Epigenetics in autoimmune diseases: Pathogenesis and prospects for therapy

    Epigenetics in autoimmune diseases: pathogenesis and prospects for therapy Zimu Zhang, Rongxin Zhang PII: DOI: Reference:

S1568-9972(15)00116-0 doi: 10.1016/j.autrev.2015.05.008 AUTREV 1722

To appear in:

Autoimmunity Reviews

Received date: Accepted date:

15 May 2015 20 May 2015

Please cite this article as: Zhang Zimu, Zhang Rongxin, Epigenetics in autoimmune diseases: pathogenesis and prospects for therapy, Autoimmunity Reviews (2015), doi: 10.1016/j.autrev.2015.05.008

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ACCEPTED MANUSCRIPT Epigenetics in autoimmune diseases: pathogenesis and prospects for therapy Zimu Zhang1 & Rongxin Zhang1,* 1

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Laboratory of Immunology and Inflammation, Department of Immunology and Research Center of Basic Medical Science; Basic Medical College; Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Immune Microenvironments and Diseases of Educational Ministry of China, Tianjin Medical University, Tianjin 300070, China;

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* Correspondence: Rongxin Zhang, Research Center of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China, Email: [email protected] or [email protected], Telephone and Fax: 86-22-83336563

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Abstract Epigenetics is the study of heritable changes in genome function without underlying modifications in their nucleotide sequence. Disorders of epigenetic processes, which involve DNA methylation, histone modification non-coding RNA and nucleosome remodeling, may influence chromosomal stability and gene expression, resulting in complicated syndromes. In the past few years, it has been disclosed that identified epigenetic alterations give rise to several typical human autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and multiple sclerosis (MS). These emerging epigenetic studies provide new insights into autoimmune diseases. The identification of specific epigenetic dysregulation may inspire more discoveries of other uncharacterized mechanisms. Further elucidation of the biological functions and clinical significance of these epigenetic alternations may be exploited for diagnostic biomarkers and therapeutic benefits.

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Keywords: autoimmune disease, DNA methylation, epigenetics, epigenetic therapy, histone modification, ncRNA

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1. Introduction Autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS) and type 1 diabetes (T1DM), are characterized by the presence of autoreactive immune cells or the development of specific autoantibodies [1]. Autoimmune diseases possess diverse epidemiology or symptoms, however, they have a common origin and it is clear that genetic predisposition is involved in the etiopathology of autoimmune disorders[2]. Genetic studies revealed that the extended major histocompatibility complex (MHC), particularly human leukocyte antigen (HLA) genes, has a strong association with autoimmune diseases[3]. In recent decades, more susceptibility genes shared by most autoimmune diseases have been uncovered, such as PTPN22, IRF5, STAT4, BANK1 and ICAM3. These associated genes point to some common pathways, expanding our understanding of autoimmune diseases[1]. Whereas the high discordance in the onset rate of autoimmune diseases was subsequently observed in detailed investigations into monozygotic twins whose genetic information and susceptibility variants are virtually identical. The fact that different phenotypes arise from the same genotype suggests that environmental factors, other than genetics, also have an essential role in the onset and progression of diseases[4]. Epigenetics refers to the study of heritable alterations in genome function without underlying modifications in their nucleotide sequence[5]. Via epigenetic mechanisms, a number of internal and external environmental risk factors, including smoking, nutrition, viral infection and the exposure to chemicals, could exert their influence on the pathogenesis of autoimmune diseases. Such factors could impact the epigenetic mechanisms, which, in turn, build relationship with the regulation of gene expression, and eventually triggering immunologic events that result in instability of immune system[6]. For example, smoking, the best-known environmental risk factor, plays a 1

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critical role in autoimmune diseases. Epidemiologic studies of RA and MS showed that heavy-smokers had a significantly increased risk of getting diseases compared with never-smokers [7, 8]. In addition, increasing levels of serum Vitamin D, a steroid hormone, could decrease the risk of MS[9]. Epstein-Barr virus (EBV) exposure was hypothesized to be related to the increased risk of RA and SLE as well[10]. To bridge the gap between environmental and genetic factors, over the past few years, great progress has been made in identifying detailed epigenetic mechanisms for autoimmune diseases. Furthermore, with rapid advances in technological development, high-throughput screening approaches and other novel technologies support the systematic investigations and facilitate the epigenetic identification. These emerging epigenetic studies provide new insights into autoimmune diseases, raising great expectations among researchers and clinicians. They might reveal not only new clinical biomarkers for diagnosis and disease progression but also novel targets for potential epigenetic therapeutic treatment. In this review, we summarize and discuss the epigenetic alterations in autoimmune diseases, with a focus on prospective candidates for ‘epigenetic therapy’, and outline the future steps to be taken in the field. 2. Roles of epigenetic alterations in the pathogenesis of autoimmune diseases Epigenetic regulations plays a critical role in determining gene function and activities, meanwhile they remain highly sensitive and could be even reversed by environmental influences. Disorders of epigenetic processes, which involve DNA methylation, histone modification and non-coding RNA expression, were found associated with the pathogenesis of autoimmune diseases. Although research progress varies in different kinds of autoimmune diseases, they could provide useful references for each other. 2.1 DNA methylation DNA methylation, a dynamic process involving methylation and demethylation events, occurs in different regions of the genome and is crucially important for embryogenesis, cellular proliferation and differentiation [11, 12]. Methylation is established and maintained by the DNA methyltransferase (DNMT) family which is composed of a group of enzymes, including DNMT1, DNMT3a, DNMT3b, and DNMT3L. Among them, DNMT1 contributes to maintaining methylation status during replication. DNMT3a and DNMT3b are de novo DNMTs that are responsible for methylation during embryonic development. DNMT3L is reported to be a cofactor for the de novo DNMT stimulating DNMT3a/3b activity. Methyl-CpG-binding domain (MBD) proteins could also regulate methylation together with DNMTs[13]. Demethylation could be passive or active. Passive demethylation is induced by inhibition of DNMTs, providing the basis for treatments with the aim of erasing abnormal hypermethylation [14, 15]. Active demethylation might depend on the activity of cytosine deaminases occurring predominantly in cell differentiation and associated with the activation of immune cells. For instance, activation-induced cytidine deaminase (AICDA) plays a critical role in activating B cells as well as generating IgG anti-nucleosomal antibodies of murine SLE[16, 17]. In mammals, DNMTs catalyze the donation of a methyl group to CpG dinucleotides, particularly CpG islands located within promoter regions, leading to the structural changes of chromatin. Methylated regions block the accessibility to transcriptional activators and thereby inhibit the gene transcription.[13] In contrast, an unmethylated state will permit the transcription [14]. 2.1.1 DNA methylation alterations in lymphocytes The analysis of epigenetic alterations in CD4+ T cells from patients has revealed that DNA methylation pattern is profoundly disrupted (Table 1). A range of autoimmune diseases, including SLE, RA and systemic sclerosis (SSc), display global hypomethylation associated with a concurrent decreased expression of methylation-related genes, such as DNMT1 and MBD4. [18, 19]. CD4+ T cells from patients with SLE, RA and SSc show a substantial reduction in the levels of DNA methylation on the promoter of CD40LG (which encodes the B cell costimulatory molecule CD40L; also known as CD154)[20-22], which is thought to be important in disease pathogenesis given that the CD40-CD40L interaction is critically associated with Th17 cells differentiation and IL-17 production [23]. It is noteworthy that 2

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the fact that demethylation of CD40L regulatory elements on the female inactive X chromosome upregulates CD40L expression in CD4+ T cells may contribute to the susceptibility of women to these autoimmune diseases [20-22]. In addition, the hypomethylation of CD70 (also known as TNFSF7 or CD27L) is observed and probably contributes to the overexpression of CD70 by CD4+ T cells from patients with SLE, SSc and primary Sjögren's syndrome (pSS)[24-26]. Moreover, with the development of the genome-wide DNA methylation analysis approach, hypomethylation patterns were observed at interferon-regulated genes, such as IRF5, IFIT2, STAT1 and USP18, in CD4+ T cells in the context of SLE and pSS. This hypomethylation exists in T cells before activation and differentiation, as well as maintains in the immune system throughout and even beyond active stages of the disease. It might provide an explanation for the mechanism of type I interferon pathway playing an important role in the pathogenesis of SLE and pSS [27-29]. Despite of the importance of B cells in the pathogenesis of autoimmune diseases, few studies have addressed methylation changes in B cells. Garaud et al. reported that the E1B promoter of CD5 is hypomethylated in SLE B cells. High levels of CD5-E1B could block the inhibitory effects of CD5 on B cell receptor signaling and antibody production, leading to the activation of B cells. Moreover, in a recent study, HRES-1, the prototype of Human Endogenous Retroviruses (HERV), is demonstrated hypomethylated in SLE B cells. This hypomethylation might provide explanations for increasing detection of HERV in patients with autoimmune diseases including SLE, pSS and MS. On the other hand, in these two studies, high level of interleukin-6 (IL-6) is also showed in SLE B cells, which could reduce the expression of DNMT1 by arresting the cell cycle at late G1 phase and thereby affect the methylation of CD5 and HRES-1, contributing to disease activity [30, 31]. However, it is noteworthy that not all overexpressed genes in autoimmune diseases are accompanied by a concurrent epigenetic alteration. It has been demonstrated that IRF5 mRNA expression and CD40L on CD4+ T cells are increased in patients with pSS, but the aberrant DNA methylation profiles are absent from corresponding putative regulatory sequences[32, 33]. Further mechanisms leading to their overexpression need to be investigated. Moreover, not all epigenetic altered genes in lymphocytes are subject to hypomethylation. It has been reported that regulatory regions of FOXP3 in peripheral blood CD4+ T cells from patients with SSc, RA and T1DM is hypermethylated, affecting the expression of key transcription factor which is required for the generation of regulatory T cells[34-36]. These findings might indicate an epigenetic alteration responsible for the loss of immune homeostasis in development of SSc, T1DM and RA. 2.1.2 DNA methylation alterations in other cell types In addition to lymphocytes, a number of evidences support that DNA methylation pattern in other cell types could be altered (Table 1). Fibroblasts from patients with SSc showed increased DNA methylation in the promoter region of FLI1 that encodes an inhibitor of collagen expression [37]. Hypermethylation also silences the expression of the DKK1 and SFRP1, which are antagonists of Wnt signaling, permitting the profibrotic pathway in RA and SSc [38, 39]. These findings suggest that epigenetic mechanisms might mediate the fibrotic manifestations of RA and SSc. Peripheral blood mononuclear cells (PBMCs) from patients with RA showed hypomethylation of the promoter of IL6 encoding IL-6, which is instrumental in B cell response and hyperactivation of the inflammation circuit[40]. In addition, specifically hypermethylation is observed in synovial cell, which is a major pathology contributing to development of RA, silencing the expression of DR3 whose encoded protein is associated with cell apoptosis[41]. In the context of MS, discordance in CD4+ T cells of monozygotic twins has been studied but found no evidences for epigenetic differences[42]. Whereas Mastronardi et al. demonstrated hypomethylation at the promoter of PAD2 in normal-appearing white matter (NAWM) from patients [43]. MS is a chronic inflammatory disease characterized by myelin destruction followed by progressive neurodegeneration. Overexpression of PAD2 3

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plays a critical role in citrullination process of myelin basic protein (MBP) followed by increased production of immunodominant peptides, promoting the protein autocleavage responsible for proteolytic digestion, myelin instability and chronic inflammation response[44]. The different conclusions between two studies mentioned above suggest that perhaps microglia, oligodendrocyte or other cell types should be studied for MS, and emphasize the importance of selecting appropriate cell types or tissues for study. 2.2 Histone modifications Histones, involved in packaging and organizing nucleosomes, could be grouped into core histones (H2A, H2B, H3, and H4) and linker histones (H1 and H5). As another major source of epigenetic alteration, histone post-translational modifications, including acetylation and methylation, could modulate the chromatin structure, influencing its accessibility to transcription factors at gene promoters and enhancers. The balance of histone modifications are established and maintained by a range of enzymes including, but not limited to histone methyltransferases (HMT) and demethylases (HDM), and histone acetyltransferases (HAT) and deacetylases (HDAC)[45]. In some cases, the correlation of specific histone modifications and gene expression associated with autoimmune diseases has been well investigated (Table 2). 2.2.1 Histone acetylation Histone acetylation is catalyzed by HAT, adding an acetyl group on lysine residues in the N-terminal tail and promoting a more open chromatin structure. Generally, H3 and H4 hyperacetylation (H3Ac, H4Ac) are characteristic of active genes. Whereas histone deacetylation catalyzed by HDAC removes the acetyl groups, causing the DNA wrapped around nucleosome more tightly, thereby repressing gene expression[45]. SLE, as a systemic disease characterized by autoantibodies against nuclear or cytoplasmic self-antigens resulting in inflammation-mediated multiorgan damage, displays global H3 and H4 hypoacetylation in the CD4+ T cells of patients[46]. However, gene-specific histone modification patterns might differ from those of genome-wide ones. By investigating the alterations of histone modification on CD70 gene transcription, Zhou et al. showed that H3 acetylation was significantly increased in patients with active SLE and was correlated positively with disease activity[47]. Furthermore, histone modification patterns could vary in different cell types. Owing to the decreased HDAC2 and HDAC7, histone H4 hyperacetylation was demonstrated in B cells from patients with SSc[48], while in fibroblast, increased H3 and H4 deacetylation downregulate FLI1 gene expression, contributing to excessive synthesis of collagen and other extracellular matrix components[37]. In addition, in context of MS, histone H3 deacetylation could inhibit oligodendrocyte differentiation[49]. As for T1DM, it has been investigated that the increased H3K9 acetylation (H3K9Ac) could upregulate HLA-DRB1, HLA-DQB1 gene expression[50]. These aforesaid altered histone acetylation patterns might provide potential therapeutic targets for the treatment. 2.2.2 Histone methylation Histone methylation, which adds a methyl group to the histone subunit, is catalyzed by HMT, while demethylation occurs as a result of interaction of HDM. In most cases, histone hypermethylations, such as histone H3 lysine9 methylation (H3K9me) or trimethyl-lysine27 (H3K27me3), is present in repressed genes[45]. H3K9 hypomethylation has been detected in B cells from patients with SSc, which is correlated with downregulated histone methyltransferase SUV39H2 and upregulated histone demethylase JHDM2A[48]. Moreover, H3K27me3 is inhibited in fibroblast from patients with SSc, upregulating FOSL2 gene expression involved in fibroblast activation and fibrosis [51]. Similarly, CLTA4, a susceptibility gene of T1DM, displays increased promoter histone H3 lysine 9 dimethylation (H3K9me2), which is associated with T cells activation[52]. Synovial fibroblasts from patients with RA show increased histone methyltransferase EZH2 which could silence SFRP1 gene expression and thereby abrogate its inhibitory role on aberrant collagen deposition by such fibroblasts [39]. However, not all histone methylation are markers for inactive regions of chromatin. Typically, methylation of 4

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lysine 4 of histone H3 denotes an activation mark and is predominantly found at promoters of active genes[45]. For instance, CD4+ T cells from patients with SLE show a significantly elevated level of dimethylated H3 lysine 4 (H3K4me2) upregulating CD70 gene expression associated with excessive autoimmune responses[47]. 2.3 Non-coding RNA Only around 2% of mammalian transcripts could be translated into functional proteins. The rest of the remaining transcripts were defined as non-coding RNAs (ncRNAs) which serve as epigenetic modifiers and play a critical role on gene regulation. These ncRNAs could be further classified into several groups including microRNAs and long non-coding RNAs [53]. 2.3.1 MicroRNAs MicroRNAs (miRNAs) are approximately 22 nucleotides short, single-stranded ncRNAs involved in regulating the gene expression in a posttranscriptional manner. Via binding to complementary sequences on target mRNA, the miRNA-induced silencing complex (miRISC) could lead to translational repression, mRNA destabilization and degradation. It has been revealed that miRNA expression could control dynamic aspects of immune responses which are important for maintaining homeostasis and defending the host against antigens [54]. Functional studies subsequently demonstrated that a common set of aberrantly expressed miRNAs exert a significant role in pathogenesis of most autoimmune diseases by modulating multiple autoimmunity-related genes and other epigenetic processes (Table 3). Aberrant miR-21 expression has been reported to correlate with the progress of a range of autoimmune diseases. The expression of miR-21 is increased in CD4+ T cell from patients with SLE and has been revealed to target an important autoimmune gene RASGRP1, which mediates the Ras-MAPK pathway, downregulating DNMT1 expression and thereby contributing to the DNA hypomethylation[55]. Similarly, it has been demonstrated that increased levels of miR-21 contribute to the B cell hyperresponsiveness and aberrant T cell response in SLE by targeting the gene PTEN and PDCD4 respectively[56, 57]. Overexpression of miR-21 has been also found to negatively regulate SMAD7, enhancing TGF-β signaling and upregulating fibrosis-related genes, such as ACTA2, COL1A1, COL1A2 and FN1, in fibroblasts from patients with SSc [58]. Similarly, by depleting SMAD7 and therefore enhancing TGF-β signaling, miR-21 could limit the autocrine inhibitory effects of IL-2, and thereby promotes Th17 differentiation and mediates MS [59]. By contrast, CD4+ T cells show a significant reduction in levels of miR-21 in the context of RA, accompanied by increased STAT3 expression, decreased STAT5/pSTAT5 protein and Foxp3 mRNA levels which might contribute to the imbalance of Th17 and Treg cells[60]. Given that decreased Foxp3 expression has been also found in CD4+ T cell from patients with T1DM[35] and furthermore miR-21 has been reported to prevent T1DM[61], the association between its abnormal differentiation of regulatory T cells and miR-21 expression could be further investigated. The miR-29 family, including miR-29a, miR-29b and miR-29c, play a critical role in the pathogenesis of autoimmune diseases. It has been demonstrated that miR-29b targets directly DNMT3A and DNMT3B, inducing DNA hypomethylation, while CD4+ T cells in patients with SLE show that miR-29b downregulates DNMT1 indirectly by targeting Sp1 which is a transactivator of DNMT1 gene [62]. In the context of MS, miR-29b is upregulated in CD4+ T cells where it targets T-bet and IFN-γ, controlling Th1 differentiation [63]. Furthermore, in fibroblasts from patients with SSc, miR-29a serves as an antifibrotic miRNA that opposes the profibrotic activity of miR-21[64]. As well as prominent roles of miR-21 and miR-29 in regulating the autoimmune responses, aberrant expressions of other miRNAs, such as miR-30, miR-146a and miR-155, could also dysregulate epigenetic processes and, consequently, contribute to the pathogenesis of autoimmune diseases (Table 3). In addition to well-established roles of these miRNAs, the detection of specific circulating miRNAs that correlate with disease severity could provide reliable and accurate biomarkers of autoimmune diseases. For instance, altered circulating miR-223 expression has been found in T cells or other samples, such as plasma and PBMCs, from patients with SLE, RA and MS [65-67], however, further studies on their induced epigenetic alterations and molecular 5

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mechanisms are clearly warranted. 2.3.2 Long non-coding RNAs Long non-coding RNAs (lncRNAs), which are larger than 200 nucleotides and lack of protein-coding function, have been found to be pervasively transcribed but highly diverse in sequence, structure and function. Presently, according to their position relative to protein-coding mRNAs, lncRNAs are classified into the long intergenic ncRNA (lincRNA), intronic lncRNA, antisense lncRNA, transcribed pseudogene lncRNAs and enhancer RNA (eRNA)[68]. Compared to widely-investigated miRNAs, however, studies of autoimmune diseases in search of lncRNA expression are just beginning. Understanding of lncRNAs involving immunity, inflammation and degeneration might provide useful references for investigating this type of defect in autoimmune diseases. The largest group of lncRNAs is the lincRNAs which are located between protein-coding genes. Pin et al. has demonstrated that lnc-DC regulates differentiation of human and mouse monocytes into dendritic cells. Lnc-DC knockdown results in altered expression of 664 protein-coding genes, affecting the antigen uptaking, allogenic CD4+ T cells production and cytokine release which might have potential relevance to autoimmune diseases involving monocytes and DC dysfunction[69]. Moreover, lincRNA-Cox2 mediates the induction or repression of immune response-related genes, including Il6, Stat1 and Irf7, in mouse macrophages [70]. LncRNA-ATB, which is activated by TGF-β involved in hepatocellular invasion-metastasis cascade as well as profibrotic activity, could promote autocrine induction of IL-11 and trigger STAT3 signaling [71]. NRON, an intronic lncRNA transcribed from intronic regions in the sense or antisense orientation, serves as the non-coding repressor of nuclear factor of activated T cells[72]. Lethe, a transcribed pseudogene lncRNA arising from a gene losing its intrinsic protein-coding ability through mutation or inaccurate duplication, blocks NF-κB-driven inflammatory responses in mouse fibroblasts[73]. Whether these lncRNAs are associated with the pathogenesis of autoimmune diseases deserves further investigations. Antisense lncRNAs, which are transcribed across the exons of protein-coding genes from the opposite strand, could form sense-antisense pairs by pairing with the protein-coding strand to regulate epigenetic silencing, transcription or mRNA stability. BDNF-AS has been found to normally repress brain-derived neurotrophic factor (BDNF). Inhibition of this antisense lncRNA leads to increased BDNF level, promoting neuronal outgrowth and differentiation [74]. Carrieri et al. identified a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1) involved in brain function and neurodegeneration given that oxidative inactivation of UCHL1 protein has been reported in Parkinson’s and Alzheimer’s disease brains. Via a 5' overlapping sequence and an embedded inverted SINEB2 element-dependent manner, antisense Uchl1 could upregulate mouse ubiquitin carboxy-terminal hydrolase L1 (UCHL1) protein synthesis, furthermore, this lncRNA is under the control of stress signaling pathways, particularly the mTORC1 pathway[75]. These data reveal novel mechanisms of gene expression control at post-transcriptional level and might expand the potential roles of lncRNA for neuroprotection in MS. Of note, it is relatively easy to extrapolate miRNA functions in humans from experiment results in mouse given that miRNAs and their targets are generally well conserved throughout evolution. In contrast, lncRNAs demonstrate low evolutionary sequence conservation, for less mouse lncRNAs have homologues in humans[76]. Non-coding RNAs open up new avenues for epigenetic research, however, multiple challenges still need to be appropriately addressed. 2.4 Cross-talk between multiple epigenetic mechanisms It is noteworthy that all these aforementioned epigenetic mechanisms act together at the same time rather than regulate gene expression separately. DNA methylation status is established and maintained by DNMTs, whereas methyl-CpG-binding domain (MBD) proteins associated with methyl-cytosine could also recruit silencing complexes participating in transcriptional repression. These silencing complexes which contain histone deacetylases (HDACs) and histone methyltransferases (HMTs) contribute to a cross-talk among the DNA methylation, histone modifications and 6

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nucleosome remodeling[12]. Meanwhile, cross-talks among various ncRNAs or between ncRNAs and other epigenetic mechanisms exist extensively as well[77]. Typically, a recent study by Hedrich et al. showed an epigenetic cross-talk contributing the pathogenesis of SLE. Reduced DNA methylation of the IL10 gene promoter leads to Stat3 and Stat5 recruitment to and trans-activation of the IL10 promoter and an intronic enhancer which is referred to as intronic Stat-responsive element (I-SRE). The effects of Stat3 on the I-SRE were found higher compared with Stat5. In T cells from patients with SLE, increased Stat3 phosphorylation enhances its recruitment to the IL10 promoter and the replacement of Stat5 at the I-SRE, accompanied by the recruitment of histone acetyltransferase p300 to those regions, resulting in trans-activation and nucleosome remodeling which thereby elevates IL-10 level inducing autoantibody production and tissue damage in SLE[78]. 3. Prospects for epigenetic therapy Rapid progress in understanding epigenetic dysregulation in autoimmune diseases has enabled us to determine the general mechanisms of epigenetic alterations and attract intensive investigation aimed at identifying specific clinical markers for early diagnosis, furthermore, embark on the development of novel therapeutic drugs and other therapeutic approaches. 3.1 Drugs with epigenetic effects Many epigenetic aetiology-related human diseases, including cancer, encourage the development of a new therapeutic approach termed ‘epigenetic therapy’. Drugs with the ability to alter methylation patterns on DNA or the modification of histones are a means of inducing large-scale recovery in the epigenome. Repurposing drugs approved for cancer could be a promising orientation, as these drugs might have properties that allow them to be beneficial for multiple diseases. Among them, HDAC inhibitors and DNMT inhibitors are currently the most widely studied epigenetic therapeutics for autoimmune diseases (Table 4). Trichostatin A (TSA), which is an HDAC inhibitor inducing growth arrest and apoptosis in tumors, has demonstrated its inhibitory effect on fibroblast activation in fibrosis-related autoimmune diseases such as RA and SSc. TSA could restore the expression of FLI1 which is the negative regulator of collagen expression but usually repressed in SSc by promoter hypoacetylation[37]. Furthermore, the inhibition of HDAC-7 by TSA downregulates the expression of fibrosis-related genes COL1A1 and COL3A1[79]. In the context of RA, TSA significantly impairs the stability of IL-6 mRNA and thereby disrupts inflammatory cytokine production in synovial cells [80]. Apart from its anti-fibrosis effects, TSA reverses the aberrant increased CD40L and IL10 expression as well as the decreased IFN-γ expression in T cells from patients with SLE[81]. As for T1DM, it is suggested that TSA could increase the expression of IFN-γ and its transcription factor Tbx21 in T lymphocytes, reducing the cellular infiltration of islets [82]. In clinical trials, orally active histone deacetylase inhibitor givinostat (also termed ITF2357) was performed on 17 patients with systemic juvenile idiopathic arthritis. Although 6 adverse events exhibited in 3 patients, two thirds of the patients achieved significant therapeutic benefit after 12 weeks, particularly in terms of the arthritic component of the disease[83]. Decreased production of IL-6 by givinostat might contribute to this therapeutic benefit [80]. With regard to DNMT inhibitors, 5-aza-2'-deoxycytidine reverses the aberrant hypermethylation of DKK1, SFRP1 and FLI1 in fibroblasts from patients with SSc, normalizing the Wnt signalling and type I collagen expression respectively[37, 38]. In the mice with experimental autoimmune encephalomyelitis (EAE) which resemble the human demyelinating disease MS in both clinical course and histopathology, 5-aza-2'-deoxycytidine treatment increases the population and immunosuppressive function of Foxp3+ regulatory T cells as well as inhibits the effector cells in the periphery, preventing EAE development and suppressing CNS inflammation[84]. Of note, because of the large number of genes targeted by each HDAC, even a very enzyme-specific HDAC inhibitor is still unavoidable to alter expression of many genes which is likely to lead to unacceptable toxicity[85]. Therefore, HDAC inhibitors might act as a double edged sword in epigenetic therapy and the same is true for the 7

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risk of DNMT inhibitors in clinical applications. Further investigations into targets downstream of HDACs and DNMTs, such as proteins that bind to modified histones, could be carried out to identify safer targets for therapy. Moreover, in hypomethylation-related autoimmune disease, including SLE and RA, DNMT inhibitors would not be appropriate to restore their DNA methylation patterns, thus drugs should be further designed to specifically increase methylation without triggering hypermethylation-related tumorigenesis. 3.2 MicroRNAs and lncRNAs-targeting therapeutics MicroRNAs have been demonstrated closely associated with autoimmune diseases in terms of diagnosis marks as well as therapeutic targets. The modulation of miRNA expression by using miRNA mimics or inhibitors might, therefore, represent attractive future opportunities for autoimmune disease treatment. A study by Tang et al. has demonstrated that reduced level of miR-146a, which directly targets IRF5 and STAT1, increases the induction of type I interferon in PBMCs from patients with SLE. In clinical trials, 5 patients with SLE who had high interferon scores were transfected with miR-146a expression plasmid. After 24 hours, PBMCs from the patients were tested for the mRNA levels of the selected interferon-inducible genes. As expected, the expressions of selected genes were notably reduced by miR-146a, indicating that manipulation of miR-146a levels could potentially provide a therapeutic benefit to SLE patients, even though the alleviation of related symptoms need to be further determined[86]. As for strategies of miRNA inhibition, miRNA sponges, small-molecule inhibitors and antisense oligonucleotides are main approaches that have been taken (Figure 1). Termed miRNA sponges, also known as vector-based strategies, are constructed based on mRNA sequences containing multiple artificial miRNA-binding sites which could act as decoy or ‘sponges’. When vectors encoding these sponges are transfected into cultured cells, sponges could selectively deplete endogenous miRNAs and thereby allow translation of the target mRNAs[87]. A study by Du et al. has shown that in vivo silencing of miR-326 by miRNA sponges caused a decrease in the number of Th17 cells and disease severity and vice versa, indicating that miR-326 plays an important role in Th17 polarization and the pathogenesis of MS[88]. Interestingly, it seems that some lncRNAs could serve as natural sponges to abrogate miRNA availability and lead to upregulation of downstream target genes. For instance, lnc-ATB was found to be competing endogenous RNAs (ceRNA) binding the miR-200 family, which might provide inspiration for further investigations [71]. Small-molecule inhibitors might target multiple steps of miRNA assembly and function, including transcription of primary miRNAs, the formation of miRNA-induced silencing complex (miRISC) and interactions between miRISC and target mRNA. It has been reported that azobenzene could affect miR-21 expression and several compounds exhibit inhibitory effect on miR-122 expression [89, 90]. Small-molecule inhibitors targeting miRNAs are less used in research on autoimmune diseases but provide alternative experimental methods. The miRNA-targeting antisense oligonucleotides (anti-miRs) are designed to be high complementary to the target miRNA. When delivered into cultured cells, the anti-miRs could bind to and specifically suppress target miRNAs function, blocking their inhibitory effect on the expression of endogenous target genes. Moreover, many anti-miRs have been demonstrated to induce degradation of targeted miRNAs. Recently, anti-miRs delivered by lentiviral vectors have been widely used to investigate miRNA function. For instance, recombinant lentiviral vectors silencing miR-223 was constructed and intraperitoneally injected into mice with collagen-induced arthritis. The anti-miR-223-treated mice showed the amelioration of score and incidence of arthritis, histopathologic examination, as well as the attenuation of osteoclastogenesis and bone erosion in joints[91]. In clinical trials, miR-122-targeted therapy in chronic hepatitis C patients has been reported to be long-term safe and effective [92, 93]. Furthermore, miRNA-masking (miR-mask) technology is another sort of antisense oligonucleotides approaches. Unlike anti-miRs directly binding to the target miRNAs, a miR-mask covers up the sites on mRNAs where are fully complementary to target miRNA, derepressing its target mRNAs [94]. In summary, antisense oligonucleotides technology provides valuable experimental platforms and opens up new avenues of clinical treatments of autoimmune diseases. 8

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4. Conclusion & future perspective Ranging from candidate-gene to genome-wide association studies, a large number of susceptibility genes have been identified in the pathogenesis of autoimmune diseases. However, genetic factors are unable to fully account for the risk of autoimmune diseases. A growing series of evidences suggest that gene function depends on not only DNA sequences, but also the epigenetic modifications, including DNA methylation, histone modification, non-coding RNA and their cross-talks, which are thought to be crossroads between environment and genetics. The endeavor to completely understand molecular mechanisms governing epigenetic alterations will provide more research orientations. It is noteworthy that epigenetic alterations vary among different tissue and cell types. Each cell type could be characterized by a particular epigenome that are associated with a specific gene expression profile, for instance, epigenome-wide profile analysis showed DNA methylation patterns varying dramatically between T and B lymphocytes[95]. Furthermore, different environment exposed and roles in pathogenesis could also contribute to their distinction in epigenetic alterations, so that appropriate selection of cell or tissue types for analysis is particularly required. Autoimmune diseases share many features, and a multitude of studies have revealed the existence of many common susceptibility genes and epigenetic alterations. It is viable that different kinds of autoimmune diseases could acquire useful references from each other when it comes to researching into pathogenesis. Meanwhile, the discovery of shared aspects of disease pathogenesis could offer the opportunity to use known drugs or therapeutic strategies across multiple diseases. Drugs with epigenetic effects, such as HDAC inhibitors and DNMT inhibitors, have been applied on animal models and even in clinical trials. Furthermore, with the rapid development of basic and clinical studies, microRNAs-targeting therapeutics by mimics or inhibitors has become a valuable and promising way for the modulation of multiple aspects of human diseases. We are optimistic that taking individual epigenetic variation into account will lead to a more personalized application of treatment for autoimmune diseases in the coming years.

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Conflict of interest The authors declare that they have no competing financial interests.

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Take-home messages ·Aberrant epigenetic alterations, including DNA methylation, histone modification and non-coding RNAs expression which could lead to direct effects on expression of susceptibility genes as well as cross-talks with each other, occur in lymphocytes and other cell types from patients, contributing to the pathogenesis of autoimmune diseases. ·Drugs with epigenetic effects, such as HDAC inhibitors and DNMT inhibitors, have been applied on animal models and even in clinical trials, displaying positive therapeutic effect. ·The microRNAs-targeting therapeutics by mimics or inhibitors provides a valuable and promising way for the modulation of multiple aspects of human diseases. A range of miRNA-targeting therapeutic is in now at preclinical stage or even in clinical trials. ·With the rapid development of microRNAs-targeting therapeutics, taking individual epigenetic variation into account will lead to a more personalized application of treatment for autoimmune diseases in the coming years. Acknowledgment This work is supported by the Ministry of Science and Technology of China through Grants No 2012CB932503 and 2011CB933100; the National Natural Science Foundation of China through Grants No 91029705, 81172864 and 81272317. Grant TD12-5025 and 14JCTPJC00487 from Tianjin city. References 9

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[119]

T

CE P

[122]

AC

[121]

TE

D

[120]

IP

[117]

SC R

[116]

NU

[115]

Ann Rheum Dis 2014. K.M. Pauley, C.M. Stewart, A.E. Gauna, L.C. Dupre, R. Kuklani, A.L. Chan, et al. Altered miR-146a expression in Sjogren's syndrome and its functional role in innate immunity. Eur J Immunol 2011; 41: 2029-39. C.S. Moore, V.T. Rao, B.A. Durafourt, B.J. Bedell, S.K. Ludwin, A. Bar-Or, et al. miR-155 as a multiple sclerosis-relevant regulator of myeloid cell polarization. Ann Neurol 2013; 74: 709-20. A. Junker, M. Krumbholz, S. Eisele, H. Mohan, F. Augstein, R. Bittner, et al. MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47. Brain 2009; 132: 3342-52. J. Stanczyk, D.M. Pedrioli, F. Brentano, O. Sanchez-Pernaute, C. Kolling, R.E. Gay, et al. Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis. Arthritis Rheum 2008; 58: 1001-9. J. Gillespie, S. Savic, C. Wong, A. Hempshall, M. Inman, P. Emery, et al. Histone deacetylases are dysregulated in rheumatoid arthritis and a novel histone deacetylase 3-selective inhibitor reduces interleukin-6 production by peripheral blood mononuclear cells from rheumatoid arthritis patients. Arthritis Rheum 2012; 64: 418-22. Z. Ge, Y. Da, Z. Xue, K. Zhang, H. Zhuang, M. Peng, et al. Vorinostat, a histone deacetylase inhibitor, suppresses dendritic cell function and ameliorates experimental autoimmune encephalomyelitis. Exp Neurol 2013; 241: 56-66. Q.Y. Choo, P.C. Ho, Y. Tanaka and H.S. Lin. Histone deacetylase inhibitors MS-275 and SAHA induced growth arrest and suppressed lipopolysaccharide-stimulated NF-kappaB p65 nuclear accumulation in human rheumatoid arthritis synovial fibroblastic E11 cells. Rheumatology (Oxford) 2010; 49: 1447-60. S.J. Saouaf, B. Li, G. Zhang, Y. Shen, N. Furuuchi, W.W. Hancock, et al. Deacetylase inhibition increases regulatory T cell function and decreases incidence and severity of collagen-induced arthritis. Exp Mol Pathol 2009; 87: 99-104. E.C. Lewis, L. Blaabjerg, J. Storling, S.G. Ronn, P. Mascagni, C.A. Dinarello, et al. The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet beta cells in vivo and in vitro. Mol Med 2011; 17: 369-77.

MA

[114]

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ACCEPTED MANUSCRIPT Table 1. Epigenetic alterations in autoimmune diseases at the level of DNA methylation Genes

Epigenetic alteration

CD40LG

Hypomethylation

CD40L, B cell costimulatory molecule encoded [20-22] on the X chromosome

CD70

Hypomethylation

CD70, B cell costimulatory molecule associated [24-26] with overproduction of IgG

SSc, T1DM, RA,

FOXP3

Hypermethylation Forkhead box protein 3, involved in quantitative [34-36] defects of regulatory T cells.

MS

HLA-DRB1

Hypomethylation

HLA class II beta chain

SLE

IL10, IL13

Hypomethylation

Involved in autoantibody production and tissue [78, 97] damage

SLE

IRF5, IFIT2

Hypomethylation

Involved in type I interferon pathway

[27]

SLE

ITGAL

Hypomethylation

Integrin α-L, associated with cell-cell adhesion

[98]

SLE

PRF1

Hypomethylation

Perforin 1, involved in autoreactive killing

[99]

SLE, pSS

STAT1, IFI44L, USP18

Hypomethylation

Involved in type I interferon pathway

[28, 29]

pSS

LTA

Hypomethylation

pSS

RUNX1

Hypermethylation Transcription factor associated to lymphoma

SLE

CD5

Hypomethylation

SLE

HRES-1

Hypomethylation

RA

IL6

SLE

IFNGR2, MMP14

B cell

PBMC

SLE

CD14+ monocyte

T

IP

SC R

NU

Lymphotoxin-α

[96]

[28] [28]

CD5, involved in activation and expansion of [30] autoreactive B cells Human endogenous retroviruses proteins, involved [31] in induction of cross-reactive autoantibodies

Hypomethylation

IL-6, involved in B cell response

Hypomethylation

IFN-γ receptor 1, Matrix metalloproteinase-14, [100] involved in inflammation

LCN2

Hypomethylation

Neutrophil gelatinase-associated lipocalin, iron [100] transporter and marker for SLE

SHP-1

Hypermethylation A negative regulator of cytokine signaling through [101] NF-κB and STATs

AC

MS

MA

Naïve CD4 T cell

+

References

D

Peripheral SLE, RA, blood CD4+ SSc T cell SLE, SSc, pSS

Product and/or function

TE

Disease

CE P

Cell type

[40]

T1DM

HLA-DQB1

Hypomethylation

HLA class II

[102]

T1DM

RFXAP

Hypomethylation

HLA class II regulating element

[102]

T1DM

NFKB1A

Hypomethylation

Regulator of apoptosis and inflammation

[102]

T1DM

GAD2

Hypomethylation

GAD65, a major autoantigen involved in T1D

[102]

T1DM

TNF

Hypermethylation Key inflammatory cytokine

T1DM

CD6

Hypermethylation Involved in lymphocyte activation and differentiation [102]

Fibroblast

SSc

FLI1

Hypermethylation Involved in type I collagen expression

[37]

Fibroblast, PBMC

SSc

DKK1, SFRP1

Hypermethylation Wnt signaling antagonists

[38]

Synovial fibroblast

RA

SFRP1, SFRP4

Hypermethylation Wnt signaling antagonists

[39, 103]

RA

DR3

Hypermethylation Death receptor 3, associated with cell apoptosis

[41]

NAWM

MS

PAD2

Hypomethylation

LSG

pSS

DST

Hypermethylation BP230, bullous pemphigoid antigen 1 protein

[102]

Peptidyl argininedeiminase type II, responsible for [43] the increased citrullinated myelin basic protein [104]

Abbreviations: SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; MS, multiple sclerosis; SSc, Systemic sclerosis; T1DM, type 1 diabetes; pSS, primary Sjögren's syndrome; PBMC, peripheral blood mononuclear cell; NAWM, normal appearing white matter; LSG, labial salivary gland. 17

ACCEPTED MANUSCRIPT Table 2. Epigenetic alterations in autoimmune diseases at the level of histone modification Disease Epigenetic alteration

Gene affected and expression change

References

T cell

SLE

H3 and H4 hypoacetylation, H3K9 hypomethylation

ND

[46]

SLE

Increased histone H3 acetylation at lysine 18 (H3K18ac)

IL10, upregulation

[78]

SLE

Increased H3 acetylation, dimethylated H3 lysine4 (H3K4me2)

T1DM

Increased H3K9me2

B cell

SSc

H4 hyperacetylation, decreased HDAC2 and HDAC7; ND H3K9 hypomethylation, decreased SUV39H2 (member of HMT), increased JHDM2A (member of HDM)

Monocyte

T1DM

Increased H3K9 acetylation (H3K9Ac)

SLE

Global H4 hyperacetylation

SSc

Increased H3 and H4 deacetylation

SSc

Inhibition of H3K27me3

RA RA

Synovial fibroblast

Oligodendrocyte MS

IP

CD70, upregulation

NU

SC R

CTLA4, downregulation

[47] [52] [48]

HLA-DRB1, HLA-DQB1, [50] upregulation IRF1, RFX1 and BLIMP1, [105] upregulation FLI1, downregulation

[37]

FOSL2, upregulation

[51]

Increased zeste homologue 2 (EZH2, member of HMT)

SFRP1, downregulation

[39]

Increased sirtuin 1 (Sirt1, member of HDAC)

ND

[106]

Increased histone H3 deacetylation

ND

[49]

MA

Fibroblast

T

Cell type

TE

D

Abbreviations: SLE, systemic lupus erythematosus; T1DM, type 1 diabetes; SSc, Systemic sclerosis; RA, rheumatoid arthritis; MS, multiple sclerosis; ND, not determined; HMT, histone methyltransferases; HDAC, histone deacetylase; HDM, histone demethylase.

Table 3. Common miRNAs dysregulated in autoimmune diseases Cell type

let-7a

Fibroblasts

Epigenetic alteration / Function

Disease

References

Downregulation

Increased type I collagen expression

SSc

[107]

Upregulation

Decreased E2F5 expression, increased IL6 expression, regulated by E2F2 or NF-κB

SLE

[108]

CD4+ T cell Upregulation

Repressing DNMT1 expression by targeting RASGRP1

SLE

[55]

CD4+ T cell Upregulation

Targeting and depleting enhancing TGF-β signaling

CD4+ T cell Downregulation

Increased STAT3 expression, decreased expression and STAT5/pSTAT5 protein

B cell

Upregulation

Downregulating PTEN expression, contributing to B SLE cell hyperresponsiveness

[56]

PBMCs

Upregulation

Targeting PDCD4 involved in aberrant T cell response

SLE

[57]

Fibroblasts

Upregulation

Directly downregulating SMAD7 and increasing fibrosis-related genes expression

thereby SSc

[58]

miR-29

Pancreatic islets cell

Upregulation

Decreased antiapoptotic protein Mcl1

T1DM

[109]

miR-29a

Fibroblasts

Downregulation

Targeting COL3A1 involved in collagen expression

SSc

[64]

miR-29b

CD4+ T cell Upregulation

Repressing DNMT1 expression

SLE

[62]

Mesangial cells

AC

miR-21

Expression change

CE P

miRNA

+

SMAD7,

and

thereby MS

[59]

Foxp3 RA

[60]

CD4 T cell Upregulation

Targeting T-bet and IFN-γ, controlling Th1 differentiation MS

[63]

miR-30a

B cell

Upregulation

Targeting LYN involved in B cell hyperactivity

SLE

[110]

miR-30a-3p

Fibroblasts

Downregulation

Targeting BAFF involved in autoimmune responses

RA, SSc

[111]

miR-30b

Fibroblasts

Downregulation

Targeting PDGFR-β involved in fibroblast proliferation

SSc

[112]

18

ACCEPTED MANUSCRIPT Targeting STAT1 involved in phenotype of regulatory T cells

pro-inflammatory

RA

[113]

PBMCs

Upregulation

Involved in phagocytic activity and pro-inflammatory pSS cytokine production

[114]

PBMCs

Downregulation

Targeting and IRF5 and STAT1 involved in activation SLE of type I IFN pathway

[86]

Monocytes, microglia

Upregulation

Targeting CD47, increased TNFα and IL-6 secretion, MS increased CD80 and CCR7 expression

[115, 116]

Synovial fibroblasts

Upregulation

Downregulation of MMP-3 involved in inflammation

T

Downregulation

RA

[117]

SC R

miR-155

Regulatory T cells

IP

miR-146a

Abbreviations: SSc, Systemic sclerosis; SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; MS, multiple sclerosis; T1DM, type 1 diabetes; pSS, primary Sjögren's syndrome; PBMCs, peripheral blood mononuclear cells; DNMT, DNA methyltransferase.

Cell type

Effects

Disease

References

Trichostatin A (HDAC inhibitor)

Fibroblasts

Increased acetylation of FLI1, silencing HDAC-7

SSc

[37, 79]

Macrophages, Synovial fibroblasts

Modulate NF-κB signaling, impairing the stability of IL-6 mRNA

RA

[80]

T cell

Downregulating CD40L and IL10 expression, upregulating IFN-γ expression

SLE

[81]

Entinostat (HDAC inhibitor)

D

[82]

Inhibiting TNF and IL-6 production

RA

[118]

Dendritic cell

Decreased expression of CD80 and CD86, the MS matural marker CD83, and HLA-DR

[119]

Synovial fibroblasts

Suppressing NF-κB pathway

RA

[120]

Regulatory T cell

Increasing regulatory T cell activity and expansion RA of FOXP3+Treg cells

[121]

Synovial fibroblasts

Involved in induction of p21 and suppression of RA the NF-κB pathway

[120]

Macrophages, Synovial fibroblasts

Modulate NF-κB signaling, impairing the stability of IL-6 mRNA

RA

[80]

Macrophages, Splenocytes

Inhibiting the production of TNFα and IFNγ

T1DM

[122]

Fibroblasts

Hypomethylation of FLI1, DKK1, SFRP1

SSc

[37, 38]

Synovial fibroblasts

Hypomethylation of SFRP4

RA

[103]

Regulatory T cell

Increasing the population and immunosuppressive MS function of Foxp3+ regulatory T cells

CE P

PBMCs

AC

Vorinostat (HDAC inhibitor)

Valproic acid (HDAC inhibitor)

Increasing the expression of IFN-γ and its T1DM transcription factor Tbx21

TE

T cell MI192 (HDAC3-selective inhibitor)

Givinostat (ITF2357) (HDAC inhibitor)

5-aza-2'-deoxycytidine (DNMT inhibitor)

MA

Drug (Type)

NU

Table 4. Examples of chemicals modulating DNA methylation and histone modification in the context of autoimmune diseases

[84]

Abbreviations: SSc, Systemic sclerosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; MS, multiple sclerosis; T1DM, type 1 diabetes; PBMCs, peripheral blood mononuclear cells.

19

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

Figure 1. Mechanisms of miRNA inhibition strategies. A. miRNA sponges could selectively deplete endogenous miRNAs based on high complementation to multiple artificial miRNA-binding sites, thereby allowing translation of the target mRNAs. B. Small-molecule inhibitors could target multiple steps of miRNA assembly and function, derepressing its inhibitory effect on mRNA translation. C. anti-miRs are designed to be high complementary to the target miRNA, binding to and specifically suppressing target miRNAs function. D. miR-mask covers up the sites on mRNAs where are fully complementary to target miRNA, preventing the inhibitory effect of miRNA on target mRNA translation. Abbreviations: miRISC, miRNA-induced silencing complex; Anti-miRs, miRNA-targeting antisense oligonucleotides.

20