Most scientists and clinicians would agree that the genetic code (DNA) plays a central role in metabolism and the health propensity, and trajectory of individuals. For years, the mention of “genes” involved in health and metabolism promoted the classical thinking of the genome encoding mRNA, which is translated into protein. However, the ability of scientists to conduct large scale genomic analysis has shed a bright light on epigenetic factors that include the involvement and control of the metabolism by the non-coding region of DNA, referred to as microRNA (miRNA).1-2
What Is “circRNA”?
A new type of non-coding RNA, previously considered “junk”, is now referred to as circular RNA (circRNA), and is part of a complicated network involving mRNA, miRNA, and proteins.3-4 CircRNA formation is tissue-specific and changes across various stages of cell differentiation. CircRNA is preferentially expressed in neural tissues and some are found at synapses, suggesting possible functions in the nervous system.3 Several circRNAs have been shown to function as microRNA “sponges” to counteract microRNA mediated repression of mRNA. CircRNA has been confirmed to be associated with varied cellular processes and is involved in the biogenesis and development of many diseases, especially cancer. The potential to measure circRNA as a diagnostic or as a predictive biomarker of disease has become a hot topic in cancer research.
New insights into the non-protein coding region of DNA has determined that the majority of the non-protein coding region of DNA (introns or intergenic sequences) is transcribed into long non-coding RNA or small non-coding RNA which orchestrates the regulation of protein expression at the level of transcription (generating messenger RNA) and translation (converting mRNA into an amino acid sequence of protein).5-6 Over 60 percent of human protein-coding genes are regulated by miRNA, and it is estimated that about 1800 high confidence miRNAs are encoded in the human genome which accounts for only about 1.8 percent of the transcriptional output.7-8 Interestingly, even though 75 percent of the human genome is transcribed, the protein-encoding portion of the genome only accounts for one and a half percent.9
What Is miRNA?
MiRNAs were first discovered in 1993.6,10 MiRNAs consist of approximately 22 nucleotides and regulate gene expression by binding to their complementary sites within the 3’-untranslated regions (3’UTRs) of target mRNAs, or coding sequences of 5’UTR of target mRNAs, leading to the inhibition of translation or mRNA degradation.6, 11-12 A uniform system for miRNA annotation was developed by a collaboration of scientists in 2003 in order to keep track of the vast array of miRNAs that have been discovered since 1993.13 MiRNAs are initially transcribed by RNA polymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts that are processed by the RNase III enzymes, Drosha and Dicer, to generate 18-24-nucleotide mature miRNAs.14
MiRNA pathways are evolutionarily conserved control mechanisms that use RNA molecules to inhibit gene expression at the level of mRNA degradation, translational repression, or chromatin modification and silencing.15 The degree of base pair binding or complementarity of miRNA to mRNA dictates whether there is repression of mRNA translation or transcript degradation.1 Perfect complementarity leads to targeted cleavage and degradation. However, imperfect complementarity triggers mRNA silencing by distinct mechanisms which may involve translational repression, slicer-independent mRNA degradation, and/or sequestration in cytoplasmic processing bodies. Thus, miRNAs hybridize with complementary sequences in mRNA and silence genes by destabilizing mRNA or preventing translation of mRNA.
MiRNA May be Delivered to Other Cells via Microparticles
MiRNA may be delivered to other cells via vesicular structures called microparticles. Microparticles represent an intercellular communication and delivery mechanism for the efficient and effective transfer of biological information. Contents of microparticles are derived from their parent cell during formation and include proteins, lipids, and genetic material (mRNA, miRNA) in a structured and regulated process.16 Human phagocytes are stimulated by microparticles which results in macrophage-mediated efferocytosis and stimulation of pro-resolving mediator biosynthesis.17 In particular, microparticles enriched in alpha-2-macroglobulin (A2MG) enhanced pro-resolving responses and promoted survival in a sepsis model.18 A2MG enriched microparticles delivered to mice with microbial sepsis enhanced survival, protected against hypothermia, reduced bacterial titers, elevated immunoresolvent lipid mediator levels in inflammatory exudates, and reduced systemic inflammation. Microparticles were also used to deliver A2MG to human leukocytes resulting in enhanced bacterial phagocytosis, reactive oxygen species production, release antimicrobial peptides called cathelicidin, prevent bacterial endotoxin-induced CXCR2 (an IL-8 receptor needed for neutrophil recruitment), and downregulate and preserve neutrophil chemotaxis in the presence of lipopolysaccharide. Collectively, the research identified A2MG enrichment in microparticles as an important host protective mechanism in sepsis.
Microparticles That Contain Cytokines are Involved in the Initiation and Resolution of Inflammation
Cytokines, such as interleukin-1 (IL-1), are a constituent of microparticles used to communicate with other cells in response to inflammation. The cytokine proteins are key mediators in the regulation of inflammatory responses. Following cellular activation, cytokines are released where they may either propagate the inflammatory response or promote resolution.16 IL-1β in platelet microparticles lead to the activation of endothelial cell adhesion molecules that may lead to increased leukocyte adhesion. Neutrophil microparticles may also contain annexin A1 protein that upon interaction with its receptor on the surface of neutrophils and/or endothelial cells inhibits neutrophil transmigration and recruitment. The composition of a microparticle is very complex and includes protein (cytokines, receptors, regulators, and enzymes), lipids (arachidonic acid, eiocosapentaenoic acid, and docosahexaenoic acid, the metabolome (resolvins), and genetic material.16
Microparticles Involved in Immune Response Contain Morphogens that May be Involved in Wound Repair
Microparticles may be considered “morphogens.” Morphogens describe a type of signaling molecule that acts on cells directly to induce distinct cellular responses in a concentration-dependent manner.19-22 Sonic Hedgehog (Shh) is one of the regulatory proteins in microparticles that support the resolution of inflammation and is considered a morphogen.
The Shh protein is involved in wound repair, including neo-angiogenesis, and is involved in the re-establishment of homeostasis.16 Interestingly, research has demonstrated that leukocyte-derived microparticles carrying the Shh morphogen can induce angiogenesis, in part by upregulating adhesion molecules and stimulating angiogenic factors in endothelial cells.23 Additionally, Shh in microparticles stimulates nitric oxide production in endothelial cells which promotes endothelial repair.24 Accumulatively, the research suggests that Shh containing microparticles are involved in wound repair and cancer metastasis.
Morphogens also have been found to have a role in regulating cell fate and determination in self-renewing tissues in adults, such as the immune system and haematopoietic system.25 Proteins such as Shh and Wnt are part of a group of signal transduction pathways made of protein that pass signals into a cell through transmembrane cell surface receptors, and regulate the proliferation of cells.26 Wnt proteins are highly conserved in evolution and comprise a major family of signaling molecules that orchestrate and influence a myriad of cell biological and developmental processes.26 Mutated Wnt pathway components are causative to multiple growth-related pathologies and to cancer.27-28
Wnts are secreted from cells in a lipid-bound configuration, likely bound to endosomes and secreted on exocytic vesicles. Wnt signaling, through β-catenin (a cytoplasmic protein), has a positive role in the control of T-cell development. An absence or reduction in the Wnt signal leads to a reduction in cell number and cell proliferation rate, and differentiation to the CD4+CD8+ double-positive stage.25 While the mechanics of the Wnt/β-catenin signaling pathway are still under investigation, research has led to an understanding that miR-21 translationally represses the Wnt1 gene which has implications for cellular signaling as part of the Wnt/β-catenin signaling pathway.29-30 Antagonism of the miR-21 inhibits human monocyte-derived dendritic cell (MDDC) differentiation which is a component of the innate immune system.29,31
MiRNA is Involved in the Regulation of Many Cellular Processes
MiRNA is involved in the regulation of many cellular processes such as cellular proliferation, apoptosis, and cellular metabolism.32 Circulating miRNA is usually associated with exosomes, lipoproteins, and protein complexes and is protected from degradation from RNAses. Modification of circulating miRNA are associated with cancer, cholesterol metabolism, type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), insulin sensitivity, endothelial function, neurodegenerative diseases, autoimmune diseases, inflammation and aging.6 The involvement of miRNAs in physiological and pathological processes in the lung has been well characterized and includes lung homeostasis and development.33
MiRNA has been discovered in the lymphatic endothelium and has been shown to be a regulator of cell lineage plasticity, inflammation, and regulatory function.34 Researchers have determined that miRNA is a key determinant of lymphatic endothelial cell (LEC) differentiation and inflammatory responses. This could be a very important role for miRNA because transcription factors of LEC have crucial roles in wound healing, inflammation, infection, and cancer.
Non-coding regions of RNA have been found to be a regulator of nuclear factor-κB (NF-κB) which plays an essential role in the regulation of inflammatory responses, immune function, and malignant transformation.35 Dysfunctional activity of the NF-κB signaling pathway may lead to inflammation, autoimmune diseases, and oncogenesis. Key enzymes responsible for the formation of leukotrienes (5-lipoxygenase), prostaglandins, and thromboxanes (cyclooxygenase-2), have been shown to be regulated by miRNAs.36 In addition polyphenols, (curcumin, resveratrol, quercetin, genistein, caffeic acid, ferulic acid and chlorogenic acid) found in fruits, tea, coffee, and wine, may modulate chronic disease prevention through modulation of the expression of miRNA.14
Dietary MiRNAs are Bioavailable from Plants and Foods of Animal Origin
MiRNAs ubiquitously exist in microrganisms, plants, and animals, and have been shown to modulate a wide range of critical biological processes.37 MiRNA has also been discovered in various fractions of human milk, with the highest concentrations found in the cell and lipid fractions, and the lowest in skim milk.38 No definitive conclusion has been reached regarding the uptake of exogenous dietary small RNAs into mammalian circulation and organs and cross-kingdom regulation. However, evidence suggests that miRNAs are not only synthesized endogenously, but also might be obtained from dietary sources, and that food derived miRNA alters the expression of endogenous miRNA genes.8
Researchers have identified pathways for femtomolar (10-15) concentrations of dietary miRNAs that may elicit biological effects through binding to toll-like receptors (TLRs) or by surface-antigen-mediated delivery of exosomes to immune cells.39-40 Furthermore, investigators have identified 50 plant-borne miRNAs in human plasma.41 MiRNA is abundant in milk, animal meats, and dried extracts derived from bovine sources such as adrenal tissue.42 Researchers identified 198 different homologous human miRNAs in food grade animal sources. Consumption of miRNAs in the diet may interact with mRNAs encoding transcription factors, protein receptors, transporters, and immune-related proteins. Strategic supplementation of targeted miRNAs may be a clinical tool to modify epigenetic regulation in the future.
MiRNA Variants are Epigenetic Regulators in Cardiovascular Diseases
Observational meta-analysis from a 7,187 participants from the randomized clinical PREDIMED trial (Prevenćion con Dieta Mediterránea) identified a gain-in-function microRNA-410 target site polymorphism in the 3’ untranslated region of the lipoprotein lipase (LPL) gene (rs13702). LPL is an enzyme that resides on the epithelial lining of the vasculature and is responsible for hydrolyzing the triglyceride-rich core of circulating lipoproteins; primarily chylomicrons and very low density lipoprotein (VLDL). The synthesis and secretion of LPL from adipocytes in cell culture have been shown to be modulated by dietary fatty acids.43
The single nucleotide polymorphism (SNP) of the rs13702 gene is a T nucleotide (thymidine) that is replaced by a C nucleotide (cytidine). The nomenclature for this type of polymorphism is rs13702T>C. The C allele of the LPL gene was associated with lower triglycerides, which was also influenced by the monounsaturated and saturated fat intake from the Mediterranean diet. The rs13702T>C SNP was also associated with a lower stroke risk, but only in the group randomized to the high unsaturated fat intake Mediterranean diet.44 LPL transcripts with the SNP lack translational inhibition mediated by miR-410. The SNP apparently alters microRNA binding by either creating a new binding site, or destroying an existing target.45 LPL-SNPs have also been associated with type 2 diabetes, premature atherosclerosis, and the risk of CVD, mainly stroke.44,46 The research by Corella et al., is the most thorough report that analyzed the effects of a miRNA target site SNP, and its interaction with diet on triglycerides and stroke.
It has been proposed that the plasma membrane lipid composition, which is influenced by diet, could directly impact the miRNA mediated regulation of inflammation.47 Long chain fatty acids such as EPA, DHA and Arachidonic acid are released from membrane bound phospholipids via sn-2 lipase. Inability or diminished potential to convert EPA, DHA and Arachidonic acid fatty acids into resolvins may be involved in the promotion of vascular diseases such as atherosclerosis, sepsis and an array of vascular dysfunction.48 The relative abundance of omega-3 to omega-6 fatty acids within membranes may impact the type and amount of miRNA expression of monocytes and endothelial cells leading to altered signal transduction, via cytokine-induced NFκB activation.
MiRNA is a Crucial Regulator of Innate and Adaptive Immune System Function and Autoimmune Diseases
MiRNAs have been shown to function as crucial regulators of immune response in both normal physiological and pathological conditions.49 In the case of cancer, the role of the immune system and the response that is mounted against a cancerous cell involves the innate and acquired immune system. Various miRNAs have been found to target key cancer-related immune pathways, which concur to mediate the secretion of immunosuppressive or immunostimulating factors by cancer or immune cells. In the future, the modulation of individual or multiple miRNAs has the potential to enhance or inhibit specific immune subpopulations supporting anti-tumor immune responses to reduce the risk of tumor formation. As research develops in this exciting area, miRNA strategies may be developed for more effective immunotherapeutic interventions in cancer. Table 1 identifies a limited view of the most relevant miRNAs involved in innate and adaptive immunity as well as the impact of resolvin RvD1 (300ng or 15µg/kg) in regulating miRNAs in a model of self-limited acute inflammatory exudates.49-51
RvD1-miRNAs have been shown to target select cytokines and protein involved in the immune system, e.g., miR-146b targeted NF-κB signaling and miR-219 targeted 5-lipoxygenase, and reduced leukotriene production. The relative abundance of miRNA’s differed depending on the time course of the inflammation exudates. At four hours, miR-219, miR-302, miR-142-5p and miR-142-3p were highly expressed, whereas miR-203, miR-146b,miR-21 and miR-208 were more highly expressed at 12 hours. The research by Recchiuti et al., demonstrates that RvD1 controls acute inflammation, accelerates resolution and regulates leukocyte miRNA expression over a time course via receptor interaction.
In particular, IL-10 protein levels were increased by RvD1. IL-10 is an anti-inflammatory cytokine, which inhibits the activity of Th1 cells, NK cells and macrophages.52 Interestingly, miR-219, specifically targeted TNF-α, TNF-αR,IL-1 and IL-1R accessory protein.50 Collectively, these findings indicate that target genes of miR-146b, miR-208a, and miR-219 are involved in the immune system and form RvD1-dependent networks that govern inflammation and resolution.
MicroRNAs Are Regulators of Cancer Related Immunity in Solid Tumors
Differential expression patterns of miRNAs are associated with several human pathologies, including cancer in all its stages. In the field of immunology, miRNAs have been classified as either oncogenic (miR-155, miR-21), or having a tumor suppressor role (miR-34, miR-15a). Deregulation of a single miRNA or distinctive miRNA profiles have been correlated with survival, clinical outcome, and response to therapy in various solid tumors.49 For example, miR-155 upregulation has been demonstrated to be required in the myeloid cell compartment for the promotion of anti-tumor immunity in early stages of breast cancer carcinogenesis. In Melanoma and Lewis lung cancer, a decrease in miR-155 in immune cells leads to tumor growth. Current understanding of miR-155 suggests that it plays a role in fine tuning the regulation of lymphocyte subsets such as B cells, CD8+, CD4+ T helper cells type 1, (Th1), Th2, Th17 and regulatory T cells. Given the scope of involvement of miR-155, researchers have reason to believe that miR-155 shapes the balance between tolerance and immunity.53 While beyond the scope of this document, there are many more examples of deregulated miRNA/targets having a significant biological role in immune and cancer-related pathways in solid tumors.49
The role of miRNA in inflammation and autoimmunity is an intensive area of research that may lead to therapeutic options to regulate genes at the post-transcriptional level in the management of inflammatory and autoimmune diseases.54-56 There is evidence for the role of miRNA’s in asthma, contact dermatitis, rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome, multiple sclerosis as well as the expression of gender and sex hormones. Estrogen regulates numerous miRNAs and participates in disease pathogenesis. Interestingly, the ability of estrogen to modulate miRNA that regulates genes involved in autoimmunity may explain, in part, why females are more susceptible to these diseases. Unraveling the targets of miRNAs and the influence of sex hormones will define their role in inflammation and autoimmunity and provide opportunities for therapeutic application.
The discovery that miRNA is an epigenetic regulator of many genes, and that mutations or SNPs in genes may be targeted by miRNA and alter the phenotype of an individual is potentially very powerful information for developing miRNA therapeutics.11, 57-58 Additionally, when miRNA is mutated or improperly expressed, the control of a gene may be adversely affected. Disease-associated miRNA may have the potential to represent a new class of therapeutic applications. However, there is much work to be done because the system is quite complex. A single miRNA can regulate hundreds of targets, and the biological function of miRNAs is not easily discerned from an examination of their targets.11 Additional research will shed light on the utility of miRNA for therapeutic applications.59
An excellent review of the role of miRNA in autoimmunity and autoimmune diseases details the complex consequences of abnormal miRNA regulation in terms of immune cell development, B and T cell function and innate and adaptive (acquired) immune responses.54 A recent publication indicated that CD-4 T lymphocyte activation requires tight regulation of miRNA expression via enzymatic 3’ uridylation of terminated non-templated nucleotides.60 The research emphasizes the precise control of post-transcriptional uridylation as a mechanism to fine-tune miRNA levels during T-cell activation.
Read part 7 of the Immune System Series: Oral Tolerance to Foods.