MicroRNAs in inflammatory bowel disease - pathogenesis, diagnostics and therapeutics

Abstract

The pathogenesis of inflammatory bowel disease (IBD) is complex and largely unknown. Until recently, research has focused on the study of protein regulators in inflammation to reveal the cellular and molecular networks in the pathogenesis of IBD. However, in the last few years, new and promising insights have been generated from studies describing an association between an altered expression of a specific class of non-coding RNAs, called microRNAs (miRs or miRNAs) and IBD. The short (approximately 22 nucleotides), endogenous, single-stranded RNAs are evolutionary conserved in animals and plants, and regulate specific target mRNAs at the post-transcriptional level. MiRNAs are involved in several biological processes, including development, cell differentiation, proliferation and apoptosis. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the genes in the human genome. Thus, miRNA deregulation often results in an impaired cellular function, and a disturbance of downstream gene regulation and signaling cascades, suggesting their implication in disease etiology. Despite the identification of more than 1900 mature human miRNAs, very little is known about their biological functions and functional targets. Recent studies have identified dysregulated miRNAs in tissue samples of IBD patients and have demonstrated similar differences in circulating miRNAs in the serum of IBD patients. Thus, there is great promise that miRNAs will aid in the early diagnosis of IBD, and in the development of personalized therapies. Here, we provide a short review of the current state-of-the-art of miRNAs in IBD pathogenesis, diagnostics and therapeutics.

INTRODUCTION

Inflammatory bowel disease (IBD), whose incidence is increasing[1], comprises a number of intestinal chronic relapsing inflammatory disorders[2-4], among which ulcerative colitis (UC)[5] and Crohn’s disease (CD)[6] are the two main entities. The pathogenesis of IBD remains largely unknown[7,8], but involves a complex interaction between genetic, environmental, and immunological factors[9,10]. In this context, research on microRNAs (miRs or miRNAs) is a promising new research, providing novel insights into the pathogenesis of IBD, biomarker identification, and treatment. Since their discovery and initial characterization in 1993[11,12], the number of miRNA sequences deposited in the miRBase database (Sanger Database) has grown continuously[13]. Numerous investigations have uncovered the key roles of miRNAs in several physiological networks[14-18]. Given their ability to target mRNAs and their predicted regulation of the expression of nearly one-third of all human transcripts, miRNAs have been linked to critical processes underlying development and tissue homoeostasis. Thus, dysregulation of miRNAs is implicated in the pathogenesis of several human diseases[18-26], including gastrointestinal disorders, such as cancer and inflammation[27-29]. Actually, loss of intestinal miRNAs in mouse models has been shown to impair differentiation of intestinal cells and epithelial barrier function, resulting in inflammation[30].

The discovery and role of miRNAs in IBD, particularly their role in cell signaling, seems to offer a new way of understanding this chronic disease and gives rise to new diagnostic tools and potential therapeutic strategies. Thus, it is possible that some miRNAs could serve as biomarkers for IBD, aiding early diagnosis, and lead to the development of personalized therapies.

Here we provide a short review of the current research in miRNAs in IBD; including pathogenesis, diagnostics and therapeutics.

MiRNA BIOGENESIS

MiRNAs are a recently discovered class of short (18-24 nucleotides in length), endogenous, non-coding single-stranded RNAs that regulate gene expression by controlling the stability and translation of protein-coding mRNAs[26,31,32]. A number of biological processes are regulated by miRNAs, including cell differentiation, proliferation, apoptosis, and cell cycle control[30,33,34]. Long primary miRNA transcripts (pri-miRNA) are initially transcribed by RNA polymerase II or III in the nucleus, and are then cleaved to precursor hairpin (pre-miRNA) by the microprocessor complex, Drosha and DiGeorge syndrome critical region gene 8[35-37]. Next, the pre-miRNAs are exported into the cytoplasm and further cleaved to mature miRNAs by the RNase III endonuclease, Dicer, in a complex with a trans-activator RNA binding protein[38-40]. One RNA strand from the mature miRNA is incorporated into the RNA-induced silencing complex, and guides this complex to the 3’ untranslated regions of “target” mRNA sequences, which in mammalian cells induces target mRNA degradation and suppresses protein expression[31,32,41-44].

ABBERANT EXPRESSION OF MiRNAS IN INFLAMMATORY BOWEL DISEASE

In 2008, Wu et al[45] were the first to report miRNA expression in colonic mucosa samples from IBD patients. They identified 11 miRNAs that were differentially expressed in active UC vs controls (Table 1)[45] and demonstrated an inverse relationship between macrophage inflammatory peptide-2α (previously shown to be implicated in IBD[46]) and miR-192. Similarly, Bian et al[47] found miR-150 to be significantly upregulated in inflamed colonic mucosa of UC patients, as compared to controls (Table 1), and they established an inverse correlation between miR-150 and its target, c-Myb[48], a proto-oncogene that is involved in apoptosis. Consequently, these two studies have exposed new and important insights into the pathogenesis of IBD.

Table 1.

Overview of dysregulated miRNAs reported to date in tissues from inflammatory bowel disease patients

miRNAs	Sample type	Population (n)	Approach	Reference
miRs-192, 375, 422b, 16, 21, 23a, 24, 29a, 126, 195, and let-7f	Sigmoid colon biopsies	Active UC (n = 15) vs healthy controls (n = 15)	Microarray and qRT-PCR	Wu et al[45]
miR-21 and miR-155	Sigmoid colon biopsies	Active UC (n = 12) vs healthy controls (n = 12)	Microarray and qRT-PCR	Takagi et al[49]
miRs-19b, 629, 23b, 106a, and 191	Sigmoid colon biopsies	Active CD (n = 5) vs healthy controls (n = 13)	Microarray and qRT-PCR	Wu et al[50]
miRs-16, 21, 223, and 594	Terminal ileum biopsies	Active CD (n = 6) vs healthy controls (n = 13)	Microarray and qRT-PCR	Wu et al[50]
miRs-188-5p, 215, 320a, 346, 7, 31, 135b, 223, 29a, 29b, 126*, 127-3p, and 324-3p	Colon biopsies	Active UC (n = 8) vs healthy controls (n = 8)	qRT-PCR	Fasseu et al[51]
miRs-188-5p, 215, 320a, 346, 196a, 29a, 29b, 126*, 127-3p, and 324-3p	Colon biopsies	Inactive UC (n = 8) vs healthy controls (n = 8)	qRT-PCR	Fasseu et al[51]
miRs-9, 126, 130a, 181c, 375, 26a, 29b, 30b, 34c-5p, 126*, 127-3p, 133b, 155, 196a, 324-3p, 21, 22, 29c, 31, 106a, 146a, 146b-3p, and 150	Colon biopsies	Active CD (n = 8) vs healthy controls (n = 8)	qRT-PCR	Fasseu et al[51]
miRs-9*, 30a*, 30c, 223, 26a, 29b, 30b, 34c-5p, 126*, 127-3p, 133b, 155, 196a, 324-3p, 21, 22, 29c, 31, 106a, 146a, 146b-3p, and 150	Colon biopsies	Inactive CD (n = 8) vs healthy controls (n = 8)	qRT-PCR	Fasseu et al[51]
miRs-150, 196b, 199a-3p, 199b-5p, 223, and 320a	Colon biopsies	Inactive UC (n = 8) vs Inactive CD (n = 8)	qRT-PCR	Fasseu et al[51]
miR-7	Colon biopsies	Active CD (n = 8) vs healthy controls (n = 6)	qRT-PCR	Nguyen et al[53]
miR-150	Colon biopsies	Active UC (n = 5) vs healthy controls (n = 4)	qRT-PCR	Bian et al[47]
miR-196	Colon biopsies	Active CD (n = 83) vs healthy controls (n = 67)	qRT-PCR and ISH	Brest et al[54]
miR-143 and miR-145	Colon biopsies	Active UC (n = 8) vs healthy controls (n = 8)	qRT-PCR	Pekow et al[52]

Both downregulated miRNAs (miRs) and upregulated (bold characters) are shown. CD: Crohn’s disease; ISH: In situ hybridization; UC: Ulcerative colitis; qRT-PCR: Quantitative real-time polymerase chain reaction.

In 2010, three studies[49-51] identified altered miRNA expression in IBD tissue but without concomitant functional analyses. In a cohort of 12 controls and 12 active UC patients, Takagi et al[49] reported miR-21 and miR-155 to be significantly upregulated in inflamed colonic UC tissue (Table 1). In a second study, Wu et al[50] assayed the expression of 467 miRNAs in patients with sigmoid CD and in patients with active terminal ileal CD. They found five miRNAs to be associated with active sigmoid CD, and four miRNAs were significantly increased in active ileal CD, as compared to control tissues (Table 1)[50]. These reports were followed by a similar study by Fasseu et al[51] evaluating the expression of more than 300 miRNAs in colonic tissue samples of UC and CD patients using quantitative real-time polymerase chain reaction analysis. Several miRNAs were differentially expressed in accordance with disease type, but with a large number in common to both groups (Table 1). They identified a set of eight miRNAs (miR-26a, miR-29a, miR-29b, miR-30c, miR-126*, miR-127-3p, miR-196a, and miR-324-3p) defining quiescent IBD vs controls, and a distinct subset of 15 miRNAs that could differentiate between quiescent UC and CD (n = 16) (Table 1)[51]. These three studies illustrate the potential use of miRNAs as biomarkers and the possibilities of developing miRNA profile-based diagnostic tools.

Other recent studies have focused on certain specific miRNAs and their association with target genes. Hence, Pekow et al[52] reported an inverse correlation of the tumor suppressors miR-143 and miR-145 with their target genes, IRS-1 (miR-145), and K-RAS, API-5 and MEK-2 (miR-143). Similarly, Nguyen et al[53] demonstrated decreased levels of miR-7 in inflamed CD colonic tissue, where expression of its target, CD98, was upregulated compared with control tissue. Dysregulation of CD98 interferes with the natural proliferation and differentiation of enterocytes. These two studies[52,53] not only provided new information on the pathophysiology of the well known, but poorly understood, inflammation-driven neoplastic progress in the colonic mucosa of UC, but also provided targets for future therapeutic interventions.

Some studies have focused on single miRNAs and their relation to single-nucleotide polymorphisms (SNPs). Brest et al[54] found increased expression of miR-196 restricted to intestinal epithelial cells within inflamed CD as compared to controls. They showed that miR-196 binds to, and correlates with, a decreased expression of the immunity-related GTPase M (IRGM) protective variant (c.313C) during inflammatory conditions, but not with the IRGM c.313C>T polymorphism, IRGM^T, which is strongly associated with CD in European populations[55]. In a similar study, Zwiers et al[56] found that the mutation (rs10889677 C>A) in the IL-23R gene, associated with IBD, results in loss of its binding sites for let-7e and let-7f miRNAs, leading to sustained IL-23R expression in vitro, which contribute to the chronicity of IBD. Thus, these studies indicate that single SNPs located at miRNA-binding sites are likely to affect the expression of their targets and that they might contribute to the pathogenesis of IBD.

In a semi-invasive approach and with diagnostic intentions, few studies have used whole blood instead of colonic tissue. As a proof of concept, Wu et al[57] performed a microarray-based study on whole blood from IBD patients and found a panel of differentially expressed miRNAs (Table 2) that enabled them to distinguish active IBD subtypes from each other and from controls. In a similar study by Zahm et al[58], higher concentrations of 11 miRNAs (Table 2) were found in the sera of pediatric CD patients vs controls. Receiver operating characteristic analyses resulted in a diagnostic sensitivity above 80% for CD[58]. Recently, Paraskevi et al[59] identified that 11 circulating miRNAs that were differentially expressed in blood samples from active CD vs controls, and identified a set of six miRNAs that were significantly elevated in active UC vs healthy controls (Table 2). Similarly, Duttagupta et al[60] found seven circulating miRNAs that were differentially expressed in UC patients vs controls (Table 2).

Table 2.

Overview of dysregulated circulating miRNAs reported to date in sera of inflammatory bowel disease patients

miRNAs	Sample type	Population (n)	Approach	Reference
miRs-149*, miRplus-F1065, 199a-5p, 362-3p, 340*, 532-3p, and miRplus-E1271	Peripheral blood	Active CD (n = 14) vs healthy controls (n = 13)	Microarray and qRT-PCR	Wu et al[57]
miR-149* and miR-340*	Peripheral blood	Inactive CD (n = 5) vs healthy controls (n = 13)	Microarray and qRT-PCR	Wu et al[57]
miRs-505*, 28-5p, 151-5p, 103-2*, 199a-5p, 340*, 362-3p, 532-3p, and miRplus-E1271	Peripheral blood	Active UC (n = 13) vs healthy controls (n = 13)	Microarray and qRT-PCR	Wu et al[57]
miRs-505*, 28-5p, 103-2*, 149*, 151-5p, 340*, 532-3p, and miRplus-E1153	Peripheral blood	Active UC (n = 10) vs active CD (n = 14)	Microarray and qRT-PCR	Wu et al[57]
miRs-195, 16, 93, 140, 30e, 20a, 106a, 192, 21, 484, and let-7b	Serum	Active CD (n = 46) vs healthy controls (n = 32)	LDA qRT-PCR	Zahm et al[58]
miRs-16, 23a, 29a, 106a, 107, 126, 191, 199a-5p, 200c, 362-3p, and 532-3p	Peripheral blood	Active CD (n = 128) vs healthy controls (n = 162)	qRT-PCR	Paraskevi et al[59]
miRs-16, 21, 28-5p, 151-5p, 155, and 199a-5p	Peripheral blood	Active UC (n = 88) vs healthy controls (n = 162)	qRT-PCR	Paraskevi et al[59]
miRs-188-5p, 422a, 378, 500, 501-5p, 769-5p, and 874	Peripheral blood	UC (n = 20) vs healthy controls (n = 20)	qRT-PCR	Duttagupta et al[60]

Downregulated miRNAs (miRs) as well as upregulated (bold characters) are shown. CD: Crohn’s disease; LDA: Low-density array; UC: Ulcerative colitis; qRT-PCR: Quantitative real-time polymerase chain reaction.

Although these studies are preliminary, they demonstrate the possibilities of developing a much-needed semi-invasive test based on differentially expressed peripheral blood miRNAs.

FUTURE USE IN DISEASE DIAGNOSTICS AND THERAPEUTICS

The studies described above have significantly increased our knowledge regarding the pathogenesis of IBD and have demonstrated the usefulness of miRNAs, not only as potential biomarkers, but also as latent targets for therapeutic interventions. However, a range of obvious challenges lie ahead, especially as identification of new miRNAs is an ongoing process. In 2008, Wu et al[45] used a miRNA microarray containing 553 known human miRNA genes, but currently more than 1900 human miRNA sequences are known[13], and new ones are identified on an almost daily basis. Consequently, this area of research is in its infancy, and future studies in the field of IBD need to focus on miRNA sequencing and using larger cohorts to address two important challenges: (1) identification of all of the miRNAs that are consistently dysregulated in IBD; and (2) to identify all of the targets of the miRNAs involved in IBD. If these challenges are met, the subsequent possibilities regarding diagnostics and therapeutics seem endless.

The potential clinical use of miRNAs is best illustrated by the most investigated and well-described miRNA, miR-21, also classified as an “oncomiR”[61,62]. The almost omnipresent overexpression of miR-21 in human cancers[62-64], including colorectal cancer, provides new options for cancer therapy[65]. Interestingly, as listed in Tables 1 and 2, miR-21 is the only miRNA commonly found to be upregulated in inflamed tissue or sera of IBD patients[45,49-51,58]. Thus, miR-21 is a potential biomarker for active IBD. It may also be of significant importance for the pathogenesis of IBD, as expression of miR-21 is mediated by nuclear factor-κB[66] - a key transcription factor involved in the pathogenesis of various human diseases, including IBD[67,68]. Unfortunately, the importance of these miRNA pathways in disease pathogenesis is unknown. Thus, functional studies are needed to address the role of miRNAs in IBD and particularly their role in cell signaling.

CONCLUSION

Currently, several clinical trials are testing the therapeutic efficacy of miRNA-based therapies; one such miRNA-target drug is “miravirsen”[69], a specific inhibitor of miR-122 that is currently in phase II clinical trials for Hepatitis C infections[70]. Within the next several years, further studies will undoubtedly provide a basis for more successful clinical trials and provide more insights in to the efficacy of miRNA-based therapeutics, including IBD.