Roles of microRNAs in inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract that mainly affects young people. IBD is associated with various gastrointestinal symptoms, and thus, affects the quality of life of patients. Currently, the pathogenesis of IBD is poorly understood. Although intestinal bacteria and host immune response are thought to be major factors in its pathogenesis, a sufficient explanation of their role in its pathophysiologic mechanism has not been presented. MicroRNAs (miRNAs), which are small RNA molecules that regulate gene expression, have gained attention as they are known to participate in the molecular interactions of IBD. Recent studies have confirmed the important role of miRNAs in targeting certain molecules in signaling pathways that regulate the homeostasis of the intestinal barrier, inflammatory reactions, and autophagy of the intestinal epithelium. Several studies have identified the specific miRNAs associated with IBD from colon tissues or serum samples of IBD patients and have attempted to use them as useful diagnostic biomarkers. Furthermore, some studies have attempted to treat IBD through intracolonic administration of specific miRNAs in the form of nanoparticle. This review summarizes the latest findings on the role of miRNAs in the pathogenesis, diagnosis, and treatment of IBD.


Introduction
Inflammatory bowel disease (IBD) is a complex and multifactorial condition characterized by chronic gastrointestinal tract inflammation. There are two main forms of IBD: Crohn's disease (CD) and ulcerative colitis (UC). CD is manifested as patchy transmural inflammatory patterns that affect all layers of the intestinal wall in any portion of the gastrointestinal tract, whereas UC is limited to the innermost layers of the mucosa in the large intestine.
CD and UC mainly affect young people, causing bloody diarrhea, abdominal pain, malabsorption, fatigue, and impaired quality of life [1]. Long-lasting inflammation also increases the risk of colorectal cancer in patients with IBD, which has a mortality rate of 10-15% [2].
Rapid modernization and urbanization seem to be related to the increasing incidence of IBD, which affects approximately 0.5% of the general population Ivyspring International Publisher in developed nations [3,4]; however, the exact pathogenesis of IBD remains poorly understood. Based on speculations, IBD is presumed to be the result of complex interactions among genetic, microbial, and environmental factors [1]. Genomewide association studies (GWASs) have identified the genetic susceptibility loci for IBD, and more than 240 of these loci have been reported [5][6][7][8]. Substantial differences have been found between different ethnic groups [9,10]. Moreover, the existence of IBD susceptibility mutations is not sufficient to explain the entire disruption of intestinal homeostasis; this is because twin studies revealed low concordance between genetic variations and clinical presentation of the disease [11]. The role of the microbiome and lifestyle factors in the pathogenesis of IBD is unquestionable; however, these conditions only cause disease in genetically susceptible individuals [12].
Recent molecular research has shown that microRNAs (miRNAs or miR-) play important roles in the pathophysiology of IBD [13]. miRNAs are evolutionarily conserved, endogenous, singlestranded, non-coding RNAs that bind to the 3' untranslated region (UTR), 5' UTR, or partially translated region of a target mRNA, inhibiting mRNA translation and blocking its expression [14]. miRNAs are major regulators of cell function and homeostasis, and their abnormal activity has been demonstrated in various diseases, including IBD. Thus, novel treatment options could be developed to alter imbalances in miRNA expression by targeting certain molecular pathways [15]. In this study, we evaluated the role of miRNAs in IBD by examining published results of studies conducted in the past 2-3 years and suggest the clinical potential of miRNAs as biomarkers and/or new therapeutic strategies for IBD.

Biosynthesis of microRNA
miRNAs are small, 18-21 nucleotide-long, evolutionarily conserved RNA molecules involved in the regulation of gene expression. The primary nuclear transcripts of miRNAs, or pri-miRNAs, are typically a few thousand nucleotides long and are located in introns, exons, or intergenic regions of the human genome. Pri-miRNAs are processed into 70 nucleotide-long, stem-loop structured pre-miRNAs by the RNAse-3-type enzyme Drosha. Pre-miRNAs are then translocated to the cytosol from the nucleus and cleaved by the endoribonuclease Dicer. The remaining miRNA duplex binds with Argonaute to form an RNA-induced silencing complex (RISC). The guidance strand directs the RISC to the complementary region in the 3'-UTR of the target mRNA, and the RISC makes post-transcriptional modifications. When an exact match is found, the target mRNA is degraded; however, when an incomplete match occurs, translation is destabilized [14]. Figure 1 briefly summarizes the biosynthesis of miRNAs and their mechanisms of action.
The seed region responsible for identifying target mRNAs is a 6 nucleotide-long sequence from the 2nd to the 7th nucleotide of the miRNA. Based on similar seed sequences, miRNAs are classified into miRNA families. Members of a given miRNA family share almost the same pool of mRNAs as their target as they possess the same seed region. Numerous miRNAs form clusters in the genome and are transcribed as one common pri-miRNA; therefore, their transcription is regulated simultaneously. Notably, the role of miRNAs is redundant; multiple different miRNAs can control a single mRNA, and a given miRNA can inhibit numerous mRNAs. miRNA regulation does not act as a switch; rather, it functions as a synchronized tuner of gene expression [16].  In intestinal homeostasis, commensal bacteria and dietary metabolites, such as RA and AhR ligands, induce immune tolerance through the action of Treg cells and anti-inflammatory cytokines, including IL-10 and TGF-β1. Various immune cells are recruited into the intestinal lamina propria but are not activated. However, IgA produced by plasma cells, and anti-microbial peptides, including REG3 and defensin, which are secreted from ILC3 and Th17 cells, prevent the pathogenic colonization of intestinal bacteria and invasion through the intestinal epithelium. In intestinal inflammation, direct invasion of commensal or pathogenic bacteria through the broken intestinal barrier causes inflammatory responses. Various immune cells and cytokines induce inflammatory reactions and augment anti-microbial activity. AhR: aryl hydrocarbon receptor; IFN: interferon; IL-: interleukin; ILC: innate lymphoid cell; NK T cell: natural killer T cell; PMN: polymorphonuclear leukocyte; RA: retinoic acid; REG3: Regenerating islet-derived protein 3; TGF β1: transforming growth factor β1; Th1: T helper type 1; Th2: T helper type 2; Th17: T helper type 17; TNF-α: tumor necrosis factor-α; Treg cell: regulatory T cell.

Brief description of the pathophysiology of IBD
Commensal bacteria are essential components in the maintenance of intestinal homeostasis. They provide essential nutrients, assist in energy metabolism, and suppress the growth of pathogenic bacteria by competitive inhibition in the intestine [17]. Nevertheless, they can cause opportunistic infections and are under the surveillance of host immunity. Consequently, the human immune system has adapted to maintain a commensal relationship by establishing a balance between immune suppression and activation. The intestine is continuously exposed to large amounts of toxins and antigenic proteins from ingested foods. Therefore, a high level of immune tolerance is essential for intestinal homeostasis [18]. However, well-controlled immune responses can be aberrantly activated by altered states of intestinal bacteria or the intestinal barrier, which are two important factors that maintain immune tolerance. From our perspective, a disruption in physiological balance can result in the development of IBD ( Figure  2).
Many immune cells, such as neutrophils, dendritic cells, macrophages, and lymphoid cells, reside in the intestinal epithelium and the underlying lamina propria. These cells are regulated by immune tolerance mechanisms. When macrophages phagocytose the antigen from commensal bacteria, interleukin (IL)-10 is secreted from macrophages [19], resulting in the promotion of the response of a cluster of differentiation 4 + (CD4 + ) Treg cells to suppress inflammatory T cell reactions. CD4 + Treg cells produce IL-10 and transforming growth factor β1 (TGF-β1) to change the subtype of macrophages into an anti-inflammatory phenotype and to facilitate peripheral development of Treg cells [20]. Serum amyloid A is an important acute reactant protein produced in the intestine and liver. A previous study demonstrated that serum amyloid A had a higher increase in the presence of intestinal microbiota than in the germ-free state. Serum amyloid A enhances the recruitment of neutrophils to the intestine and increases pro-inflammatory gene expression in neutrophils, but decreases bactericidal capability and inflammatory tones [21,22]. These contradictory responses would not damage the intestinal microbiota during the prevention of opportunistic infections by commensal bacteria. CD4 + T cells secrete IL-21 and TGF-β1 to stimulate immunoglobulin (Ig) class switching in B cells. The secreted IgAs bind to commensal bacteria in the intestinal lumen, which prevents bacteria from invading the intestinal epithelium, and suppress the inflammatory reaction of the intestinal barrier against commensal bacteria [23]. Retinoic acid, a metabolite of vitamin A, is an important nutritional factor that is closely related to the immune response. Retinoic acid plays an important role in maintaining intestinal homeostasis, specifically inducing intestinal immune tolerance through the actions of CD103 + dendritic cells and innate lymphoid cells (ILCs)3 [24]. However, based on a recent study, commensal bacteria reduced retinoic acid and IL-22 by inhibiting retinol dehydrogenase 7 (Rdh7) in intestinal epithelial cells. Consequently, the anti-microbial reaction was downregulated, and a symbiotic state could be maintained [25]. Such findings indicate that retinoic acid has both tolerogenic capacity and antimicrobial activity. Thus, intestinal immune tolerance does not simply mean a decrease in intestinal immunity, but rather a balanced state in which anti-inflammatory and anti-microbial activities coexist. The aryl hydrocarbon receptor (AhR) is a transcription factor that induces anti-inflammatory reactions at the gene level. Previous studies have indicated that AhR reduces intestinal inflammation through anti-inflammatory mediators, such as IL-10 and IL-22. Several AhR ligands exist in the intestine, the majority of which are metabolites of commensal bacteria. Therefore, intestinal microbiota contributes to AhR-induced immunomodulation [26,27]. Taken together, host immunity has dual effects on the intestinal microbiota to maintain intestinal homeostasis. The intestinal immune system should maintain an appropriate balance between inflammatory and anti-inflammatory responses. Dysbiosis, which is an imbalance in microbes in the intestine, disrupts intestinal homeostasis and should thus be considered as a major cause of IBD [28].
The intestinal epithelium is one of the key components that sustain homeostasis of the intestine. The membranous barrier separates intestinal germs from the submucosal lymphoid tissue to prevent inflammatory responses and integrates molecular signals from dietary metabolites, commensal bacteria, and pathogens to regulate immune reactions [29]. Resident lymphoid cells, such as NK T cells, γδ T cells, ILCs, intraepithelial lymphocytes, and CD8 + mucosal-associated invariant T cells produce IL-6, IL-17, and IL-22 to activate the signal transducer and activator of transcription (STAT) 3 pathway in response to disruption of the intestinal barrier. The STAT3 pathway promotes the recovery of barrier function and increases mucus secretion and epithelial repair [30]. Macrophages secrete type I interferon (IFN) and activate STAT1 and STAT2 pathways to prevent epithelial apoptosis, promote epithelial differentiation, and maintain the integrity of the intercellular barrier [31]. CD4 + T cells produce IL-17 and IL-22 in response to IL-23 and serum amyloid A, which results in the activation of the STAT3 pathway in the epithelium [32]. These interactions enhance the intestinal barrier against pathogenic luminal bacteria.
When the mucus barrier is broken, permeability through the epithelium increases. These situations are mostly due to the dysfunction of the mucosal barrier itself, or inflammatory damage. Mucous membrane disorder, tight junction disorder, increased intestinal permeability, and increased binding of bacteria to epithelial cells can induce intestinal inflammation. Penetration of intestinal bacteria, including intestinal microbiota, into the lamina propria leads to recognition by phagocytes and the production of inflammatory cytokines by macrophages. Macrophages secrete IL-1β to promote the differentiation of CD4 + T cells into T helper 17 (Th17) cells and the production of IFN-γ [33,34]. Damaged epithelium releases IL-18, which disrupts goblet cell maturation and impairs its function, leading to further damage of the membranous barrier [35]. Macrophages enhance the survival of pro-inflammatory T cells and stimulate epithelial apoptosis through the tumor necrosis factor-α (TNF-α) signaling pathway [36,37]. IL-6 from macrophages and granulocyte-macrophage colony-stimulating factor (GM-CSF) from ILCs recruit polymorphonuclear leukocytes, which secrete pro-inflammatory cytokines for pathogenic T cell responses [38][39][40]. Repeated cycles of dysregulated immune responses result in chronic intestinal injury and fibrotic remodeling in patients with IBD [41].
IBD is a multifactorial disease. Although dysbiosis of commensal bacteria and breakdown of the intestinal barrier are considered as major pathological mechanisms in the development of IBD, other important factors such as genetic aberrations also contribute to its development. In this regard, miRNAs are an emerging risk factor for IBD. miRNAs are involved in the maintenance of intestinal barrier integrity, as well as a large number of intracellular signaling pathways involved in inflammatory or anti-inflammatory actions [42].

Role of miRNAs in the pathophysiology of IBD
miRNAs have been reported to be associated with several pathophysiological factors of IBD. miRNAs affect the intestinal barrier and inflammatory reactions through various pathological mechanisms. The pathogenic roles of miRNAs in IBD are summarized in Table 1.

miRNAs and the intestinal barrier in IBD
Disrupted intestinal membranes are one of the most significant factors in the pathogenesis of IBD. TNF-α is known to be a major pro-inflammatory cytokine in the pathogenesis of IBD. Therefore, several in vitro experiments have been conducted using intestinal epithelial cells to induce injury by TNF-α [43]. miR-191a and miR-212 are known to damage intestinal barriers. In fact, in vitro studies have shown that their mimics downregulate the expression of zonula occludens (ZO)-1, one of the major components of the tight junction between the intestinal epithelium [44,45]. By measuring the binding of miR-675 in colon cells in vitro, Zou et al. found that miR-675 destabilized the mRNA of ZO-1 and cadherin E, causing reduced expression of key molecules for intercellular tight junctions [46]. Aquaporin 3 is another important protein in the intestinal membrane. The overexpression of miR-874 in vitro induced by pre-miR-874 transfection in intestinal epithelial cells was demonstrated to decrease the expression of aquaporin 3 [47]. Epidermal growth factor receptor (EGFR) was identified as the target gene of miR-122a. The overexpression of miR-122a was found to increase zonulin expression and intestinal permeability [48]. Further, the overexpression of miR-21 in vitro was found to cause an increase in intestinal barrier defects and was suggested to target the phosphatase and tensin homolog (PTEN)/PI3K (phosphoinositide 3-kinase)/Akt signaling pathway to enhance the paracellular permeability of the intestinal epithelium [49]. As the miR-21 mimic suppressed the level of PTEN and increased the level of phospho-Akt (p-Akt) in vitro, Zhang et al. reported the impairing of intestinal permeability through the PTEN/PI3K/Akt pathway as a potential mechanism [49].
Some miRNAs have been reported to strengthen the intestinal barrier. Transfection of the miR-200b precursor in TNF-α-treated intestinal epithelium in vitro inhibited the damage to trans-epithelial electrical resistance and intercellular tight junctions. Further, c-Jun and myosin light chain kinase (MLCK) were demonstrated to be the targets of miR-200b [50]. Haines et al. revealed that silencing the expression of protein tyrosine kinase 6 (PTK6) with miR-93 in the intestinal epithelium increased the resistance to TNF-α-induced injury [51]. miRNAs and immune response in IBD miRNAs are known to contribute to the immunological reactions that lead to IBD. Shi et al. compared miR-21 knockout mice to wild-type mice after the induction of intestinal damage by dextran sulfate sodium (DSS); miR-21 knockout mice demonstrated reduced weight loss, intestinal inflammation (confirmed by histopathology), serum leukocyte levels, and TNF-α and macrophage inflammatory protein 2 (MIP2) levels in colon culture supernatants compared to wild-type mice [52]. miR-124 was reported to target the 3'-UTR of AhR to suppress its expression in Caco-2 cells and HT-29 cells in vitro [53]. Intracolonic administration of miR-124 inhibitors and precursors to 2,4,6-trinitrobenzen acid (TNBS)-induced colitis mice was found to alleviate and aggravate intestinal inflammation, respectively [53].
Some miRNAs have been reported to suppress intestinal inflammation.
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is one of the genes clearly associated with CD [54]. In fact, Pierdomenico et al. reported its relationship with miR-320 [55]. HT29 cells transfected with exogenous miR-320 displayed a downregulated expression of NOD2, while miR-320 inhibitor increased NOD2 expression as well as its downstream signaling pathway, leading to nuclear factor κB (NF-κB) and inflammatory cytokines [55]. Wu et al. used human monocyte-derived dendritic cells and CD4 + T cells isolated from the peripheral blood of IBD patients treated with the miR-10a precursor. The transfection caused decreased expression of IL-12/23p40 (p40 subunit of IL-12 and IL-23) in dendritic cells and suppression of IFN-γ, TNF-α, and IL-17A in CD4 + T cells, suggesting inhibited differentiation into Th1 and Th17 cells [56]. C-X-C motif chemokine ligand 12β (CXCL12β) is an important mediator of leukocyte migration. Huang et al. used TNBS-induced colitis mice, IL-10 knockout colitis mice, and colon biopsy samples of CD patients to identify an inverse relationship between miR-141 expression and CXCL12β levels [57]. Intracolonic administration of miR-141 precursors and inhibitors to both TNBS-colitis mice and IL-10 knockout mice alleviated and aggravated colitis, respectively [57].

miRNAs and autophagy in IBD
Autophagy is a cellular response that degrades and recycles internal structures, such as organelles and macromolecules. Therefore, its purpose is to maintain cell homeostasis and regulate inflammatory responses [58]. Defects in autophagy can cause epithelial dysfunction and immune disruption, which play important roles in the pathogenesis of IBD [59].
Recent studies suggest that miRNAs target autophagy-susceptibility genes to regulate intestinal autophagy and control intestinal inflammation in IBD [60]. ATG16L1 is an autophagy-related gene that forms autophagosomes during autophagy [61]. miR-346 was reported to downregulate the expression of the vitamin D receptor, glycogen synthase kinase 3 beta (GSK3B), to increase the level of ATG16L1 in colon biopsy samples of IBD patients [62]. miR-665 represses X-box binding protein 1 (XBP1) and ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), which also stimulates autophagy [63].

Role of miRNAs in IBD diagnosis
Diagnosis and evaluation of IBD have always been challenging. IBD is diagnosed based on clinical manifestations and endoscopy with histopathological examination [78]; however, various clinical manifestations make diagnosis difficult, and endoscopy with histopathology requires the expertise of clinicians [79]. As miRNAs are known to be associated with the pathogenesis of IBD, several studies have suggested that miRNAs are non-invasive and inexpensive biomarkers. A list of possible candidates is provided in Table 2.
Among the miRNAs included in the differential analysis of IBD colon tissues, miR-21, miR-31, and miR-141 are suggested to be valuable targets for the diagnosis of IBD. miR-21 is known to be an important factor in the pathogenesis of IBD as it modulates T-cell responses [90] and disrupts intercellular tight junctions in the intestinal epithelium [91]. miR-21 was found to be elevated in the mucosal tissue of both UC and CD [92,93], and the knockout of miR-21 in a mouse model of DSS-induced colitis inhibited the inflammatory response and increased survival rate, indicating the importance of miR-21 in IBD [52]. miR-31 was upregulated in the colon tissues of both UC and CD patients [88]; this miRNA is known to target IL-25 to stimulate the Th1/Th17-mediated pro-inflammatory molecular pathway in mouse colitis models [94]. miR-31 has also been suggested to be capable of distinguishing IBD from microscopic colitis, as its expression is elevated in IBD compared to microscopic colitis [95]. miR-141 inhibits CXCL5 and CXCL12β, and its downregulation in IBD is expected to stimulate leukocyte recruitment and the consequent inflammatory response. A decrease in miR-141 was observed in the colon tissues of patients with UC [87].
Gallo et al. suggested that miRNAs in human saliva and serum samples mainly exist in the form of exosomes (encapsulated microvesicles) [104]. Vickers et al. presented evidence that miRNAs are transported by high-density lipoprotein (HDL) to recipient cells [105]. Exosomes and HDL can protect miRNAs from ribonucleases in the blood, which makes it easier to measure miRNA levels in serum samples.

Role of miRNAs in the treatment of IBD
As in vitro studies have confirmed the importance of miRNAs in the pathophysiology of IBD, some researchers have attempted to administered miRNAs in vivo to mice with experimental colitis. Intracolonic administration of miRNA mimic molecules and antagonists led to the overexpression and inhibition of miRNA expression, respectively [106].

Nanoparticle-mediated approach
Tian et al. found that the delivery of the miR-31 mimic into the colon of DSS-colitis mice alleviated the inflammatory response. miR-31 was bound to encapsulating microspheres and administered via enema, resulting in a slower loss of body weight and colon length, as well as increased epithelial proliferation and reduced inflammation, as confirmed by colon biopsy [107]. Neudecker et al. also reported the attenuation of occult bleeding, weight loss, and edema in DSS-colitis mice after intracolonic administration of synthetic murine miR-223 mimetic in nanoparticle lipid emulsion [108]. The intracolonic administration of miR-141 precursors in mice with TNBS-induced colitis resulted in a decrease in CXCL12β expression and leukocyte infiltration in the intestine [57]. miR-146b was also found to alleviate intestinal inflammation when administered into the peritoneum of DSS-induced colitis mice with expression vectors [74]. By using a mouse model of TNBS-induced colitis, Cheng et al. demonstrated that the intracolonic administration of pre-miR-19b can suppress the inflammatory process, with in vitro results suggesting that miR-19b inhibits suppressor of cytokine signaling 3 (SOCS3) to reduce inflammation of the intestinal epithelium in IBD [109]. miR-200b bound to microvesicles was administered to intestinal epithelial cells in vitro and the colon of TNBS-colitis mice in vivo; this administration resulted in the attenuation of intestinal fibrosis with suppression of TGF-β1 mediated epithelium-to-mesenchyme transition [110].
Other routes of administration have been attempted to deliver miRNA mimic molecules to mice with colitis. Zhang et al. purified nanoparticles from edible ginger and found that their oral administration by colitis mice reduced the severity of colitis and promoted wound healing. The nanoparticles contained around 125 miRNAs. Further, the colon samples of mice administered the nanoparticles had a decrease in pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), increase in anti-inflammatory cytokines (IL-10 and IL-22), and increase in the proliferation of the epithelium based on the biopsy [111]. Fukata et al. attempted to intravenously administer miR-29a-3p and miR-29b-3p bound to supercarbonate apatite. These researchers reported a decrease in the inflammatory response in DSS-colitis mice and revealed that the subcutaneous injection of miR-29b bound to supercarbonate apatite inhibited the immune response by targeting CD11c + dendritic cells in a DSS-colitis model [112].

Conclusion
IBD is a multifactorial disease associated with environmental and genetic factors. Dysbiosis of commensal bacteria and breakdown of the intestinal barrier integrity are considered to be major factors in the development of IBD. However, recent studies have indicated that genetic factors, including miRNA dysregulation, play a major role in the pathophysiology of IBD. Numerous miRNAs participate in the complex regulatory system of intestinal inflammation. Further, the number of molecular interactions seems to be uncountable; however, recent findings suggest the targeting of certain miRNAs as clinical biomarkers or treatment options. Additional in vivo studies should be conducted to validate data from in vitro studies and assess the practicality of manipulating miRNA expression in IBD.