multiple target genes or signaling pathways.

In prokaryotes, this molecular signaling is typically referred to as quorum sensing, whereas in eukaryotic cells, the molecular communication occurs through hormones and cytokines. Recently various publications report that microRNAs can also be transmitted from one species to another, inducing signal interference in distant species, even in a cross-kingdom manner. This new mode of cross-species communication might mediate symbiotic and pathogenic relationships between various organisms.

This can be of enormous importance in the inter-species communication of microorganisms and their hosts or by diet-derived small RNAs. Higher organisms are constantly under attack from pathogens, resulting in severe consequences on global human or veterinary health. Recently also the uptake of plant microRNA is discussed.
Hence small RNA mediated RNA interference (RNAi) is a conserved regulatory mechanism that is involved in almost all eukaryotic cellular processes, including host immunity and pathogen virulence. Recent evidence supports the significant contribution of small RNAs and RNAi to the communication between hosts and some eukaryotic pathogens or symbiotic microorganisms. Mobile silencing signals - most likely small RNAs - are capable of translocating from the host to its interacting organism, and vice versa.

• Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
• Secreted microRNAs -- a new form of intercellular communication.
• Exosomes -- vesicular carriers for intercellular communication.
• REVIEW -- New roles for microRNAs in cross-species communication.
• Cardiovascular extracellular microRNAs: emerging diagnostic markers and mechanisms of cell-to-cell RNA communication.

• Gap junctional shuttling of miRNA--A novel pathway of intercellular gene regulation and its prospects in clinical application.
• Gap junction mediated miRNA intercellular transfer and gene regulation: A novel mechanism for intercellular genetic communication.

The communication between different organisms:

Exosomes in detail ... and more!

Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).

Cardiovascular diseases are a leading cause of morbidity and mortality in Western societies. It is now well established that microRNAs (miRNAs) are determinant regulators in various medical conditions including cardiovascular diseases. The recent discovery that miRNAs, while associated with different carriers, can be exported out of the cell, has triggered a renewed interest to analyze the potential to use extracellular miRNAs as tools for diagnostic and therapeutic studies. Circulating miRNAs in biological fluids present a technological advantage compared to current diagnostic tools by virtue of their remarkable stability and relative ease of detection rendering them ideal tools for non-invasive and rapid diagnosis. Extracellular miRNAs also represent a novel form of inter-cellular communication by transferring genetic information from a donor cell to a recipient cell. This review briefly summarizes recent insights in the origin, function and diagnostic potential of extracellular miRNAs by focusing on a select number of cardiovascular diseases.

FIGURE: Schematic representation of cellular release (A) and inter-cellular communication (B) of miRNAs.
(A) In the nucleus, miRNA genes are mainly transcribed by the RNA polymerase II (Pol II) into primary miRNAs (pri-miRNAs) and processed to precursor miRNAs (pre-miRNAs) by the Drosha complex. Pre-miRNAs are exported to the cytoplasm and cleaved by Dicer to produce a double stranded miRNA duplex. The duplex is separated and a mature miRNA is incorporated into the RNA-induced silencing complex (RISC) while the other strand is likely subject to degradation. Within the RISC complex, miRNAs bind to their target messenger RNAs (mRNAs) to repress their translation or induce their degradation. In addition, miRNAs can be exported out of the cells and transported by various carriers, membrane-derived vesicles (exosomes, microvesicles, apoptotic bodies), miRNA-binding protein complexes (RBP), or high density lipoproteins (HDL).
(B) Extracellular miRNAs can be transferred to recipient cells where they alter gene expression.

Intercellular genetic communication is an essential requirement for coordination of cell proliferation and differentiation and has an important role in many cellular processes. Gap junction channels possess large pore allowing passage of ions and small molecules between cells. MicroRNAs (miRNAs) are small regulatory RNAs that can regulate gene expression broadly. Here, we report that miRNAs can pass through gap junction channels in a connexin-dependent manner. Connexin43 (Cx43) had higher permeability, whereas Cx30 showed little permeability to miRNAs. In the tested connexin cell lines, the permeability to miRNAs demonstrated: Cx43 > Cx26/30 > Cx26 > Cx31 > Cx30 = Cx-null. However, consistent with a uniform structure of miRNAs, there was no significant difference in permeability to different miRNAs. The passage is efficient; the miRNA level in the recipient cells could be up to 30% of the donor level. Moreover, the transferred miRNA is functional and could regulate gene expression in neighboring cells. Connexin mutation and gap junctional blockers could eliminate this miRNA intercellular transfer and gene regulation. These data reveal a novel mechanism for intercellular genetic communication. Given that connexin expression is cell-specific, this connexin-dependent, miRNA intercellular genetic communication may play an important role in synchronizing and coordinating proliferation and differentiation of specific cell types during multicellular organ development.

microRNAs (miRNAs) have been identified as a fine-tuner in a wide array of biological processes, including development, organogenesis, metabolism, and homeostasis. Deregulation of miRNAs causes diseases, especially cancer. This occurs through a variety of mechanisms, such as genetic alterations, epigenetic regulation, or altered expression of transcription factors, which target miRNAs. Recently, it was discovered that extracellular miRNAs circulate in the blood of both healthy and diseased patients. Since RNase is abundant in the bloodstream, most of the secretory miRNAs are contained in apoptotic bodies, microvesicles, and exosomes or bound to the RNA-binding proteins. However, the secretory mechanism and biological function, as well as the significance of extracellular miRNAs, remain largely unclear. In this article, we summarize the latest and most significant discoveries in recent peer-reviewed research on secretory miRNA involvement in many aspects of physiological and pathological conditions, with a special focus on cancer. In addition, we discuss a new aspect of cancer research that is revealed by the emergence of "secretory miRNA."

CONCLUSIONS: Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.

BACKGROUND: MicroRNAs (miRNAs) are small, noncoding RNAs that play an important role in regulating various biological processes through their interaction with cellular messenger RNAs. Extracellular miRNAs in serum, plasma, saliva, and urine have recently been shown to be associated with various pathological conditions including cancer.

METHODS: With the goal of assessing the distribution of miRNAs and demonstrating the potential use of miRNAs as biomarkers, we examined the presence of miRNAs in 12 human body fluids and urine samples from women in different stages of pregnancy or patients with different urothelial cancers. Using quantitative PCR, we conducted a global survey of the miRNA distribution in these fluids.

RESULTS: miRNAs were present in all fluids tested and showed distinct compositions in different fluid types. Several of the highly abundant miRNAs in these fluids were common among multiple fluid types, and some of the miRNAs were enriched in specific fluids. We also observed distinct miRNA patterns in the urine samples obtained from individuals with different physiopathological conditions.

MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

Cancer-cell communication is an important and complex process, achieved through a diversity of mechanisms that allows tumor cells to mold and influence their environment. In recent years, evidence has accumulated indicating that cells communicate via the release and delivery of microRNAs (miRNAs) packed into tumor-released (TR) exosomes. Understanding the role and mode of action of miRNAs from TR exosomes is of paramount importance in the field of cancer biomarker discovery and for the development of new biomedical applications for cancer therapeutics. In this review, we focus on miRNAs secreted via TR exosomes, which by acting in a paracrine or endocrine manner, facilitate a diversity of signaling mechanisms between cancer cells. We address their contribution as signaling molecules, to the establishment, maintenance, and enhancement of the tumor microenvironment and the metastatic niche in cancer. Finally, we address the potential role of these molecules as biomarkers in cancer diagnosis and prognosis and their impact as a biomedical tool in cancer therapeutics.

CONCLUSIONS: In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.

Anticancer treatments aiming at killing malignant cells have been applied for decades but have been unsuccessful at curing the disease. The modern concept of tumor microenvironment, especially angiogenesis, suggests that the tumor is not only composed of malignant cells, but also consists of other groups of cells that work together. Recently, genetic message transfer has been revealed between tumor cells and their microenvironment. The latest cell-derived vector, extracellular membrane microvesicles (EMVs), has been found to provide membrane protection and allowed to deliver genetic information beyond the cells. Additionally, EMV-associated microRNAs are involved in a variety of cellular pathways for tumor initiation and progression. Previous published reviews have focused on miRNA that included EMVs as a sensitive marker for tumor monitoring in clinical applications that are based on the alteration of their expression levels in conjunction with disease occurrence and progression. From the aspect of cellular crosstalk, this article will review the role of EMV-mediated microRNA transfer in tumor pathogenesis, including tumor treatment obstacles, history and features, and current research in inflammatory/immune pathologies, as well as in solid tumors and hematological malignancies. This nascent crosstalk model will provide a novel insight into complementing the classic mechanisms of intercellular communication and contribute to the potential therapeutic strategy via small RNA molecule-carrying EMVs for multimodality treatment of cancer.

Communication between cells ensures coordination of behavior. In prokaryotes, this signaling is usually referred to as quorum sensing, while eukaryotic cells communicate through hormones. In recent years, a growing number of reports have shown that small signaling molecules produced by organisms from different kingdoms of nature can facilitate cross-talk, communication, or signal interference. This trans-kingdom communication (also termed as trans-kingdom signaling or inter-kingdom signaling) mediates symbiotic and pathogenic relationships between various organisms (e.g., microorganisms and their hosts). Strikingly, it has been discovered that microRNAs (miRNAs)--single-stranded noncoding RNAs with an average length of 22 nt--can be transmitted from one species to another, inducing posttranscriptional gene silencing in distant species, even in a cross-kingdom fashion. Here, we discuss several recent studies concerning miRNA-mediated cross-kingdom gene regulation.

Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, participate in intercellular communication, and particularly, in paracrine and endocrine signalling. The EVs and their specific contents have been considered hallmarks of different diseases. It has been recently discovered that EVs can co-transport nucleic acids such as DNAs, ribosomal RNAs, circular RNAs (circRNAs), long noncoding RNAs (lnRNAs) and microRNAs (miRNAs). miRNAs are important regulators of gene expression at the post-transcriptional level, although they may also play other roles. Recent evidence supports the hypothesis that miRNAs can activate Toll-like receptors (TLRs) under certain circumstances. TLRs belong to a multigene family of immune system receptors and have been recently described in the nervous system. In the immune system, TLRs are important for the recognition of the invading microorganisms, whereas in the nervous system, they recognise endogenous ligands released by undifferentiated or necrotic/injured cells. In the neuronal disease field, TLRs activity has been associated with amyotrophic lateral sclerosis (ALS), stroke, Alzheimer's and Parkinson's disease. Herein, we reviewed the current knowledge of the relationship between miRNA release by EVs and the inflammation signalling triggered by TLRs in neighbouring cells or during long-distance cell-to-cell communication. We highlight novel aspects of this communication mechanism, offering a valuable insight into such pathways in health and disease.

The role of circulating free microRNAs (cfmiRNAs) as promising tools for cancer screening, prognosis and monitoring of anticancer therapies has been widely studied in the past decades. cfmiRNAs have all the characteristics of the perfect biomarkers owing high stability under storage and handling conditions and being detectable not only in plasma, but in almost all body fluids. Moreover, their levels in plasma are likely to resemble ones in the primary tumor. Recently, viral and plant miRNAs have been found in plasma of healthy individuals through deep sequencing technique, and subsequently the same ones were deregulated in patients. Growing body of literature is recently focusing on understanding the potential cross-kingdom regulation of human mRNAs by miRNAs most likely absorbed with food ingestion. In this article we will review the literature concerning the xenomiRs detected in plasma and their role in influencing cancer onset and progression. XenomiRs could potentially be used not only as early screening tool, but also for patients' prognosis.

A new class of RNA regulatory genes known as microRNAs (miRNAs) has been found to introduce a whole new layer of gene regulation in eukaryotes. The intensive studies of the past several years have demonstrated that miRNAs are not only found intracellularly, but are also detectable outside cells, including in various body fluids (e.g. serum, plasma, saliva, urine and milk). This phenomenon raises questions about the biological function of such extracellular miRNAs. Substantial amounts of extracellular miRNAs are enclosed in small membranous vesicles (e.g. exosomes, shedding vesicles and apoptotic bodies) or packaged with RNA-binding proteins (e.g. high-density lipoprotein, Argonaute 2 and nucleophosmin 1). These miRNAs may function as secreted signaling molecules to influence the recipient cell phenotypes. Furthermore, secreted extracellular miRNAs may reflect molecular changes in the cells from which they are derived and can therefore potentially serve as diagnostic indicators of disease. Several studies also point to the potential application of siRNA/miRNA delivery as a new therapeutic strategy for treating diseases. In this review, we summarize what is known about the mechanism of miRNA secretion. In addition, we describe the pathophysiological roles of secreted miRNAs and their clinical potential as diagnostic biomarkers and therapeutic drugs. We believe that miRNA transfer between cells will have a significant impact on biological research in the coming years.

In prokaryotes, small noncoding RNAs (snRNAs) of 50-500 nt are produced that are important in bacterial virulence and response to environmental stimuli. Here, we identified and characterized snRNAs from the endosymbiotic bacteria, Wolbachia, which are widespread in invertebrates and cause reproductive manipulations. Most importantly, some strains of Wolbachia inhibit replication of several vector-borne pathogens in insects. We demonstrate that two abundant snRNAs, WsnRNA-46 and WsnRNA-49, are expressed in Wolbachia from noncoding RNA transcripts that contain precursors with stem-loop structures. WsnRNAs were detected in Aedes aegypti mosquitoes infected with the wMelPop-CLA strain of Wolbachia and in Drosophila melanogaster and Drosophila simulans infected with wMelPop and wAu strains, respectively, indicating that the WsnRNAs are conserved across species and strains. In addition, we show that the WsnRNAs may potentially regulate host genes and Wolbachia genes. Our findings provide evidence for the production of functional snRNAs by Wolbachia that play roles in cross-kingdom communication between the endosymbiont and the host.

By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems.

MicroRNAs (miRNAs) are small noncoding RNA gene products that commonly regulate mRNA expression by repression of translation and/or transcript decay. Whereas common and unique types of miRNAs are expressed by the placenta during pregnancy, the functions of most placental miRNA species are unknown. In addition to their intracellular silencing function, miRNAs are also released to the extracellular space and systemic circulation, where they can potentially target cells to regulate mRNA and protein expression, providing a non-hormonal means of intercellular communication that contributes to tissue homeostasis and disease pathophysiology. This review centers on extracellular miRNAs that originate in trophoblasts and that could mediate crosstalk between the feto-placental unit and the mother during pregnancy. We specifically detail the function of miRNAs from the primate-specific chromosome 19 miRNA cluster. These miRNAs are highly expressed in human placentas and in the serum of pregnant women. They are also packaged into extracellular vesicles of diverse sizes, including exosomes, and endow non-trophoblastic cells with resistance to a variety of viruses.

A newly uncovered means of communication between cells involves intercellular transfer of nano-sized extracellular vesicles (EV), composed of lipids, proteins, and genetic material. EV released by cells of the immune system can play a regulatory role in the induction and suppression of immune responses. These functions may be mediated not only by the bioactive lipids and proteins present in EV but also by EV-associated RNAs. The RNA in EV mainly consists of microRNAs and a large range of other small non-coding RNA species. Since many of these small RNAs have the potential to regulate gene expression, intercellular transfer of these RNAs via EV may cause long-term changes in the function of EV-targeted cells. Several types of innate immune cells release EV that affect innate immune responses and other (patho)physiological processes. Additionally, the innate immune system is influenced by EV released by non-immune cells and EV found in body fluids. In this review, we focus on how EV-associated RNAs contribute to these immune regulatory processes.

Humans, animals, and plants are constantly under attack from pathogens and pests, resulting in severe consequences on global human health and crop production. Small RNA (sRNA)-mediated RNA interference (RNAi) is a conserved regulatory mechanism that is involved in almost all eukaryotic cellular processes, including host immunity and pathogen virulence. Recent evidence supports the significant contribution of sRNAs and RNAi to the communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. Mobile silencing signals—most likely sRNAs—are capable of translocating from the host to its interacting organism, and vice versa. In this review, we will provide an overview of sRNA communications between different kingdoms, with a primary focus on the advances in plant-pathogen interaction systems.

Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals.

Comments:

• Reply to Lack of detectable oral bioavailability of plant microRNAs after feeding in mice.   [Nat Biotechnol. 2013]
• Lack of detectable oral bioavailability of plant microRNAs after feeding in mice.   [Nat Biotechnol. 2013]
• Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: underestimated effects of contamination in NGS.  [RNA. 2014]

The detection of exogenous plant microRNAs in human/animal plasma/sera lies at the foundation of exploring their cross-kingdom regulatory functions. It is necessary to establish a standard operation procedure to promote study in this nascent field. In this study, 18 plant miRNAs were assessed in watermelon juice and mixed fruits by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). CT values, no-template controls and standard curves for each miRNA were used to evaluate the specificity and sensitivity of qRT-PCR and to obtain concentrations. Sixteen miRNAs were selected and measured in human plasma from volunteers after drinking juice. The CT values of 6 plant miRNAs in human plasma fell outside the linear ranges of their standard curves. The remaining 10 miRNAs were present at high basal levels, and 6 of them showed a dynamic physiological pattern in plasma (absorption rates of 0.04% to 1.31%). Northern blotting was used to confirm the qRT-PCR results. Critical issues such as RNA extraction and internal controls were also addressed.

Evidence that exogenous dietary miRNAs enter the bloodstream and tissues of ingesting animals has been accompanied by an indication that at least one plant miRNA, miR168, participates in "cross-kingdom" regulation of a mammalian transcript. If confirmed, these findings would support investigation of miRNA-based dietary interventions in disease. Here, blood was obtained pre- and post-prandially (1, 4, 12 h) from pigtailed macaques that received a miRNA-rich plant-based substance. Plant and endogenous miRNAs were measured by RT-qPCR. Although low-level amplification was observed for some plant miRNA assays, amplification was variable and possibly non-specific, as suggested by droplet digital PCR. A consistent response to dietary intake was not observed. While our results do not support general and consistent uptake of dietary plant miRNAs, additional studies are needed to establish whether or not plant or animal xenomiRs are transferred across the gut in sufficient quantity to regulate endogenous genes.

Food turns out to be not only the nutrient supplier for our body but also a carrier of regulatory information. Interestingly, a recent study made the discovery that some plant/food-derived microRNAs (miRNAs) accumulate in the serum of humans or plant-feeding animals, and regulate mammalian gene expression in a sequence-specific manner. The authors provided striking evidence that miRNAs could function as active signaling molecules to transport information across distinct species or even kingdoms. Although the mechanism of how miRNAs are shuttled between different organisms is still not well characterized, initial results point to the involvement of microvesicles and specific RNA-transporter-like proteins. These findings raise both speculation about the potential impact that plants may have on animal physiology at the molecular level, and an appealing possibility that food-derived miRNAs may offer us another means to deliver necessary nutrients or therapeutics to our bodies.

If validated, diet-derived foreign microRNA absorption and function in consuming vertebrates would drastically alter our understanding of nutrition and ecology. RNA interference (RNAi) mechanisms of Caenorhabditis elegans are enhanced by uptake of environmental RNA and amplification and systemic distribution of RNAi effectors. Therapeutic exploitation of RNAi in treating human disease is difficult because these accessory processes are absent or diminished in most animals. A recent report challenged multiple paradigms, suggesting that ingested microRNAs (miRNAs) are transferred to blood, accumulate in tissues, and exert canonical regulation of endogenous transcripts. Independent replication of these findings has been elusive, and multiple disconfirmatory findings have been published. In the face of mounting negative results, any additional positive reports must provide the proverbial "extraordinary proof" to support such claims. In this article, we review the evidence for and against a significant role for dietary miRNAs in influencing gene expression, and make recommendations for future studies.
Also watch the Video Abstract.

Both pleiotropic and ubiquitous, microRNAs (miRNAs) exert control over a wide range of cellular functions. They have been detected in virtually every extracellular fluid in the mammalian body, and many circulate substantial anatomical distances in plasma. Thus, secreted miRNAs are valuable not only as diagnostic tools but also may serve as novel biological effectors that can be transmitted between source and recipient tissue.
Design: This review will discuss the possibility of delivering functional miRNAs from exogenously derived dietary sources. We will examine prior research interrogating the existence and relevance of such a mechanism. Findings: Recent findings have reported cross-kingdom transfer of specific plant-derived miRNAs to mammalian tissue following consumption of plant-based foods. These exogenous miRNAs were reported to be active in the recipient organisms, directing changes in gene expression at distant organ sites. In spite of this, subsequent studies have been unable to find evidence of substantial exogenous diet-derived miRNAs in mammalian circulation or tissues, regardless of diet.
Conclusion: Further examination of diet-derived miRNA uptake is ongoing, but it does not appear that horizontal delivery of miRNAs via normal dietary intake is a generalizable or frequent process to maintain robust expression of these miRNAs in most higher-order animal organisms.

RESULTS: In this paper, we predicted human and plant target genes using RNAhybrid and set some criteria to further filter them. Then we built the cross-species regulatory network according to the filtered targets, extracted central nodes by PageRank algorithm and built core modules. We summarized the functions of these modules to three major categories: ion transport, metabolic process and stress response, and especially some target genes are highly related to ion transport, polysaccharides and the lipid metabolic process. Through functional analysis, we found that human and plants have similar functions such as ion transport and stress response, so our study also indicates the existence of a close link between exogenous plant miRNA targets and digestive/urinary organs.

Genetic information is traditionally thought to be transferred from parents to offspring. However, there is evidence indicating that gene transfer can also occur from microbes to higher species, such as plants, invertebrates, and vertebrates. This horizontal transfer can be carried out by small RNAs (sRNAs). sRNAs have been recently reported to move across kingdoms as mobile signals, spreading silencing information toward targeted genes. sRNAs, especially microRNAs (miRNAs) and small interfering RNAs (siRNAs), are non-coding molecules that control gene expression at the transcriptional or post-transcriptional level. Some sRNAs act in a cross-kingdom manner between animals and their parasites, but little is known about such sRNAs associated with plants. In this report, we provide a brief introduction to miRNAs that are transferred from plants to mammals/viruses and siRNAs that are transferred from microbes to plants. Both miRNAs and siRNAs can exert corresponding functions in the target organisms. Additionally, we provide information concerning a host-induced gene silencing system as a potential application that utilizes the transgenic trafficking of RNA molecules to silence the genes of interacting organisms. Moreover, we lay out the controversial views regarding cross-kingdom miRNAs and call for better methodology and experimental design to confirm this unique function of miRNAs.

Since a plant miRNA (miR168) cross-regulating a mammalian transcript was reported, miRNA-mediated cross-kingdom communication has become one of the most compelling but controversial topics. In the present study, we used silkworm and mulberry, which is a model for studies on the interactions between the insect and its host plant, to address whether miRNA-mediated cross-kingdom communication is a common phenomenon. The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues. Synthetic miR166b was also detected in hemolymph and fat body. However, the ingestion of synthetic miR166b did not play roles in silkworm physiological progress, which was revealed by RNA-seq analyses, RT-PCR, and phenotypic investigations. Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable. The results provided a new aspect of cross-kingdom miRNA transfer.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as efficient post-transcriptional regulators of gene expression. In 2012, the first cross-kingdom miRNA-based interaction had been evidenced, demonstrating that exogenous miRNAs act in a manner of mammalian functional miRNAs. Starting from this evidence, we defined the concept of cross-kingdom functional homology between plant and mammalian miRNAs as a needful requirement for vegetal miRNA to explicit a regulation mechanism into the host mammalian cell, comparable to the endogenous one. Then, we proposed a new dedicated algorithm to compare plant and mammalian miRNAs, searching for functional sequence homologies between them, and we developed a web software called MirCompare. We also predicted human genes regulated by the selected plant miRNAs, and we determined the role of exogenous miRNAs in the perturbation of intracellular interaction networks. Finally, as already performed by Pirrò and coworkers, the ability of MirCompare to select plant miRNAs with functional homologies with mammalian ones has been experimentally confirmed by evaluating the ability of mol-miR168a to downregulate the protein expression of SIRT1, when its mimic is transfected into human hepatoma cell line G2 (HEPG2) cells.