Nutrition, microRNAs, and Human Health^,,,

Abstract

MicroRNAs (miRs) hybridize with complementary sequences in mRNA and silence genes by destabilizing mRNA or preventing translation of mRNA. Over 60% of human protein-coding genes are regulated by miRs, and 1881 high-confidence miRs are encoded in the human genome. Evidence suggests that miRs not only are synthesized endogenously, but also might be obtained from dietary sources, and that food compounds alter the expression of endogenous miR genes. The main food matrices for studies of biological activity of dietary miRs include plant foods and cow milk. Encapsulation of miRs in exosomes and exosome-like particles confers protection against RNA degradation and creates a pathway for intestinal and vascular endothelial transport by endocytosis, as well as delivery to peripheral tissues. Evidence suggests that the amount of miRs absorbed from nutritionally relevant quantities of foods is sufficient to elicit biological effects, and that endogenous synthesis of miRs is insufficient to compensate for dietary miR depletion and rescue wild-type phenotypes. In addition, nutrition alters the expression of endogenous miR genes, thereby compounding the effects of nutrition-miR interactions in gene regulation and disease diagnosis in liquid biopsies. For example, food components and dietary preferences may modulate serum miR profiles that may influence biological processes. The complex crosstalk between nutrition, miRs, and gene targets poses a challenge to gene network analysis and studies of human disease. Novel pipelines and databases have been developed recently, including a dietary miR database for archiving reported miRs in 15 dietary resources. miRs derived from diet and endogenous synthesis have been implicated in physiologic and pathologic conditions, including those linked with nutrition and metabolism. In fact, several miRs are actively regulated in response to overnutrition and tissue inflammation, and are involved in facilitating the development of chronic inflammation by modulating tissue-infiltrated immune cell function.

Introduction

Over 60% of human protein-coding genes are regulated by microRNAs (miRs)⁹, and 1881 high-confidence miRs are encoded in the human genome (1, 2). Loci coding for miRs (miR genes or introns) are usually transcribed by RNA polymerase II to yield primary miRs (3, 4). The endoribonuclease DROSHA liberates hairpins from primary miRs in the cell nucleus. The resulting precursor hairpins, precursor miRs, are exported into the cytoplasm for processing by DICER, which removes the loop joining the 3′ and 5′ arms in precursor miRs to release a miR:miR duplex. Nonconditional knockout of DROSHA and DICER is embryonic lethal in mice (5–7).

Mature miRs are ∼22 nucleotides long, hybridize with complementary sequences in the 3′-untranslated regions in mRNA, and silence genes by destabilizing mRNA or preventing translation of mRNA (8–10). The sequence complementarity in the seed region (nucleotides 2–8) in miRs is of particular importance for binding to target transcripts (11). The miR-dependent degradation of mRNA takes place in the RNA-induced silencing complex. If the miR nucleotide sequence has a high degree of complementarity to the sequence in the mRNA target, both miR and mRNA are degraded (4, 12) (Figure 1). In contrast, if the sequence complementarity is imperfect, the binding of miR to mRNA will halt mRNA translation without causing miR or mRNA degradation (4, 13).

FIGURE 1.

miRs bind to complementary sequences in the 3′-untranslated sequence in mRNA, triggering mRNA degradation (high degree of sequence complementarity) or inhibition of mRNA translation (imperfect complementarity). The 100% compared with 66% complementarity is solely for illustrative purposes. miR, microRNA; UTR, untranslated.

Exosomes, microvesicles, and apoptotic bodies are extracellular vesicles, which are distinguished by size, biogenesis, and cargos (14). Exosomes are of particular biological importance, because their loading with cargos is not a random process but involves sorting mechanisms that favor some cargos over others (15, 16). In addition, exosomes play essential roles in cell-to-cell communication (17–19). Exosomes contain diverse cargos, e.g., various species of RNA, proteins, and lipids (18, 20, 21). Encapsulation is of particular importance for RNAs, because it confers protection against degradation and provides a vehicle for cellular uptake of RNAs by endocytosis (20, 22–27).

Evidence suggests that mature miRs may be obtained from dietary sources in addition to endogenous synthesis, and that endogenous synthesis is altered by bioactive compounds in foods (28, 29). These observations have far-reaching implications for gene regulation by food compounds, the analysis of miR-dependent gene networks with the use of bioinformatics protocols, and studies of nutrition and disease, and were the focus of the “Nutrition, microRNAs, and Human Health” symposium sponsored by ASN (J Zempleni and SA Ross, unpublished results, 2016).

Dietary miRs

In 2012, evidence emerged that miRs may not be obtained exclusively from endogenous synthesis, but may also be absorbed from rice (28). The study attributed changes in LDL-receptor adapter protein 1 expression to dietary (rice) miR -168 in humans and mice. The report was met with initial skepticism (30). However, evidence is accumulating that RNAs from plant sources are bioavailable (19, 31–39). Since the report by Zhang et al. (28), much of the research activity on the field of dietary miRs has shifted away from miRs in plant-borne foods to miRs in foods of animal origin, particularly cow milk. This transition was justified by observations that a large proportion of milk miRs is encapsulated in extracellular vesicles such as exosomes (40, 41). miRs encapsulated in milk exosomes are stable under harsh conditions, such as low pH and exposure to ribonucleases (22), and Americans consumed 195 pounds fluid milk/person in 2012 (42).

A number of studies from Zempleni's laboratory (20, 22–27) refuted the paradigm that miRs are derived exclusively from endogenous synthesis. Strong evidence has been provided that 1) humans absorb biologically effective amounts of miRs from nutritionally relevant doses of cow milk, 2) physiologic concentrations of milk miRs affect human gene expression in vivo and in cell cultures, and 3) endogenous synthesis of miRs does not compensate for dietary miR deficiency in mice (29). This discovery was challenged by Title et al. (43), who detected only trace amounts of milk miR-375 in mouse plasma after milk feeding. The authors acknowledged that “it remains possible that a small level of [microRNA and exosome] uptake does occur” but cautioned that “[i]t is unlikely that milk miRs function through canonical microRNA silencing.” Note that the study disregarded the possibility that, upon intestinal absorption, miR-375 binds to transcript targets in the intestinal mucosa and liver, followed by rapid degradation (44), the classic “first passage elimination” effect, which is consistent with the use of miR “sponges” in miR research (13, 45). Importantly, the laboratory of Randy Shekman reported an miR sequence motif [(A/U)(C_2–4)(A/U)] that is essential for miR packaging into exosomes and is missing in miR-375 (M Shurtleff, unpublished results, 2015). Pathways have been identified that explain how low femtomolar (10⁻¹⁵) concentrations of (dietary) miRs elicit biological effects through binding to Toll-like receptors (TLRs) or by surface antigen-mediated delivery of exosomes to immune cells (19, 46). The discovery that dietary miRs have biological activities in humans has been confirmed by many independent laboratories. Investigators detected numerous dietary nonhuman miRs in 6.8 billion sequencing reads from 528 human samples (R Kitchen, unpublished results, 2015), and identified 50 plant-borne miRs in human plasma (47) with the use of next-generation sequencing (NGS). Consistent with these observations, a miR-like oligonucleotide from honeysuckle was detected in rodent plasma with the use of digital droplet PCR (34). Milk exosomes cross the intestinal mucosa in mice (48), which is consistent with observations that 1) milk exosomes enter intestinal cells by glycoprotein-dependent endocytosis (27), 2) basolateral secretion of miRs is a nucleotide sequence-specific process in humans and rats (27), 3) human vascular endothelial cells transport milk exosomes by endocytosis (49), and 4) fluorophore-tagged milk exosomes accumulate in the liver and spleen after oral administration in mice (48). With the use of NGS, bovine-specific bta-miR-181, -150, -378, 380–3p, and -1839 were detected in human plasma after a milk meal (50). When foreign exosomes were injected into mice, they accumulated in resident macrophages in the liver and spleen (51, 52). Apparently, milk exosomes also accumulate in macrophages (48). Finally, some genetically modified organisms utilize small interfering RNAs (siRNAs) to achieve gene knockdown in pests (53). For example, Smart Stax Pro corn expresses an siRNA that targets corn rootworm–resistant Vacuolar Sorting Protein 7, the rootworm ortholog of the Vacuolar Sorting Protein Snf7 (54). It is beyond reasonable doubt that the siRNAs in these organisms are biologically active, i.e., kill pests upon absorption. Note that ongoing hypothesis-generating phenotype screens have revealed alterations in the composition of the gut microbiome, purine metabolism, fecundity, and immune function in mice (M Sadri and J Zempleni, unpublished results, 2016). It remains to be determined whether miRs are the sole exosome cargo eliciting these phenotypes, or whether cargos other than miRs also play a role. We propose that dietary miRs elicit biological effects through the canonical RNA-induced silencing complex (4, 12), but also through binding to TLRs (19, 46) and modulation of the composition of the gut microbiome (F Zhou and J Zempleni, unpublished results, 2016).

Effects of Dietary Compounds on the Expression of Gene Coding for Endogenous miRs

One molecular mechanism that may inform how diet and dietary pattern may influence health and disease concerns the role of diet in modulating the activity and function of endogenous miRs. In fact, several bioactive dietary factors have been found to modulate miRs in vitro. For example, polyphenols, found in fruits and in beverages such as tea, coffee, and wine, may modulate chronic disease prevention through modulation of the expression of miRs (55). The miRs that are modulated by polyphenols appear to regulate mRNA that are involved in various biological functions, such as apoptosis, inflammation, lipid metabolism, and cell migration. The mechanism of polyphenol regulation of miR is currently unknown, but may include transcription factor regulation, epigenetic modulation and other actions. Future in vitro studies intending to study the influence of dietary polyphenols on the expression of miR should consider the use of primary cells to verify findings in transformed cell lines, circulating metabolites of polyphenols, and appropriate physiologically relevant concentrations of these compounds.

Studies of dietary influence on miR expression also have been conducted in animals. As an example, Chapkin and colleagues (56) demonstrated that the combination of dietary fish oil (containing n–3 FAs) plus pectin (fermented to butyrate in the colon) upregulated a subset of putative tumor suppressor miRs in intestinal mucosa, with concomitant downregulation of their predicted target genes after carcinogen exposure compared with control (corn oil plus cellulose) in animals after carcinogen exposure. More recently, these investigators studied the effects of this diet on miRs and mRNAs in colonic stem cells by exploiting leucine-rich repeat-containing G-protein–coupled receptor 5 (Lgr5) as a marker of fast-cycling intestinal stem cells (57). Specifically, Lgr5–enhanced Green Fluorescent Protein–internal ribosome entry site–cre-inducible estrogen receptor knock-in mice were used to visualize and isolate intestinal stem cells. They found that colonic stem cells exhibit a unique miR signature and the protective effect of the combined fish oil and pectin feeding, which antagonizes the oncogenic effects of carcinogen, modulated the expression of select miRs. The data indicated that select dietary factors can influence stem cell regulatory networks in part by modulating the steady-state levels of miRs. Although animal studies have elucidated dietary factor modulation of miRs in vivo, additional probing functional studies that use emerging controls and tools are needed. Considerations about the bioavailability and metabolism of the dietary constituent also need to be explored. In addition, studies are needed to determine the appropriate timing of dietary exposure that is critical for maintaining physiologic miR expression for health maintenance and disease prevention or to disrupt or reverse aberrant miR expression during the early stages of disease development.

Human studies relating dietary influences on miR expression and how such influences may relate to signaling pathways, health, and disease are now emerging. Using a cross-sectional study design, Tarallo et al. (58) examined the association between dietary habit (by food questionnaire) and miR expression in both plasma and stool samples. They analyzed the expression levels of a panel of 7 human miRs in plasma and stool samples of a group of 24 healthy individuals whose diets were characterized by either a vegan, vegetarian, or omnivorous dietary habit. They found that those who consumed a vegan diet had higher expression levels of miR-92a in both stool and plasma than did vegetarians and people who were following an omnivorous diet. miR-92a also was associated with low BMI in both plasma and stool samples. miR-92a is part of the miR-17–92 cluster, which is located at the 13q22 region and encodes for 7 miRs processed from a single polycistronic primary transcript (59). It is interesting to note that miR-92a is thought to regulate immune function, but it has not been found to be consistently overexpressed in cancers. Such contradictions may be due to differences in dietary habits. This pilot study suggests that miR may be modulated by diet and other factors, and that they can be detected consistently in both plasma and stool samples.

To identify molecular signatures of human zinc deficiency, a combination of transcriptome, cytokine, and miR analyses was used in a dietary zinc depletion and repletion study of 9 young male human subjects (60). The authors used a qPCR-based array for the identification of zinc-responsive signature miRs circulating in pooled and individual serum samples. Of the 85 miRs screened, 9 specific serum miRs were downregulated in response to zinc depletion, including miR-204, miR-296–5p, and miR-375; after zinc repletion, this aberrant miR signature was reversed. These results were of additional interest because miRs that were found to respond to zinc depletion have been associated with inflammation, suggesting a mechanism for zinc deficiency that contributes to cancer. It is also interesting to note that lower levels of miR-375 have been observed in the serum of patients with esophageal squamous cell carcinoma (61).

Another clinical study identified a gene-nutrient interaction involving miR regulation of lipid metabolism. Evidence suggested that the function of the lipoprotein lipase variant rs13702 disrupted a miR recognition element seed site for the human miR-410, which resulted in a gain of lipoprotein lipase function (62). The variant allele was associated with lower serum TGs, and the association was modulated by fat intake. To extend the findings, Corella et al. (63), who used the Prevención con Dieta Mediterránea cohort, assessed the interaction between the rs13702 polymorphism and fat intake on serum TGs at baseline and longitudinally. They also examined the association of this variant (C carriers) with cardiovascular disease incidence and its modulation by dietary intervention group. The miR target site single nucleotide polymorphism interacted with dietary intervention in that C carriers had lower TG concentrations and lower stroke incidence in the Mediterranean diet intervention groups. The results suggest the influence of gene, miR, and diet interaction on the regulation of mRNA targets and their influence in disease modulation.

In one of the more controlled clinical studies to date, investigators examined whether consumption of a diet high in lean red meat (HRM) altered miR expression in rectal mucosa tissue, and if an HRM diet supplemented with butyrylated resistant starch could protect against this aberrant miR expression by increasing butyrate levels in the colorectum (64). To test this, a randomized crossover design trial was conducted in which markers of colorectal cancer risk were measured in 23 healthy human volunteers who engaged in four 4-wk dietary interventions: an HRM diet (300 g lean red meat/d) and an HRM diet plus 40 g butyrylated high amylose maize starch/d, preceded by an entry diet, and separated by a washout. The HRM diet was found to increase the miR17–92 cluster miR levels in rectal mucosa, which was associated with a decrease in mRNA levels of target genes, particularly the cell cycle inhibitor cyclin-dependent kinase inhibitor 1A. The increase in miR17–92 miRs and miR21 with the HRM diet was thought to contribute to the corresponding increase in cell proliferation through target gene regulation. Importantly, supplementation with butyrylated resistant starch to the HRM diet restored miR17–92 levels to baseline. Thus, regulation of miR expression may partially explain some of the preventive effects of resistant starch and the increased colorectal cancer risk associated with the intake of an HRM diet.

Nutritional status during pregnancy may influence offspring susceptibility to the development of metabolic risk factors, partly through miR action. A recent review of studies conducted in animals evaluated the effect of maternal diet on the modulation of the expression of miRs in the offspring and its role in metabolic health in later life (65). The authors suggest that miRs may fine-tune cellular and biological processes by regulating the expression of genes related to cardiometabolic risk factors that influence the phenotype of the animal later in life. How diet and dietary patterns influence miR expression through the life span and in different physiologic contexts requires further study.

Bioinformatics Databases and Tools on Dietary miR

Given the increased appreciation for dietary miR research, one pressing bioinformatics need in this community is to develop a repository that centralizes the dietary miR sequences and annotations, miR bioavailability, and experimental data available in the existing publications and public databases. Cui and colleagues (66) were motivated to develop the first system of its kind, named the Dietary MicroRNA Database, which archives information on 5865 miRs that were reported in 15 dietary species from either plant or animal sources. The information on miR sequences, structures, experimental targets in host species and predicted targets in humans, functional pathways, and annotation on gene interaction and regulation were integrated as crossreferences from public miR databases such as miRBase (67), TargetScan (68), and MirTarBase (69). The Dietary MicroRNA Database also supports sequence comparison, feature generation, and pathway and gene interaction network analysis. Another fact that warrants the efforts in bioinformatics tool development is that NGS becomes the most efficient technique in the new wave of exogenous miR discovery; for example, a large abundance of nonhuman miRs have been detected in human circulation with the use of noncoding small RNA sequencing (R Kitchen, unpublished results, 2015; 47); however, no existing NGS pipeline exists for exogenous miR detection. Tools for small RNA sequencing data analysis, such as mirdeep2 (70), CAP-miRseq (71), DSAP (72), Dario (73), omiRas (74), and srnabench (75), all are focused on miR expression profiling and novel host miR detection, whereas the challenges of exogenous sequence detection lie in fast multigenome mapping, annotation, and cross-species comparison for detection of subtle sequence differences between endogenous and exogenous miRs. A new Web system for small RNA sequencing data analysis has been developed, which allows for the detection of exogenous dietary miR from major dietary species; the beta version of the server can be accessed through Villarroya-Beltri et al. (77). Through analyzing in-house data from the milk feeding study, several bovine miR candidates, such as bta-miR-378, -380–3p, and -1839, were identified in human plasma data (50, 76).

Molecular Feature Associated with miR Transport

Another important topic related to dietary miR is identifying molecular features that contribute to sorting, packaging, and transport of miRs from cells of dietary origins to host circulation. One recent study reported that a motif (GGAG) is enriched in exosomal miRs that are secreted from T cells, through which the loading protein heterogeneous nuclear ribonucleoprotein A2B1 can specifically bind to some miRs and load them to exosomes (77). To tackle this problem with the use of a systematic approach, Shu et al. (50) conducted a bioinformatics analysis to generate 1620 features based on the miR sequence and structure. A support vector machine–based classification analysis was performed on the circulating miRs compared with other human miRs in which 8 groups of features were identified as being associated with human circulating miRs. Further motif finding analysis based on 480 known exosomal human, bovine, and mouse miRs showed that there was no significant sequence motif enriched in exosomal miRs across all species. In humans, some motifs showed a higher level of significance, but none was sufficiently reliable for the differentiation of exosomal miRs from others. Current efforts are focused on the assessment of motifs within specific groups that have the same cell type or dietary species.

Functional Analysis of Dietary miRs

Given the broad implications of miR in human health, research enthusiasm to study the biological role of exogenous dietary miR has soared. Numerous bioinformatics tools have been focused on miR target prediction, such as TargetScan (68), through which one can indirectly infer the functional processes in which a miR can participate. The DIANA miRPath tool (78) uses the comprehensive biological pathways annotated in the Kyoto Encyclopedia of Genes and Genomes (79) to provide better understandings of which biological pathways are regulated by miRs. Continued bioinformatics efforts on miR are focused on the following topics: 1) development of a new system for efficient miR target prediction that can decrease the high false positive rate with the existing methods, 2) a new system to evaluate the functional association of miRs, 3) studying miR-mediated gene regulation in a dynamic manner through network analysis. For example, miR-mediated gene regulation is a very complex, semistochastic process that integrates both cooperative and competitive binding mechanisms that are largely ambiguous.

Regulation of Immune Function in Adipose Tissue by miRs

Chronic low-degree adipose tissue inflammation induced by excess nutrients is a major contributor to the pathogenesis of insulin resistance (80–87), which is also a causal factor for a wide variety of chronic diseases, including atherosclerosis, type 2 diabetes, and certain types of cancer (88–95). Given the critical function of adipose tissue, especially the visceral fat pads, in maintaining metabolic and endocrinologic homeostasis, it is urgent that we better understand how adipose tissue immune cells respond to overnutrition stress and contribute to the development of obesity-induced chronic inflammation. The complex immune compartment within visceral adipose stromal cells consists of various dynamically interacting cell types. For example, adipose tissue macrophages (ATMs) account for 30–40% of visceral stromal cells (VSCs), and the molecular mechanisms underlying their polarized activation features have been extensively investigated (96). ATMs display a wide range of activation profiles, from alternative activation (M2) in lean adipose tissue to the predominant classic proinflammatory state (M1) in obese tissues (97–99). Upon excess nutrient stimulation, ATMs undergo a phenotypic switch toward proinflammatory activation. The classic proinflammatory response of ATMs depends on TLRs and activation of nuclear factor κB/c-Jun N-terminal kinase, leading to the production of inflammatory cytokines. In contrast, alternative activation of ATMs recruits peroxisome proliferator–activated receptor (PPAR) γ, PPARδm, or signal transducer and activator of transcription 6, and is crucial for tissue-repairing allergic response and antiparasite functions (97, 100). Recently, miR-223 was reported to act as a potent regulator for macrophage polarized activation and to be a key mediator for PPARγ-dependent activation pathway in alternative activation (96, 101). miR-223–null mice displayed exacerbated inflammatory profiles and severe insulin resistance that could not be fully restored by PPAR agonist retreatment. Several genes that are bona fide targets of miR-223 have been identified in modulating ATM polarization (96, 101). In addition, adipose tissue T cells comprise ∼10% of obese VSCs and regulate adipose tissue immunity through direct cell-cell interaction and cytokine production (102–104). Moreover, adipose tissue B cells represent >20% of VSCs in obese individuals; however, their function was poorly investigated. It was found recently that mice with miR-150 deficiency displayed enhanced obesity-associated chronic inflammation and systemic insulin resistance (105). Adoptive transfer of B-miR150^−/− B cells into B cell–deficient mice confirmed the critical role of miR-150–regulated B cell function in this context. Furthermore, a set of genes bearing miR-150 targeting sites have been identified and their involvement has been confirmed in mediating adipose tissue B cell–dependent cell-cell interaction in obese adipose tissue, which eventually elevates the inflammatory response and insulin resistance (105).

In conclusion, the above observations suggest the importance of miRs in the regulation of physiologic and pathologic conditions and the role of both endogenous and dietary-derived miRs in gene regulation, as well as the influence of dietary component regulation of endogenous miR and the need for new analytic tools for the study of nutrition and gene regulation in health and disease.