Roles of circular RNAs in immune regulation and autoimmune diseases

Circular RNAs (circRNAs), as a novel class of endogenously expressed non-coding RNAs (ncRNAs), have a high stability and often present tissue-specific expression and evolutionary conservation. Emerging evidence has suggested that circRNAs play an essential role in complex human pathologies. Notably, circRNAs, important gene modulators in the immune system, are strongly associated with the occurrence and development of autoimmune diseases. Here, we focus on the roles of circRNAs in immune cells and immune regulation, highlighting their potential as biomarkers and biological functions in autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), primary biliary cholangitis (PBC), and psoriasis, aiming at providing new insights into the diagnosis and therapy of these diseases.


Introduction
Normally, immune cells have receptors that can distinguish between self (ie, healthy native structures) and nonself or deviant self (ie, pathogens or tumor antigens), enabling these cells to discover pathogens or malignantly transformed cells. At the same time, precise regulation of certain immune-related genes is essential to an organism's ability to generate strong immunity to pathogens while limiting autoimmunity to self-antigens. Once immunodeficiency or immune dysregulation, people may suffer from immune system diseases, chronic infections, and even cancer. Typically, autoimmune diseases are a type of complex multifactorial diseases with characteristics of the presence of autoreactive immune cells and specific autoantibodies. According to statistics, there are >100 human diseases in the world that are known as autoimmune or chronic inflammatory, which are believed to affect 5-10% of individuals 1 .
Generally, circular RNAs (circRNAs) are widely studied non-coding RNA (ncRNA). Although RNA molecules are traditionally considered to be passive carriers of genetic information from DNA sequences to protein synthesis, lots of research has revealed that ncRNAs are critical participators in the process of gene expression 2,3 . To date, accumulated evidence has shown that circRNAs play an important role in various physiological and pathological processes, such as cancer 4,5 , cardiovascular diseases 6 , and neuronal diseases 7 . Intriguingly, circRNAs serve important functions in antiviral immunity 8 . Furthermore, cir-cRNAs are aberrantly expressed in patients with systemic lupus erythematosus (SLE), some of which may serve as new non-invasive biomarkers for this autoimmune disease 9 . Therefore, an in-depth study of circRNAs will not only increase our understanding of the molecular mechanisms that underlie autoimmune diseases, but also provide future potential treatment of these diseases. In this review, we emphasize the potential roles of circRNAs in certain autoimmune diseases, including SLE, rheumatoid arthritis (RA), multiple sclerosis (MS), primary biliary cholangitis (PBC), and psoriasis.

Biogenesis and functions of circRNAs
Unlike the characteristics of linear RNA molecules, circRNA has a special structure that is a covalently closed loop without 5' end caps and 3' Poly (A) tails 10,11 . This RNA species was first identified in RNA viruses in 1976 12 and subsequently discovered in the cytoplasm of eukaryotic cells 13 and yeast mitochondria 14 . With the development of high-throughput sequencing technology and microarray technique, plenty of circRNAs have been successfully discovered in various organisms in nature. In most cases, circRNAs are produced by "back-splicing" events of the precursor messenger RNAs (pre-mRNAs), in which a downstream 5' splice donor is linked to an upstream 3' splice acceptor via a 3' → 5' phosphodiester bond 15,16 . According to their components, circRNAs are mainly divided into three types: exonic circular RNAs (ecircRNAs) 17 , intronic circular RNAs (ciRNAs) 18 , and exon-intron circular RNAs (EIciRNAs) 19 , among which ecircRNAs occupy the vast majority.
Previous studies have found that RNA polymerase II (Pol II) elongation rate is associated with the efficiency and results of splicing 20,21 . The fast Pol II elongation may facilitate reverse complementary sequences across long flanking introns to pair up for back-splicing, thereby promoting circRNA formation 22 . Several possible cir-cRNA biogenetic pathways, including "complementary sequence-mediated circularization", "lariat-driven circularization" and "RNA-binding proteins-mediated circularization", have been proposed. Liang et al. 23 found that certain introns containing both splice sites and flanking inverted complementary repeats, such as Alu elements, were necessary for the circularization of the intervening exons in cells. In this process, the intronic repeat sequences must be base-paired with each other to bring the splice sites close together, thereby facilitating backsplicing. Notably, when a pre-mRNA has multiple intronic repeat sequences, the competitive pairing between the repeat sequences result in alternative circularization, thus affecting the splicing outcomes 24 . For example, this alternative circularization can cause a single gene to form multiple different circRNA transcripts 25 . Another form of circRNA generation is associated with exon skipping, in which a lariat precursor containing one or more skipped exons is first generated 26,27 . Then, the lariat removes its internal intron sequences to generate a mature circRNA and a double lariat. In some situations, the intervening introns in the encircled exons are not removed, which yields the so-called EIciRNA 16 . In addition, some RNAbinding proteins including the muscleblind, nuclear factor 90/nuclear factor 110 (NF90/NF110) and alternative splicing factor Quaking (QKI) were reported to promote back-splicing events by increasing the interaction between upstream and downstream introns [28][29][30] . CiRNAs are produced by intron lariats that fail to be degraded and debranched, and they do not contain linear 3' tails 18 . To some extent, these models explain the molecular mechanisms of circRNA biosynthesis (Fig. 1).
Usually, circRNAs work as molecular sponges for microRNAs (miRNAs), thereby regulating miRNA target gene expression 31,32 . Hansen et al. 31 first found that a highly expressed circRNA (ciRS-7) could be used as a miR-7 sponge in human and mouse brains. This circRNA contained >70 conserved binding sites for miR-7, and it was strongly inhibited miR-7 activity by binding to miR-7, resulting in elevated levels of miR-7 targets. Subsequently, increasing evidence showed that various circRNAs could adsorb miRNAs, thus participating in many physiological and pathological processes 33,34 . For example, Hsa_-circ_0009361 could regulate the expression of adenomatous polyposis coli 2 by binding to miR-582, thereby inhibiting the progression of colorectal cancer 35 . Furthermore, a class of EIciRNAs localized in the nucleus promoted the transcription of their parental genes in cis through interacting with U1 snRNP, indicating that these circRNAs could regulate gene expression via specific RNA-RNA interplay between U1 snRNA and EIciR-NAs 19 . Conn et al. 36 demonstrated that a circRNA derived from the exon 6 of SEPALLATA3 gene bound to its cognate DNA locus to form an R-loop and thus regulated the splicing of its cognate mRNA. Notably, the circular form of long intergenic non-protein-coding RNA p53induced transcript could encode an 87 amino-acid regulatory peptide, which bound to polymerase associated factor complex (PAF1c) and inhibited the transcriptional elongation in glioblastoma 37 . These findings show that there may be more biological functions of circRNA than previously predicted.

circRNAs in immunity circRNAs in immune cells
Indeed, some studies have demonstrated the diverse biological functions of circRNAs in immune cells. Hematopoietic stem cells (HSCs) can differentiate into a variety of progenitor cells, which subsequently generate all kinds of specialized blood cells, such as red blood cells, megakaryocytes, myeloid cells, and lymphocytes 38 . Nicolet et al. 39 found that circRNA showed cell-specific expression in human hematopoietic progenitors as well as differentiated lymphoid and myeloid cells. For example, during hematopoietic differentiation, the expression levels of circRNA of lymphocytes were the highest, and the high levels were reflected in abundance rather than variety. Moreover, circ-FNDC3B showed the highest expression level in natural killer cells, while circ-ELK4, circ-MYBL1, and circ-SLFN12L showed the highest expression in T cells and natural killer cells. Macrophages are an essential part of innate immunity and can be induced to diverse phenotypes under different external stimuli 40 . A recent study explored the expression of circRNAs in macrophages under two different polarization conditions (M1 macrophages induced by interferon-γ (IFN-γ) and lipopolysaccharide (LPS), and M2 macrophages induced by interleukin-4 (IL-4)) 41 . The results showed that 189 circRNAs were differentially expressed in the M1 compared with the M2 macrophages. To further elucidate the implication of the differentially expressed circRNAs, the researchers also predicted the miRNAs that interacted with them. For the overexpressed circRNA-010231 in M1, the five miRNA response elements with good scores were miR-1964-5p, miR-19b-2-5p, miR-141-5p, miR-6950-5p, and miR-145a-5p, respectively. These findings provide new ideas for the roles of circRNAs in the polarization of macrophages. Recently, Agirre et al. 42 found that 1356 new identified circRNAs were expressed in human humoral immune response, as well as plasma cells (tonsillar plasma cells and bone marrow plasma cells) had the highest average expression levels. The expression of these circRNAs was significantly negatively correlated with the levels of some RNA-binding proteins Fig. 1 Formation of circRNAs by pre-mRNA back-splicing events. a The intron complementary repeat sequences or RNA-binding proteins such as QKI, MBL, and NF90/NF110 promote the back-splicing. b A lariat containing one or more skipped exons is re-spliced to generate a circRNA and a double lariat, and the circRNA may be an EIciRNAs or an ecircRNA. c CiRNA is generated from the intron lariat including adenosine deaminase acting on RNA 1, DEAH box helicase 9, and heterogeneous nuclear ribonucleoprotein L, suggesting that these RNA-binding proteins might be involved in the biogenesis of circRNAs during terminal B-cell differentiation. Notably, the circRNAs in human plasma cells were mainly derived from immunoglobulin (Ig) genes and represented the combinatorial clonal state of Ig loci.
Calcitonin gene-related peptide (CGRP) could induce the expression of IL-6 in macrophages 43 . In this process, mmu_circRNA_007893 mediated the IL-6 expression by working as an endogenous mmu-miR-485-5p sponge. When macrophages were stimulated by CGRP, mmu_-circRNA_007893 was significantly increased. However, after silencing mmu_circRNA_007893, the level of mmu-miR-485-5p was increased while IL-6 mRNA expression was decreased. During the process of immunosenescence, there was a significant feature that the proportion of CD8 T lymphocytes lacking CD28 expression would be increased 44 . Wang et al. 45 discovered that cir-cRNA_100783 in ageing human CD8 + T cells might function as a new biomarker for CD28-related CD8 + Tcell ageing. By further investigating the circRNA_100783targeted miRNA-mRNA network, they observed that circRNA_100783 might be mainly related to alternative splicing events, the production of splice variants and the expression of phosphoprotein. Interestingly, circANRIL has been shown to disrupt pre-rRNA processing and ribosome biogenesis by binding to pescadillo homolog 1 in vascular smooth muscle cells and macrophages, leading to the nucleolar stress and activation of p53, which in turn induced apoptosis and inhibited proliferation 46 .
By measuring the expression profiles of circRNAs in 20 human tissues that were highly correlated with diseases, Maass et al. 47 demonstrated that many circRNAs showed tissue-specific expression and could be closely related to the clinical phenotypes and mechanisms of human diseases. At the same time, they found that immune-related components, toll-like receptor 6 (TLR6), and myosin 1 F (MYO1F), could produce circRNAs in neutrophils, suggesting that the circRNAs were likely to be involved in neutrophil immune responses. In addition, Li et al. 48 found that a W chromosome-linked circRNA was female-biased expression in a kind of flatfish (halfsmooth tongue sole) and tended to be expressed in some immune tissues, especially head kidney and spleen. Importantly, the expression of this circRNA in spleen was significantly upregulated after infection, indicating that it might be related to the immune response. In summary, circRNAs actively participate in various biological processes in immune cells, such as differentiation, polarization, immune response, senescence, and apoptosis (Table 1). Table 1

circRNAs in immune regulation
In recent years, increasing evidence linked circRNAs to immune regulation under multifarious physiological and pathological conditions, including anti-infection immunity 49,50 , tumor immunity 51,52 , the activation of inflammation 53 , and even organ transplantation 54 . Through next-generation sequencing technology, Ma et al. 55 found that 123 circRNAs were differentially expressed in Mockand transmissible gastroenteritis virus (TGEV)-infected porcine intestinal epithelial cell line. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the mRNAs in circRNA-miRNA-mRNA regulatory network were most significantly involved in inflammation and immune response, including retinoic acid-inducible gene-I (RIG-I)-like receptor, tumor necrosis factor (TNF), NOD-like receptor (NLR), TLR, and nuclear factor-κB (NF-κB) pathway. Of note, ssc_circRNA_009380 could promote the activation of NF-κB pathway via interacting with miR-22, thereby mediating TGEV-induced inflammation. Analogously, another study analyzed the circRNA expression profiles and circRNA-associated competing endogenous RNA (ceRNA) network of early HIV infection (EHI) patients 56 . The results indicated that 1365 circRNAs were abnormally expressed in HARRT-naive EHI patients in contrast to healthy controls, and the targeting mRNAs among the ceRNA networks were mainly related to inflammatory response, immune response and defense response to virus infection. Actually, circRNAs were closely related to the immune factors NF90/NF110 in viral infection 29 . Specifically, NF90/NF110 enhanced pre-mRNA back-splicing by stabilizing the intron complementary sequence pairs in the nucleus and interacted with mature circRNAs to form complexes in the cytoplasm. After viral invasions, NF90/ NF110 in the nucleus were transported to the cytoplasm, and then the levels of circRNAs were reduced. At the same time, NF90/NF110 could be released from the complexes and subsequently suppressed viral replication by binding to viral mRNAs.
Meaningfully, Fu et al. 57 found that 171 circRNAs were dysregulated in peripheral blood mononuclear cells (PBMCs) of patients with active tuberculosis (TB). Of these, circRNA_103017, circRNA_101128, and cir-cRNA_059914 were expected to serve as new biomarkers for active TB. What's more, circRNA_101128 could contribute to the pathogenesis of TB by regulating miRNA let-7a. In LPS-stimulated mouse macrophages, Ng et al. 58 observed that one circRNA, mcircRasGEF1B, was regulated by TLR4 pathway. Moreover, the knockdown of mcircRasGEF1B reduced the expression of mature intercellular adhesion molecule-1 (ICAM-1) via modulating the stability of ICAM-1 mRNAs. Noteworthily, ICAM-1 was related to the pulmonary neutrophil recruitment in LPS-induced airway disease 59 , and could also suppress the polarization of M2 macrophages through the blockade of efferocytosis in tumor microenvironment 60 , implicating its various roles in innate immune response.
A recent study showed that hsa_circ_0005105 could facilitate the expression of inflammatory cytokines by regulating the miR-26a-targeted nicotinamide phosphoribosyltransferase, which provided a new target for the treatment of osteoarthritis (OA) 61 . In contrast to nonlesional skin of severe acne patients, Liang et al. 62 discovered that up to 538 circRNAs were aberrantly expressed in adjacent lesional skin, and these circRNAs were mainly connected with the biological pathways such as inflammation, metabolism, and immune response. In addition, has_circ_0020397 could promote the expression of telomerase reverse transcriptase and programmed death-ligand 1 (PD-L1) by binding to miR-138, thereby regulating the viability, apoptosis and invasion of colorectal cancer cells 63 . Studies have shown that PD-L1 is closely related to tumor escape from immune control 64,65 , so has_circ_0020397 may promote tumor development by regulating tumor immunity. These findings demonstrate that circRNAs are crucial participants in immune regulation (Fig. 2). Hence, it is foreseeable that dysregulation of these functions is very likely to be involved in the development of autoimmune diseases.

circRNAs in autoimmune diseases
Autoimmune diseases, mainly characterized by a damaged immune system and the loss of immune tolerance to self-antigens, are a group of heterogeneous conditions 66 . Although the molecular mechanisms are still largely unknown, increasing evidence indicates that the complex interplay of environmental factors and epigenetic dysregulation facilitate the pathogenesis of these diseases in genetically susceptible individuals 67,68 . As described above, circRNAs are closely associated with the immune system. Meanwhile, recent studies have demonstrated that circRNAs are not only involved in the pathogenesis of autoimmune diseases, but also represent non-invasive biomarkers for them (Fig. 3).

circRNAs in SLE
SLE is a chronic autoimmune disease that predominately affects women of childbearing age. Its main features are the autoreactive B and T lymphocytes as well as the overproduction of antibodies targeting self-antigens 69 . Unfortunately, SLE can result in multi-organ pathologies and a wide range of clinical manifestations, including arthritis, central nervous system disease, renal disease and skin disease 70 . Although SLE is immune-mediated, the pathogenic mechanisms are still not fully understood.

T-cell circRNAs in SLE
Recently, 127 differentially expressed circRNAs were identified in T cells of SLE patients, and a downregulated circRNA, hsa_circ_0045272, was further verified by quantitative PCR 71 . Mechanism research revealed that the knockdown of this circRNA significantly upregulated the early apoptosis and enhanced the production of IL-2 in activated Jurkat cells. Zhang et al. 72 supported that hsa_circ_0012919 was aberrantly upregulated in CD4 + T cells of SLE patients. Meanwhile, the downregulation of hsa_circ_0012919 increased the expression of DNA methyltransferase 1 (DNMT1), whereas reduced the expression of CD70 and CD11a in CD4 + T cells from inactive and active SLE patients. The inhibition of hsa_-circ_0012919 also rescued the DNA hypomethylation of CD70 and CD11a in CD4 + T cells of SLE patients, which could be reversed by downregulation of DNMT1. Strikingly, this circRNA could regulate the expression of regulated on activation, normal T cell expressed and secreted (RANTES) and Kruppel-like factor 13 (KLF13) by bonding to miR-125a-3p. It has been confirmed that migration rate of basophils to RANTES and monocyte chemotactic protein 1 (MCP-1) is remarkably increased in SLE patients, which is possibly associated with tissue damage in SLE 73 . KLF13 could positively regulate RANTES and was related to the expression of IL-4 in CD4 + T cells 74 .

PBMC circRNAs in SLE
Wang et al. 75 found downregulation of circIBTK and upregulation of miR-29b in PBMCs of SLE patients, both of which were correlated with anti-double-stranded DNA, SLE Disease Activity Index (SLEDAI) score and complement component 3 (C3) level. Importantly, circIBTK could inhibit the DNA demethylation and activation of protein kinase B (AKT) by binding to miR-29b in SLE. Many studies have shown that AKT signaling pathway can regulate the functions of immune cells, and its The exogenous circRNA induces innate immune response by activating RIG-I, whereas the endogenous circRNA binds to different RNA-binding proteins that reflect its endogenous biogenesis. hsa_circ_0005105 facilitates the expression of inflammatory cytokines by regulating the miR-26a targeted NAMPT. has_circ_0020397 promotes the expression of PD-L1 by binding to miR-138, thereby participating in tumor immunity. In addition, mcircRasGEF1B induced by LPS is involved in anti-bacteria immunity by modulating the stability of ICAM-1 mRNAs dysregulation leads to the progression of SLE. For instance, AKT could coordinate IL-2 signaling and T-cell antigen receptor to hold the expression of adhesion molecules, cytolytic effector molecules, as well as cytokine and chemokine receptors in cytotoxic T cells 76 . Another research showed that hsa_circ_0049224 and has_-circ_0049220 were underexpressed in PBMCs of inactive and active SLE patients 77 . Moreover, the levels of these two circRNAs were negatively correlated with SLEDAI and the degree of SLE severity, which indicated that they might be regarded as markers for the activity and severity of SLE.

Plasma circRNAs in SLE
In addition, hsa_circ_400011, hsa_circ_102584, hsa_-circ_101471, and hsa_circ_100226 were abnormally expressed in plasma of SLE patients 9 . Through bioinformatics analysis, the researchers discovered multiple MREs of hsa_circ_100226, including hsa-miR-24-3p, hsa-miR-875-3p, hsa-miR-138-5p, hsa-miR-620, and hsa-miR-145-3p. Among them, decreased miR-138 could enhance NF-κB activation via suppressing the expression of p65 in the chondrocytes, triggering the inflammatory response 78 . Moreover, miR-138-5p regulated extracellular matrix catabolism and inflammation, thereby affecting the progression of OA 79 . Interestingly, the upregulated cir-cRNA_002453 level in plasma of lupus nephritis (LN) patients was related to the severity of renal involvement 80 . Although the level of circRNA_002453 had no significant correlation with disease activity, it was positively associated with renal SLEDAI score and 24-hour proteinuria.

circRNAs in RA
RA is a prevalent autoimmune disorder characterized by generalized inflammation in multiple joints, which always results in serious cartilage and bone erosion as well as articular deformation 81 . Rheumatoid factor (RF), anticarbamylated protein (anti-CarP), and anti-cyclic citrullinated peptide-2 (anti-CCP2) are the most well-known autoantibodies in this disease 82 .

PBMC circRNAs in RA
Latest studies revealed that the levels of hsa_-circ_0058794 and hsa_circ_0092285 were markedly increased in PBMCs of patients with RA, and the levels of hsa_circ_0038644 and hsa_circ_0088088 were decreased 83 . Actually, hsa_circ_0038644 was spliced from the protein kinase C beta gene, which was related to the activation of NF-κB 84 . Furthermore, the expression of ciRS-7 was significantly upregulated in RA patients, and it could reduce the inhibitory effect of miR-7 on mammalian target of rapamycin (mTOR) by inhibiting the function of miR-7 85 . As the phosphatidylinositol-3-kinase/ AKT/mTOR (PI3K/AKT/mTOR) signaling pathway played an important role in synoviocyte proliferation and inflammatory responses 86,87 , ciRS-7 might be involved in the development of RA by regulating mTOR.
Other cell type circRNAs in RA hsa_circ_0001859 was one of the differentially expressed circRNAs in synovial tissues of RA patients 88 . Mechanism studies found that this circRNA could promote activating transcription factors 2 expression and increase inflammatory activity by targeting miR-204/211. Furthermore, nuclear factor E2-related factor 2 (Nrf2), a potential therapeutic target for rheumatic diseases, could regulate many biological processes such as inflammation, immune response and cartilage and bone metabolism in the body 89 . By analyzing the circRNA expression profiles in the substantia nigra and corpus striatum of Nrf2knockout mice, Yang et al. 90 found that mmu_-circRNA_34132, mmu-circRNA-015216 and mmu_-circRNA_017077 were involved in the Nrf2-mediated neuroprotection against oxidative stress. Notably, the authors also uncovered that four mRNAs, Atp6v0a1, Atp6v0b, Atp6v0c, and Atp6v0e2, were enriched in RA pathway in the circRNA-miRNA-mRNA interaction network. Atp6v0c and Atp6v0e2 were potentially regulated by mmu_circRNA_017077 via binding to mmu-miR-346-3p, and Atp6v0e2 and Atp6v0a1 were potentially regulated by mmu_circRNA_34132 via binding to mmu-miR-346-3p as well. These results supported that mmu_-circRNA_34132 and mmu_circRNA_017077 might

circRNAs in MS
MS is a chronic disease of the central nervous system (CNS), and diffuse immune mechanisms as well as neurodegeneration are the underlying pathological processes in this disease. The peripheral immune response targeting the CNS occurs mainly in the early stage of MS, whereas immune process within the CNS dominates the progressive stage 91 . Most patients will develop permanent disability during the course of their disease, creating a huge burden for individual, family and society levels 92 .
Through the further characterization of Gasdermin B alternative splicing and back-splicing profiles, Cardamone et al. 93 found that alternative splicing isoforms and an identified ecircRNA, containing exons 4 and 5, were significantly dysregulated in PBMCs of relapsing-remitting MS patients, which suggested that the abnormal RNA metabolism was involved in the pathogenesis of this disease. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was an long non-coding RNA (lncRNA) that could regulate alternative splicing and has been shown to be associated with MS 94 . A systematic study found that the level of MALAT1 was upregulated in MS patients 95 . Meanwhile, 1114 alternative splicing events were significantly modulated and 49 circRNAs were differentially expressed in MALAT1-knockdown Jurkat T cells, a relevant cellular model for MS. Besides, the RNA-binding protein motif analysis showed a particular enrichment for the QKI in the exons modulated by MALAT1. Remarkably, QKI has been reported to regulate the formation of circRNAs 30 . These data indicate that MALAT1 dysregulation may lead to the development of MS by affecting splicing and back-splicing events.

circRNAs in other autoimmune diseases
Psoriasis is an inflammatory disease that mainly affects the skin and joints, and its pathophysiological characteristics are abnormal proliferation of keratinocytes and infiltration of immune cells in the dermis and epidermis 96 . Recently, Liu et al. 97 discovered six downregulated and 123 upregulated circRNAs in skin mesenchymal stem cells (S-MSCs) of psoriatic lesions. Pathway analysis observed that the significantly downregulated mRNAs in the lesions mainly enriched in Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling, which was reported to participate in immune regulation 98 . Of these, a circRNA chr2:206992521| 206994966 could affect the activity of T lymphocytes in local lesions by regulating the secretion of certain cytokines, including IL-6, IL-11, and hepatocyte growth factor 97 . In addition, hsa_circ_0061012, hsa_circ_0003689, chr4:121675708|121732604, and hsa_circ_0003718 were abnormally expressed in psoriatic lesions and might promote disease progression by interacting with miRNAs associated with psoriasis 99,100 .
PBC is a cholestatic, autoimmune-mediated liver disease that slowly progresses to portal fibrosis and biliary cirrhosis 101 . By carrying out the circRNA expression profiles, Zheng et al. 102 found 22 aberrantly expressed circRNAs in plasma of PBC patients. It was worth noting that the level of hsa_circ_402458 was higher in PBC patients not treated with ursodeoxycholic acid (UDCA) than in those treated with UDCA. At the same time, the authors showed that hsa_circ_402458 might target two miRNAs, hsa-miR-943, and hsa-miR-522-3p. For miR-522-3p, it might be an effective target for regulating chronic inflammatory disorder 103 . Therefore, it can be speculated that hsa_-circ_402458 may function as a miRNA sponge to regulate inflammation-related signaling pathways, thus contributing to the development of PBC.
In addition, by studying the circRNA expression profiles in atrial tissues from patients with persistent atrial fibrillation (AF) with rheumatic heart disease, Hu et al. 104 predicted the potential roles of the differentially expressed circRNAs. The results suggested that 51 circRNAs were upregulated, and 57 circRNAs were downregulated in AF tissues compared with controls, respectively. Gene Ontology (GO) analysis revealed that the most significantly enriched biological process term was muscle contraction, the most significantly enriched cellular component term was muscle myosin complex, and the most significantly enriched molecular function term was muscle alpha-actinin binding. Meanwhile, KEGG pathway analysis indicated that the main involved pathways were dilated cardiomyopathy and hypertrophic cardiomyopathy.

circRNAs as potential biomarkers in autoimmune diseases
Owing to their stability, abundance, and evolutionary conservation, as well as their differential expression in patients with autoimmune diseases, circRNAs are likely to be potential biomarkers for these diseases. circPTPN22 derived from protein tyrosine phosphatase nonreceptor type 22 (PTPN22) was downregulated in the PBMCs of patients with SLE 105 . Importantly, the receiver operating characteristic (ROC) curve analysis showed that circPTPN22 had good diagnostic value for SLE. The downregulation of circPTPN22 was strongly negatively correlated with the SLEDAI scores, suggesting that this circRNA might be a biomarker for SLE diagnosis and disease severity. Zhang et al. 106 observed that hsa_-circRNA_407176 and hsa_circRNA_001308 were downregulated in both PBMCs and plasma of patients with SLE. Also, these two circRNAs in plasma and PBMCs might be candidate biomarkers for SLE, and their combination could improve the diagnostic efficiency. Even more, the level of hsa_circRNA_001308 was associated with C reactive protein and anti-sjögren's syndromerelated antigen A in plasma, as well as leukopenia in PBMCs. Analogously, hsa_circ_0003090 and hsa_-circ_0057762 in whole blood could differentiate the patients with SLE from the healthy controls, indicating that these two circRNAs might have potential value for SLE diagnosis 107 .
By the analysis of ROC curve, Ouyang et al. 108 found that circRNA_104871 in PBMCs was a strong predictor for RA. Likewise, another study found that hsa_-circ_0044235 was significantly decreased in peripheral blood of patients with RA 109 . Meaningfully, according to the risk score based on hsa_circ_0044235, the researchers could effectively distinguish the patients with RA from those with SLE. Iparraguirre et al. 110 indicated that circ_0035560 and circ_0005402 were underexpressed in peripheral blood leukocytes of MS patients and might function as dependable biomarkers for this disease. Interestingly, these two circRNAs were derived from annexin A2 (ANXA2), whose linear form was also downregulated in MS patients. Increasing evidence has shown that ANXA2 is involved in many autoimmune diseases, including antiphospholipid syndrome and LN, suggesting that circ_0035560 and circ_0005402 may be associated with the development of MS 111,112 . In summary, these studies provide a theoretical basis for the clinical application of circRNAs in autoimmune diseases.

Conclusion and future perspectives
Indeed, increasing evidence has identified that cir-cRNAs are active participants in multiple stages of immune-cell development and immune regulation. Furthermore, circRNAs may not only be diagnostic biomarkers for human autoimmune diseases, but also represent the disease activity or severity. More importantly, circRNAs contribute to the development of autoimmune diseases by acting as miRNA sponges to regulate many biological processes, including DNA methylation, immune response, and inflammatory response (Table 2). Therefore, elucidating the roles of circRNAs in the setting of autoimmune disease will be a promising field.
Recently, Chen et al. 113 found that the purified exogenous circRNA could induce innate immune response and confer a protective effect on viral infection by activating RIG-I. Further exploration indicated that cells could distinguish between self-nonself circRNAs based on the introns that produced them, and the reason might be that mature human circRNAs always bound to different RNA-binding proteins that reflect their endogenous splicing and biogenesis. In this regard, we speculate that the abnormal circRNAs in vivo, like foreign circRNAs, are involved in autoimmune diseases by activating the immune signaling. Interestingly, Xia et al. 114 demonstrated that a novel circRNA, cia-cGAS, could protect dormant long-term-hematopoietic stem cells from cyclic GMP-AMP synthase (cGAS)-mediated exhaustion by suppressing the enzymatic activity of cGAS under homeostatic conditions. Meanwhile, the binding affinity of cia-cGAS for cGAS was stronger than that of self-DNA, thereby inhibiting cGAS-mediated generation of type I IFNs to maintain dormant HSCs. Three-prime repair exonuclease 1 (TREX1) was a major 3ʹ → 5ʹ DNA exonuclease, whose dysregulation has been associated with some autoimmune diseases 115,116 . Conspicuously, the overexpression of cia-cGAS could suppress IFN expression in TREX1-deficient bone marrow derived macrophages (BMDMs), indicating that cia-cGAS could restrain autoimmune signaling in TREX1-deficient cells 114 . Thus, cia-cGAS might act as a potential target for the treatment of autoimmune diseases by antagonizing cGAS. Recently, Liu et al. 117 found that endogenous cir-cRNAs tended to form 16-26 bp intra-molecular RNA duplexes and inhibited double-stranded RNA (dsRNA)activated protein kinase (PKR) activity by preferentially binding to PKR. The activity of RNase L in PBMCs derived from SLE patients was enhanced, accompanied by reduced circRNA expression and augmented PKR phosphorylation. Importantly, overexpression of dsRNAcontaining circRNAs could strongly attenuate the aberrant PKR activation cascade in SLE patient-derived cells, suggesting that circRNAs might serve as potential targets for the treatment of autoimmune diseases.
However, several important questions merit further resolution. To date, we have detailed the close relationship between autoimmune diseases and circRNAs, but little is known about the molecular mechanisms that trigger the pathogenesis. CircRNAs that act as endogenous miRNA sponges have been studied widely, but few circRNA/miRNA interactions have been experimentally validated in immunity 61,63,118 . It is suggested that exploring other mechanisms may improve the functional description of circRNAs in immunological contexts. Furthermore, circRNAs can exert potential biomarkers for various autoimmune diseases. Nonetheless, in many studies, the sample size of patients is relatively small, or the sample source has some limitations, which may affect the validity and universality of the conclusions that cir-cRNAs can serve as biomarkers. Moreover, in-depth studies of circRNAs' biogenesis, accumulation in the cytoplasm, and even post-transcriptional modifications may increase our understanding of their biological functions.
In conclusion, continued investigation into circRNAs may yield more discoveries in the pathogenesis of autoimmune diseases and broaden the spectra of diagnosis and therapy for these diseases in the future.