miRNAs: how many in IBD?

Abstract

Purpose of review

MicroRNAs (miRNAs), small noncoding RNA molecules of approximately 22 nucleotides, have emerged as critical mediators of gene expression. As the dysregulation of gene expression can have far reaching impact on health and disease, miRNAs are being examined as potent new mediators of disease as either biomarkers or potential therapeutic targets. The purpose of this review is to evaluate the contribution of miRNAs to inflammatory bowel disease (IBD) pathophysiology.

Recent findings

Recent studies have evaluated the expression of miRNAs in tissue and body fluid specimens from patients with of the main subtypes of IBD -Crohn’s disease (CD) and ulcerative colitis (UC). Unique miRNA expression patterns that may distinguish IBD subtypes have been uncovered.

Summary

Significant progress has been made in illuminating the complex interactive networks of miRNAs and gene targets in IBD. The potential use of miRNAs as disease biomarkers or therapeutics shows promise. However, there are still significant hurdles to overcome before miRNA-based therapeutics and diagnostics will be of clinical utility.

Introduction

The inflammatory bowel diseases (IBD) are a group of chronic inflammatory conditions affecting the gastrointestinal tract. IBD is marked by periods of active disease flare-ups followed by remissions, either of which can last variable periods of time. While Crohn’s disease (CD) and ulcerative colitis (UC) are the two major forms of IBD, there are a number of other less common forms of IBD, including indeterminate colitis and microscopic colitis. Whereas CD can affect any portion of the gastrointestinal tract and is commonly patchy with a transmural inflammatory pattern, UC is limited to the large intestine and has a continuous inflammatory pattern that involves only the mucosa. A commonality between CD and UC is a dysregulated immune response in the intestine. Based on current evidence, IBD develops in genetically susceptible individuals due to complex interactions between environmental stimuli, the genome, the microbiome, and an inappropriate mucosal immune response. Genome wide association studies (GWAS) and linkage analyses have established a strong genetic component to IBD (stronger in CD than UC) with the identification of about 200 loci associated with the development of IBD [1–15]. However, this only accounts for 16–23% of the heritability of IBD [16–18]. Lack of concordance between twin siblings (one sibling is affected with IBD while the other is disease-free) has further established that other factors-epigenetics, epistasis, and environmental factors-influence the development of IBD [19–22]. This review will discuss some of the current findings regarding the role of microRNAs (miRNAs) in the pathophysiology of IBD and the outlook for miRNA-based diagnostics and therapeutics.

Overview of miRNAs

MicroRNAs are non-coding single stranded RNAs of 21–23 nucleotides derived from primary miRNA transcripts (pri-miRNA) of intergenic or intronic origin [23]. Originally discovered in the early 1990’s, the first miRNA, Lin-4, regulates Caenorhabditis elegans development through repression of Lin-14 and Lin-28 [24,25]. Despite this observation, the significance of miRNAs continued to be largely underappreciated until the early 2000’s when they were recognized as a distinct class of RNAs. Prior to 2002, when miRNAs were named the breakthrough of the year, there were less than 5oo miRNA publications. In 2015, as evidence of the import of miRNAs in health and disease, there were over 9000 miRNA publications in PubMed.

The primary function of miRNAs is to regulate gene expression at the post-transcriptional level. This begins with the biogenesis of miRNAs. miRNAs are transcribed as longer primary miRNAs (pri-miRNAs) of several hundred nucleotides from either a distinct gene or coupled to a host gene (e.g. intronic source). This distinction is important for regulatory mechanisms as the former will operate as its own transcriptional unit while the latter’s transcription will be tied to that of the host protein coding gene. miRNA biogenesis occurs in a stepwise process using multiple RNA-binding proteins (DGCR8) and endoribonucleases (Drosha and Dicer). In the nucleus, the RNA-binding protein DGCR8 binds the RNA-specific endoribonuclease enzyme (ribonuclease type III) Drosha to form the microprocessor complex thereby initiating miRNA processing to cleave the pri-miRNA to the precursor miRNA (pre-miRNA) stem-loop structure. The pre-miRNA is exported to the cytoplasm where it undergoes further cleavage by Dicer ribonuclease to remove the stem loop. The active strand of the resulting miRNA:miRNA* duplex, of approximately 22 nucleotides in length, is incorporated into the RNA-induced silencing complex (RISC), a multisubunit complex composed of Argonaute proteins. The miRNA loaded RISC (miRISC) is now competent to induce gene silencing as the loaded miRNA provides specificity by binding to complimentary sequences on the target miRNA 3′ untranslated region (UTR). Gene silencing proceeds depending on this complementarity between the miRNA and its target mRNA: (i) mRNA degradation is favored with complete complementarity (primarily plants) [23], (ii) post-translational repression through mismatch binding of the 3′-UTR of the target gene, or (iii) mRNA destabilization via deadenylation of the poly(A) tail [23]. One of the key features of miRNAs is their redundancy-a single miRNA can regulate multiple target genes and conversely, a single target gene will have multiple miRNAs with the potential to regulate it. This is one of the great challenges in miRNA research; defining which of the many miRNAs that may regulate a specific gene is the most important goal, particularly since many of the miRNA effects are relatively mild.

It is well established now that miRNAs play essential roles in the regulation of multiple vital developmental processes from cell differentiation and organogenesis to the fine-tuning of metabolic and physiochemical responses. As with such a large biological footprint, up to 60% of genes are targeted by miRNAs, abnormalities in miRNA expression could and do pose problems that contribute to the development of disease. Chronic lymphocytic leukemia was among the first human diseases associated with miRNA dysregulation, and dysregulated miRNA expression has since been found in numerous other disease states [26]. This applies to IBD, as there have been a number of recent studies examining the expression of miRNAs in IBD. Distinct miRNA expression patterns have been observed in intestinal biopsies, peripheral blood, and saliva.

Mucosal tissue miRNA expression

A number of studies have examined intestinal biopsies as a first step to define the miRNAs that are differentially expressed at the site of IBD pathology. The most common design for these studies was to compare patients with active CD or UC versus healthy controls. A few more recent studies have included subjects with inactive CD or UC as comparison groups. Other studies have examined the miRNA expression in biopsies from areas of active disease versus areas ostensibly normal by endoscopic appearance.

The formative studies into the role of miRNAs in IBD were pioneered by an initial study aimed primarily at delineating miRNAs linked to UC. In this study, Wu et al. compared intestinal biopsies from 15 healthy controls to 15 active UC and 15 inactive UC patients; microscopic colitis, irritable bowel syndrome, and CD patients were included in the analysis as well [27]. Among 11 miRNAs identified as having differential expression, miR-21 and miR-29a were found to have elevated expression in the active UC group while miR-192 expression was reduced [27]. Macrophage inflammatory peptide 2-α was identified as a regulatory target of miR-192 and its expression was found to have an inverse relationship with miR-192 [27]. A follow up study was performed in CD colitis and ileitis patients to identify colonic- and ileal-specific miRNAs, respectively. miR-23b, miR-191, and miR-106a were elevated while miR-19b and miR-629 were reduced in CD colitis [28]. miR-16, miR-21, miR-223, and miR-594 were elevated in CD ileitis [28]. Additional studies by Bian et al., Brest et al., Coskun et al., Fasseu et al., Feng et al., Lin et al., Min et al., Takagi et al., Yang et al., and others identified additional miRNAs with dysregulated expression in IBD in comparison to healthy controls (summarized in Tables 1 & 2).

Table 1.

miRNAs with altered expression in Crohn’s disease

miRNA	Tissue/Body Fluid Type	Expression	Citation
miR-16	Intestinal biopsy, peripheral blood/serum	Elevated	Paraskevi et al.[28], Wu et al. [27]
miR-19b	Intestinal biopsy, peripheral blood/serum	Reduced	Cheng et al. [29], Lewis et al. [30]
miR-21	Intestinal biopsy	Elevated	Fasseu et al. [31], Polytarchou et al. [32], Wu et al. [27], Zahm et al. [33]
miR-23a	Peripheral blood/serum	Elevated	Paraskevi et al.[28]
miR-26a	Saliva	Elevated	Schaefer et al.[34]
miR-29a	Peripheral blood/serum	Elevated	Paraskevi et al.[28]
miR-31	Intestinal biopsy	Elevated	Fasseu et al. [31], Lin et al.[35], Peck et al.[36], Schaefer et al.[34]
miR-101	Intestinal biopsy, peripheral blood/serum, saliva	Elevated	Schaefer et al.[34]
miR-106a	Intestinal biopsy, peripheral blood/serum	Elevated	Fasseu et al. [31], Paraskevi et al.[28], Wu et al. [27]
miR-107	Peripheral blood/serum	Elevated	Paraskevi et al.[28]
miR-126	Peripheral blood/serum	Elevated	Paraskevi et al.[28]
miR-146a	Intestinal biopsy	Elevated	Béres et al. [37], Polytarchou et al. [32], Schaefer et al.[34]
miR-148a	Intestinal biopsy	Elevated	Cheng et al.[29]
miR-155	Intestinal biopsy	Elevated	Béres et al. [37]
miR-191	Peripheral blood/serum	Elevated	Paraskevi et al.[28]
miR-192	Peripheral blood/serum	Elevated	Zahm et al. [33]
miR-196	Intestinal biopsy	Elevated	Brest et al.[38]
miR-199a-5p	Peripheral blood/serum	Elevated	Paraskevi et al.[28], Wu et al.[39]
miR-200c	Peripheral blood/serum	Elevated	Paraskevi et al.[28]
miR-203	Intestinal biopsy	Reduced	Peck et al.[36]
miR-215	Intestinal biopsy	Elevated	Peck et al.[36]
miR-223-3p	Intestinal biopsy, peripheral blood/serum	Elevated	Peck et al.[36], Wang et al.[40], Wu et al. [27]
miR-301a	Intestinal biopsy, peripheral blood/serum	Elevated	He et al.[41]
miR-320a	Intestinal biopsy	Reduced	Pierdomenico et al.[42]
miR-320b	Intestinal biopsy	Reduced	Pierdomenico et al.[42]
miR-320c	Intestinal biopsy	Reduced	Pierdomenico et al.[42]
miR-375	Intestinal biopsy	Reduced	Schaefer et al.[34], Zahm et al. [33]
miR-362-3p	Peripheral blood/serum	Elevated	Paraskevi et al.[28], Wu et al.[39]
miR-532-3p	Peripheral blood/serum	Elevated	Paraskevi et al.[28], Wu et al.[39]

Table 2.

miRNAs with altered expression in ulcerative colitis

miRNA	Tissue/Body Fluid Type	Expression	Citation
let-7i	Intestinal biopsy	Elevated	Zahm et al. [33]
miR-19a	Intestinal biopsy	Elevated	Schaefer et al.[34]
miR-20b	Intestinal biopsy	Elevated	Coskun et al.[43]
miR-21	Intestinal biopsy, peripheral blood/serum, saliva	Elevated	Feng et al. [44], Paraskevi et al.[28], Polytarchou et al. [32], Schaefer et al.[34], Takagi et al. [45], Yang et al.[46], Wu et al.[26], Zahm et al. [33]
miR-23a-3p	Peripheral blood/serum	Elevated	Polytarchou et al. [47]
miR-24	Intestinal biopsy	Elevated	Zahm et al. [33]
miR-26	Intestinal biopsy	Reduced	Koukos et al. [48]
miR-28-5p	Peripheral blood/serum	Elevated	Paraskevi et al.[28], Wu et al.[39]
miR-29a	Intestinal biopsy	Elevated	Fasseu et al. [31], Wu et al.[26]
miR-31	Intestinal biopsy, saliva	Elevated	Fasseu et al. [31], Lin et al.[35], Schaefer et al.[34]
miR-101	Intestinal biopsy	Reduced	Koukos et al. [48]
miR-101	Intestinal biopsy, peripheral blood/serum	Elevated	Schaefer et al.[34]
miR-124	Intestinal biopsy (Pediatric only)	Reduced	Koukos et al. [48]
miR-125b	Intestinal biopsy	Elevated	Coskun et al.[43]
miR-126	Intestinal biopsy	Elevated	Fasseu et al. [31], Feng et al.[44]
miR-142-3p	Intestinal biopsy, peripheral blood/serum, saliva	Elevated	Schaefer et al.[34], Zahm et al. [33]
miR-142-5p	Peripheral blood/serum	Elevated	Schaefer et al.[34]
miR-146a	Intestinal biopsy	Elevated	Béres et al. , Polytarchou et al. [32], Zahm et al. [33]
miR-150	Intestinal biopsy	Elevated	Bian et al. [49]
miR-151-5p	Peripheral blood/serum	Elevated	Paraskevi et al.[28], Wu et al.[39]
miR-155	Intestinal biopsy, peripheral blood/serum	Elevated	Béres et al. [37], Min et al. [50], Paraskevi et al.[28], Takagi et al. [45]
miR-192	Peripheral blood/serum	Elevated	Zahm et al. [33]
miR-192	Intestinal biopsy	Reduced	Wu et al.[26], Zahm et al. [33]
miR-194	Intestinal biopsy	Reduced	Zahm et al. [33]
miR-199a-5p	Peripheral blood/serum	Elevated	Paraskevi et al.[28], Wu et al.[39]
miR-200b	Intestinal biopsy	Reduced	Zahm et al. [33]
miR-214	Intestinal biopsy	Elevated	Polytarchou et al. [32]
miR-223-3p	Peripheral blood/serum	Elevated	Koukos et al.[51], Polytarchou et al. [47], Schaefer et al.[34], Wang et al.[40]
miR-301a	Intestinal biopsy, peripheral blood/serum	Elevated	He et al. [41]
miR-320a	Intestinal biopsy	Reduced	Pierdomenico et al.[42]
miR-320b	Intestinal biopsy	Reduced	Pierdomenico et al.[42]
miR-320c	Intestinal biopsy	Reduced	Pierdomenico et al.[42]
miR-320e	Peripheral blood/serum	Elevated	Polytarchou et al. [47]
miR-375	Intestinal biopsy	Reduced	Zahm et al. [33]
miR-375	Peripheral blood/serum	Elevated	Schaefer et al.[34]
miR-494	Peripheral blood/serum	Elevated	Schaefer et al.[34]
miR-4284	Intestinal biopsy	Reduced	Koukos et al.[51]
miR-4454	Peripheral blood/serum	Elevated	Polytarchou et al. [47]

More recent studies in the last year have identified or confirmed additional miRNAs. In a comparison of formalin-fixed paraffin-embedded or fresh-frozen colon biopsies, Béres and colleagues identified miR-146a and miR-155 as elevated in UC and CD patients [29]. Using RNA sequencing (RNA-seq), Peck et al. identified miR-31-5p, miR-215, and miR-223-3p as elevated in CD while miR-203 was reduced [43]. Schaefer et al. identified miR-19a and miR-101 as additional miRNAs elevated in UC [47]. In contrast, Koukos et al. found miR-101 to be reduced in UC [37].

In a recent screen for novel regulators of nuclear factor kappa B (NF-κB) using miRNA inhibitors in human colonocytes, seven miRNAs (miR-7, miR-21, miR-146a, miR-181b, miR-214, miR-372, and miR-373) suppressed NF-κB phosphorylation and thus its activity [45]. In contrast, miRNA inhibitors for miR-26b and miR-199a promoted NF-κB phosphorylation and thus its activity [45]. When CD and UC colon biopsies were subsequently assayed for miRNA expression, miR-21 and miR-146a were elevated in both CD and UC while miR-214 was elevated only in UC [45]. PTEN and PDLIM2 were identified as targets of miR-214 and STAT3 as a transcriptional regulator of miR-214 expression [45].

Circulating miRNA expression

The development of an effective noninvasive and accurate test for disease has been a goal in the medical community for many diseases. This has been the case in the clinical diagnosis and treatment of IBD as well. Although endoscopic biopsies and histologic analysis remain the gold standard for diagnosing CD and UC, a number of less invasive tests are useful in the clinical management of IBD. Chief among these are fecal calprotectin levels and serum C-reactive protein levels. In an effort to improve the quality and accuracy of clinical tests of IBD, circulating miRNA expression has been explored as potential biomarkers of IBD. This is buoyed by several recent studies demonstrating that miRNAs exist in the peripheral blood, serum, and plasma in a stable RNase-resistant form [53,54].

Blood miRNA expression

An initial examination of peripheral blood miRNA expression in IBD patients relative to healthy controls revealed elevated expression of miR-199a-5p, miR-340-3p, miR-362-3p, and miR-532-3p in CD and UC [50]. miR-28-5p, miR-103-2-5p, and miR-151-5p were additionally elevated in UC while miR-505-5p and miR149-3p were reduced in UC and CD, respectively [50]. Duttagupta et al. analyzed miRNA expression after fractionating peripheral blood into microvesicle, peripheral blood mononuclear cell and platelet fractions [55]. Among 31 candidates within the platelet fraction, miR-188-5p, miR-422a, miR-378, miR-500, miR-501-55, miR-769-5p, and miR-874 were validated to have elevated expression in UC [55]. Zahm and colleagues identified 11 miRNAs with altered expression in pediatric CD (miR-16, let-7b, miR-195, miR-106a, miR-20a, miR-30e, miR-140, miR-484, miR-93, miR-192, and miR-21) [56]. Twelve miRNAs (miR-127-3p, miR-491-5p, miR-18a, miR-145, let-7b, miR-185, miR-29c, miR-19b, miR-20b, miR-106a, miR-17, and miR-222) were found to be elevated and one (miR-135a) reduced in serum in a study of CD and UC patients [57]. Paraskevi and colleagues reported eleven (miR-16, miR-23a, miR-29a, miR-106a, miR-107, miR-126, miR-191, miR-199a-5p, miR-200c, miR362-3p, and miR-532-3p) and five miRNAs (miR-16, miR-21, miR-28-5p, miR-151-5p, and miR-199a-5p) with elevated expression in CD and UC peripheral blood, respectively [42]. Another study examining the effects of anti-tumor necrosis factor-α (TNF-α) induction therapy on miRNA expression in the serum of CD patients found elevated expression of let-7d, let-7e, miR-28-5p, miR-221, and miR-224 at week 6 of treatment [58]. Patients that were in remission by week 14 of therapy had elevated levels of let-7d and let-7e compared to non-responders [58]. Several recent studies found miR-223 to be elevated in the peripheral blood and serum of CD and UC patients [46,47,49]. Polytarchou and colleagues reported that serum miR-223-3p, miR-4454, miR23a-3p, and miR320e levels correlated with UC disease activity better than serum C-reactive protein levels [46]. Lewis et al. identified miR-19a-3p and miR-19b-3p as markers of stricturing CD, both miRNAs were expressed at lower levels in the serum of CD patients with strictures in comparison to non-stricturing CD patients [39].

Salivary miRNA expression

Evaluation of oral fluid (saliva) has become an intriguing diagnostic tool for many diseases due to its ease of accessibility. A recent study examining miRNA expression in saliva found that, in UC patients, salivary miRNA expression of miR-21, miR-31, and miR142-3p was elevated in comparison to healthy controls [47]. Salivary expression of miR-26a and miR-101 was elevated in CD [47].

miRNA targeting of IBD-associated genes

GWAS, next generation sequencing, and other studies have been instrumental in identifying IBD-susceptibility loci to help understand the complex genetic and biologic pathways underlying disease development [9]. The identification of dysregulated miRNAs in IBD, and their concomitant ability to regulate gene expression, has led to the exploration of the relationship of these miRNAs to these IBD loci. The primary strategies employed have either used IBD-associated miRNAs to search for downstream gene targets or used specific IBD-associated genes to identify regulatory miRNAs. Several laboratories employed the latter candidate gene approach to identify miR-93, miR-106b, miR130a, and miR142-3p as regulators of ATG16L1, one of the most common polymorphisms associated with CD [59–63]. Cognizant of the import of NF-κB as a mediator of inflammatory signals in UC, Polytarchou et al. performed a screen in colonocytes to identify miRNAs that altered NF-κB activity; PTEN and PDLIM2 were identified as targets of miR-214 as a result [45]. Through these efforts, a number of IBD-associated genes have been demonstrated to be targets of miRNAs, including genes involved in autophagy (ATG16L1), inflammation (TNF-α), intestinal homeostasis (IL12B), and cytokine production (STAT3) (Table 3).

Table 3.

IBD-associated genes targeted by miRNAs

Gene	miRNA	Citation
ATG5	miR-30c miR181a	Nguyen et al.[59] Tekirdag et al.[63]
ATG16L1	miR-93 miR-106b miR130a miR142-3p	Lu et al.[58] Lu et al.[58]; Zhai et al.[61] Nguyen et al.[59] Zhai et al.[62]
CXCL5	miR-4284	Koukos et al.[51]
IFNG (IFNγ)	miR-29a, 29b, and 29c	Ma et al.[64]
IKBA (IκBα)	miR-126	Feng et al.[44]
IL10	miR-106a	Sharma et al.[65]
IL10RA (IL10Rα)	miR-15a miR-185 miR-211	Venza et al.[66] Venza et al.[66] Venza et al.[66]
IL12B (IL12p40)	miR-29a, 29b, and 29c miR-10a	Brain et al.[67] Wu et al.[68]
IL23R	Let-7f	Li et al.[69]
IRF5	miR-146a	Tang et al.[70]
IRGM	miR-196	Brest et al.[38]
NOD2	miR-10a miR-192 miR-320a, 320b, and 320c	Wu et al.[68] Chuang et al.[71] Pierdomenico et al.[42]
SOCS1	miR-155	Pathak et al.[72]; Wang et al.[73]; Yao et al. [74]
STAT3	miR-124	Koukos et al.[48]
TNFA (TNFα)	miR-187	Rossato et al.[75]

Biologic therapies (primarily humanized monoclonal antibodies) directed against inflammatory mediators involved in the pathophysiology of IBD have revolutionized medical treatment. Tumor necrosis factor antagonists (adalimumab, certolizumab pegol, golimumab, infliximab), anti-cell adhesion molecule antibodies [natalizumab (anti-ITGA4 antibody) and vedolizumab (anti-LPAM-1 antibody)], and IL12p40 antagonists (ustekinumab) are either currently used for treating IBD [77–82]. While these biologics have the advantage of specificity in comparsion to glucocorticoids and other immunosuppressive agents (azathioprine, methotrexate), not all patients respond to these medications and some encounter significant side effects [77–83].

Thus, miRNA-based therapeutics could represent a significant alternative to these biologics. The potential of developing miRNAs to target IBD-associated genes is intriguing. However, there are several limitations that have yet to be solved in terms of miRNA-based therapeutics. To be successful, miRNA modulators must be specific, efficient, and safely deliverable to the affected tissue. Off-target side effects are still a major concern as altering the function of a single miRNA could affect many downstream gene targets and pathways. Site-specific delivery of miRNA therapeutics remains challenging.

Nevertheless, preclinical and clinical studies have demonstrated efficacy of miRNA-based therapeutics in cancer, hepatitis C virus infection, and heart disease [84–89]. MRX34, a liposomal injection of a miR-34a mimic, is being developed as a treatment for liver cancer (Mirna Therapeutics-NCT01829971) and has progressed to the clinical trial stage. A Phase 1 clinical study was recently initiated for MRG-201, a miRNA mimic to microRNA-29b, as an anti-fibrotic agent for cutaneous and pulmonary fibrosis (miRagen Therapeutics).

Several miRNA-based therapeutic studies have reported positive results in animal models of IBD. A study using the azoxymethane (AOM)-dextran sulfate sodium (DSS) mouse model of colitis-associated colorectal cancer development reported tumor suppression in groups treated with a chemical inhibitor (anti-miR) of miR-214 [45]. Anti-miR inhibition of miR-30c and miR-130a reduced intestinal inflammation in a mouse ileal loop model [60]. In IL-10 knockout mice and TNBS models of colitis, intestinal inflammation was exacerbated or reduced after intracolonic administration of an anti-miR (for inhibition) or pre-miR miRNA mimic (for overexpression) to miR-141, respectively [90]. Overexpression of miR-146b ameliorated intestinal inflammation in DSS-treated mice [91].

Conclusion

While the exact biological mechanisms are not well understood, it is undeniable that miRNAs play a significant role in the pathogenesis of IBD. This leads to several important conclusions. The first is that several miRNAs were consistently altered across multiple studies reinforcing the import of these miRNAs in IBD. Second, these studies underscore the promise of miRNAs as biomarkers for diagnosing and monitoring disease activity in IBD as well as the potential of miRNA-based therapeutics. Future areas of investigation include continued improvement and refinement of miRNA diagnostics and therapeutics and identification of polymorphisms that impact miRNA binding sites.

Key points.

• MiRNAs are a class of small noncoding RNA that regulate gene expression while influencing many biological processes.

• Aberrant expression of miRNAs has been associated with many diseases including IBD.

• MiRNAs are promising as biomarkers and/or therapeutic targets in the management of IBD.