circRNA: Regulatory factors and potential therapeutic targets in inflammatory dermatoses

Abstract The skin is the largest organ of the human body and acts as the first line of defence against injury and infection. Skin diseases are among the most common health problems and are associated with a considerable burden that encompasses financial, physical and mental consequences for patients. Exploring the pathogenesis of skin diseases can provide insights into new treatment strategies. Inflammatory dermatoses account for a large proportion of dermatoses and have a great impact on the patients' body and quality of life. Therefore, it is important to study their pathogenesis and explore effective treatment. Circular RNAs (circRNAs) are a special type of RNA molecules that play important regulatory roles in several diseases and are involved in skin pathophysiological processes. This review summarizes the biogenesis, properties and functions of circRNAs as well as their roles in the pathogenesis of inflammatory dermatoses, including psoriasis, lupus erythematosus, atopic dermatitis, lichen planus and severe acne and their potential as therapeutic targets.


| INTRODUC TI ON
The skin, which forms an interface between the body and the environment, provides the first line of defence against exogenous insults. Skin disorders not only cause considerable economic burden to patients, but also negatively impact their physical and mental health. For example, both severe acne and psoriasis can negatively impact the quality of life. Therefore, understanding the pathogenesis of skin diseases is crucial to the development of therapeutic approaches. Circular RNAs (circRNAs) are a special type of RNA molecule involved in several diseases and can serve as biomarkers or therapeutic targets. For instance, circRNAs are diagnostic markers and potential therapeutic targets for lung, breast and colorectal cancers. 1 circRNAs also play a role in the pathological process of various cardiovascular diseases, such as myocardial infarction, heart failure and atherosclerosis. 2 Associations of circRNAs with autoimmune diseases and viral infections have also been reported. 3 In addition, circRNAs are involved in the pathogenesis of skin diseases through various molecular mechanisms. This review discusses the biological origin, properties and functions of circRNAs as well their roles in inflammatory skin disorders.

| circRNA
A circRNA is a non-coding RNA derived from back-splicing, a type of non-canonical splicing in eukaryotes. Different from linear RNAs, circRNAs form a closed ring structure with covalent bonds, which is more stable against exonucleases. Increasing evidence has proposed a regulatory role for circRNAs in gene expression.

| Brief research history of circRNAs
In 1971, 'viroids' were found in potato and they were the first cir-cRNA molecules to be discovered. 4 And, the ultrastructure of cir-cRNAs was first observed using an electron microscope in 1976.
Several circRNAs have since been discovered in abnormal splicing transcripts of some genes, including the tumour suppressor gene DCC in humans, testis-determining gene Sry in mice and the cytochrome P450 2C24 gene in rats. Supported by high-throughput RNA sequencing, numerous circRNAs in eukaryotes, including plants, fungi, protists and various types of metazoans, have been identified, and their abundance exceeds that of their associated linear mRNA in some cases, suggesting that circRNAs are old, universal and conserved in the gene expression programmes of eukaryotes.
CircRNAs were believed to result from splicing errors with unknown function at first. In 1988, Weiner et al. 5 found that circRNAs with open reading frames encode the antigenic polypeptides P24 Delta and P27 Delta in hepatitis delta virus. Later studies showed that cir-cRNAs can act as microRNA (miRNA) sponges via their binding sites, regulating the activity of miRNAs on certain target genes. More recently, the involvement of circRNAs in the physiology and pathology of various tissues has also been revealed.

| Biogenesis of circRNAs
The circRNAs are generally categorized into exonic circRNAs (ecir-cRNAs), 6 exon-intron circRNAs (EIciRNAs), 7 intronic circRNAs (ciRNAs) 8 and intergenic circRNAs. 9 EIciRNAs and ciRNAs are predominantly present in the nucleus, whereas ecircRNAs are mainly enriched in the cytoplasm. As previously mentioned, circRNAs are derived from back-splicing, a type of alternative splicing that occurs via different mechanisms from those of canonical linear splicing. The

| Lariat-driven circularization
Evidence indicates that the production of circRNAs is a result of exon-skipping events. The pre-mRNA is spliced in accordance with the classical GU-AG rule. Owing to the folded pre-mRNA, the nonadjacent exons are at close proximity to each other, resulting in exonskipping. After back-splicing, a lariat precursor containing exons and introns is formed, which can splice itself internally to remove introns and form an ecircRNA. However, EIciRNA is produced when introns are not completely spliced but remain in the newly generated circR-NAs ( Figure 1A). 10

| Intron-pairing-driven circularization
In the mechanism of intron-pairing-driven circularization, backsplicing to a circular molecule is likely triggered by intronic sequences adjacent to the exons that will be circularized. The intronic sequences can interact with each other, bringing the ends of exons into close proximity. Long flanking complementary introns are essential for the formation of circRNAs, and these sequences are usually either non-repetitive complementary sequences or intronic repeat sequences, including Alu elements and mammalian-wide interspersed repeats. 11,12 Back-splicing is driven by base pairing of these sequences. Similar to lariat-driven circularization, a lariat precursor containing both exons and introns, or EIciRNA, is formed after back-splicing, followed by the removal of introns to form an ecircRNA. In this model, certain structures such as Alu elements bring non-adjacent sequence pairs into apposition, initiating circularization, without the requirement of exon-skipping ( Figure 1B). 13

| RNA-binding protein (RBP)-driven circularization
Another mechanism for circRNA formation based on RBPs has been proposed. The splicing factors, including heterogeneous nuclear ribonucleoprotein L (HNRNPL), 14 Quaking (QKI), 15 Muscleblind (MBL), 13 FUS 16 and immune factor NF90/NF110, 17 can bind to certain motifs in the flanking introns on both sides of the exon, which will be back-spliced, bringing these introns into close proximity with each other and facilitating the generation of circRNAs. The main difference between this process and intron-pairing-driven circularization is that the former is regulated by RBPs while the latter relies on complementary sequence pairing ( Figure 1C).

| Other mechanisms
Biogenesis of ciRNAs relies on a motif that consists of a 7 nucleotide (nt) element with enriched GU ribonucleotides at the 5′ splicing end and an 11 nt element with enriched C ribonucleotides at the 3′ end near the branchpoint site. Generally, intron lariats are degraded by debranching enzymes, but the above structures can prevent such enzymes from binding to the ciRNAs ( Figure 1D). 8

| Regulation of circRNA biogenesis
During circRNA biogenesis, several regulators can influence the biogenesis rate. It has been demonstrated that the transcription elongation rate (TER) of a gene is influenced by whether it can produce circRNA; if a gene can produce circRNA, its TER will be high. 18 A mild increase in TER promotes circRNA formation, as fast elongation pairs non-adjacent complementary sequences across one or more exons and introns, 18 consequently promoting circularization. The spliceosome is also associated with the rate of circRNA formation.
Depletion or inhibition of some core spliceosome components, such as SF3b and SF3a complexes, increases the expression of circRNAs while decreasing the expression of their associated linear mRNAs, 19 suggesting a possible conversion from canonical alternative splicing to back-splicing. Moreover, some sequences, such as simple poly(A) sequences and mild distortions like G-U wobble pairs, can suppress circRNA circularization. 11 Although several mechanisms of circRNA biogenesis have been identified, the precise mechanisms remain unclear.

| Stable molecules
The circRNAs of eukaryotic cells are exceptionally stable. 20 Because circRNAs have no free 3′ poly(A) tail or 5′ cap, they are resistant to RNase R, a potent 3′-to-5′ exonuclease that can efficiently degrade linear RNAs. 21 The half-life of mRNAs is 10 h on average, whereas most exonic circRNA species have a half-life of over 48 h, which can be attributed to their resistance to RNA exonucleases. 22

| Diversity and specific expression patterns
More than 25,000 circRNAs have been identified in human fibroblasts. 23 At the individual gene level, one gene locus can produce multiple circRNAs via various mechanisms. Generally, circRNA expression is low, but some circRNAs are expressed at higher levels than those of their linear mRNAs. 24 For example, circRNA expression increases substantially compared with that of linear isoforms during central nervous system ageing. Hence, circRNAs can serve as ageing biomarkers in Drosophila. Under certain circumstances, the amount of circRNAs can be over 10-fold richer than that of the corresponding linear mRNAs. Additionally, circRNA expression is tissue-and cell-type-specific. For example, AMBRA1 RNA shows differential expression of two types of circular isoforms in different cells; the long isoform (362 nt) is expressed at higher levels in MCF-7 cells, whereas the short isoform (182 nt) shows higher expression in HepG2 cells. Moreover, the mammalian brain is enriched with circRNAs, 25 and in Drosophila, circMbl expression in the brain is considerably higher than in the ovary. 26 The circRNA expression also varies with the developmental stage. During human epithelialmesenchymal transition, the expression of circRNAs markedly increases. 15 This phenomenon has also been reported in the mouse brain, where the expression of 258 circRNAs in the cortex and 250 circRNAs in the hippocampus was higher in 22-month-old mice than in 1-year-old mice. 27

| Evolutionary conservation and variation between different species
Studies have found that some circRNAs in humans are also present in the circRNA expression profile of mice, with 10%-20% being identical. 28 In fact, 457 human circRNAs can be mapped to murine genes, 69 of which contain homologous back-splice points. The complementary sequences in introns near the back-splice points, instead of the circRNA sequences themselves, are associated with the emergence of these conserved circRNAs. Moreover, compared with species-specific circRNAs, the conserved molecules are more abundant in both humans and mice. 28

| Functions of circRNAs
Although circRNA expression in eukaryotes is generally very low, many play regulatory roles in gene expression under various physiological and pathological conditions, such as during the innate immune response, basic functioning of nerve cells and cell differentiation and proliferation.

| Acting as miRNA sponges
Competing endogenous RNAs (ceRNAs) can bind miRNAs via specific sites, acting as miRNA sponges and regulating miRNA function on their target genes. 29 As a type of ceRNA, circRNAs, such as CDR1as or ciRS-7, 30 circ-HIPK3, 31 circ-SRY, circRNA_010383, 32 hsa_ circ_0000615 33 and hsa_circ_0000735, 34 can also act as sponges for miRNA. ciRS-7, the most well-characterized circRNA, is stably and highly expressed in human and mouse brains. ciRS-7 has over 70 selective binding sites for miR-7, which is associated with Argonaute (AGO) proteins, and can significantly repress miR-7 activity, thereby increasing AGO protein levels, 35,36 suggesting a regulatory function for ciRS-7. Moreover, the expression of Fos, an immediate early gene and a direct miR-7 target, increases in CDR1as-deficient brains and the animals display impaired sensorimotor gating, suggesting a possible link between circRNA and the behavioural phenotype. 30 Particularly, circRNAs can be sponges for several miRNAs instead of containing binding sites for just one miRNA; for instance, circC-CDC66 can bind to miR-33b, miR-93 and miR-185. 37

| Interaction with proteins
The circRNAs can also act as sponges for proteins, interacting with many different RBPs to influence protein functions. For example, circPABPN1 can bind to HuR, a type of RBP, preventing the binding between HuR and PABPN1 mRNA and subsequently reducing the latter's translation. 38 Circ-Foxo3, which is associated with cell cycle progression, is highly expressed in non-cancer cells. Normally, cyclin-dependent kinase 2 (CDK2) promotes cell cycle progression by interacting with cyclin A/E, whereas cyclin-dependent kinase inhibitor 1 (p21) arrests this process. Ectopic expression of circ-Foxo3, in the presence of CDK2 and p21, leads to ternary complex formation and CDK2 repression, resulting in a blockade of cell cycle progression. 39 Another example is the MBL protein in D. melanogaster; MBL promotes the expression of circMbl, which binds to the protein itself and regulates parental gene expression. Excessive MBL protein decreases the expression of its linear mRNA by promoting circMbl expression, suggesting the presence of an autoregulatory circuit. 9 Additionally, circ-Amotl1 can bind to AKT1 and PDK1, resulting in phosphorylation of AKT1 and its translocation into the nucleus, a process involved in cardioprotection. 40

| Templates for translation
Although circRNAs have been thought to be non-coding, at least a subset of circRNAs can be translated into proteins under specific conditions. [41][42][43] During the translation of linear mRNAs, a 7-methylguanosine (m7G) cap at the 5′ end and a poly(A) tail at the 3′ end are essential elements. Due to the lack of both these structures, circRNAs are translated in a cap-independent manner. One such mechanism is driven by N 6 -methyladenosine (m6A) modification of the 5′ untranslated region. m6A can bind to eukaryotic initiation factor 3 (eIF3), facilitating recruitment of the 43S complex to initiate translation without the cap-binding factor eIF4E. 44 This process is promoted by methyltransferases (e.g. METTL3 and METTL14) and heat shock and restrained by demethylases (e.g. FTO). 42 Another mechanism of circRNA translation relies on internal ribosome entry sites, which can accelerate binding between circRNAs and ribosomes or initiation factors, thereby promoting translation. 41 CircRNAs containing repeated FLAG-coding sequences can also be translated into proteins via rolling circle amplification. 45 Through these different mechanisms, circRNA translation into functional proteins is possible.
For example, FBXW7-185aa, encoded by circ-FBXW7, has potential prognostic implications in brain cancer, 43 and the protein encoded by circ-ZNF609 can specifically control myoblast proliferation. 41

| Regulation of transcription
As mentioned above, EIciRNAs and ciRNAs mainly exist in the cell nucleus and some are involved in transcription regulation. EIciRNAs can interact with U1 small nuclear RNA to form a complex that binds Pol II at their parental gene promoters to upregulate gene expression. 7 CircSCMH1 may interact with transcription factor methyl CpG binding protein 2 (MeCP2) to restrain its transcriptional activity. circMRPS35 can elicit the acetylation of H4K5 in the promoters by recruiting the histone acetyltransferase KAT7, and it can bind to FOXO1 and FOXO3a promoters to activate transcription. In Arabidopsis, circRNAs derived from exon 6 of the SEP3 gene can bind to its cognate DNA locus to pause transcription. 46 Ci-ankrd52 has a stronger ability of R-loop formation than that of its cognate pre-mRNA, allowing the release of pre-mRNA from R-loops by ci-ankrd52 replacement and followed by ciRNA removal via RNase H1 for efficient transcriptional elongation. 47

| circRNA s IN THE S KIN
The circRNAs are critical for physiological homeostasis and function through their modulation of gene expression in various tissues, including the skin. Indeed, the regulatory role of circRNAs in cutaneous wound healing has been well demonstrated. For instance, circ-Amotl1 interacts with STAT3 to promote wound healing, 48 whereas hsa_circ_0084443 is downregulated during normal wound healing and negatively regulates keratinocyte migration. Diabetic foot ulcers exhibit increased expression of hsa_circ_0084443, suggesting that its overexpression can contribute to the delayed wound healing. 49 Knocking down circ-PRKDC enhances keratinocyte migration and promotes skin wound healing; this is achieved through the miR-31-FBN1 axis. 50 Moreover, circRNAs can regulate keratinocyte differentiation, as inhibition of circZNF91, which contains 24 miR-23b-3p binding sites, 51 reduces the expression of several proteins associated with keratinocyte differentiation. 52 circRNAs have also been shown to regulate melanin production, and the evidence suggests that they are involved in the pigmentation and melanin-related pathways. 53 For instance, the interaction between lnRNA and cir-cRNAs is associated with melanocyte development. 54 ciRS-7, which is highly expressed in melanocytes, triggers melanin production in melanocytes via regulation of the miR-7-STAT3 and AKT-FGF2 paracrine axes, suggesting ciRS-7 as a regulator for the development of pigmented skin diseases. 55 Moreover, circ_0091223 is involved in α-MSH-induced melanin production. 56 Other regulatory roles of circRNAs in cutaneous functions include their involvement in keloid formation via the interaction between hsa_circ_0001320 and miR-574-5P, 57,58 skin tissue regeneration under mechanical tension, 59 circCOL3A1-859267 inhibition of the UVA-induced downregulation of type I collagen in skin 60,61 ; and the regulation of hair growth. 62 Therefore, circRNAs can regulate multiple cutaneous functions.

| Psoriasis
Psoriasis is a common inflammatory skin disease that presents with abnormal differentiation and excessive proliferation of epidermal cells as its main pathological features. 63 Psoriasis has a great impact on the quality of life of patients. 64 Although the pathogenic roles of keratinocytes, dendritic cells, T cells, neutrophils and mesenchymal stem cells in psoriasis are appreciated, 65  promoting IFNγ signalling in Tregs. 75 These results suggest that circ_0003738 attenuates the inhibitory function of Tregs, which contributes to the onset of psoriasis.

| Lupus erythematosus
Lupus erythematosus is a diffuse connective tissue disease that affects many systems throughout the body. It is characterized by the presence of pathogenic autoantibodies and immune complexes that cause a loss of immune tolerance. Most patients exhibit cutaneous involvement. 77 80,81 ; for example, T cells from patients with SLE express reduced levels of hsa_circ_0045272, which has binding sites for hsa-miR-6127, resulting in downregulation of two mRNAs, NM_015177 (DTX4) and NM_003466 (PAX8). 82 In activated Jurkat cells, knockdown of hsa_circ_0045272 significantly enhances IL-2 production and promotes early apoptosis. 82 Studies have also shown that in CD4+ T cells of SLE, reduced expression of hsa_circ_0012919, which can bind to miR-125a-3p, contributes to the methylation of CD70, CD11a and MDA5. 83,84 These findings suggest that circRNAs play a role in the abnormal autoimmune response of SLE.
In addition to T cells, altered levels of circRNA in the peripheral blood or peripheral blood mononuclear cells (PBMCs) have also been reported in patients with SLE. Endogenous circRNAs can form incomplete RNA duplexes (16-26 bp) that can suppress double-stranded RNA-dependent protein kinase (PKR), which is associated with innate immunity. 85 PBMCs from patients with SLE show reduced circRNA expression and abnormal activation of PKR, suggesting a link between circRNAs and the abnormal autoimmune response in SLE. 85 In addition, hsa_circ_0000479 expression is markedly increased in PBMCs from patients with SLE compared with healthy individuals. Bioinformatic analysis indicated that hsa_circ_0000479 regulates the Wnt signalling pathway and metabolic pathways, influencing SLE progression. 86 Moreover, the levels of hsa_circRNA_001308 and hsa_circRNA_407176 are lower in both PBMCs and the plasma of patients with SLE than in healthy controls. 87 In the peripheral blood of patients with SLE, expression levels of hsa_circ_0082688 and hsa_circ_0082689 are markedly increased while those of hsa_circ_0044235 and hsa_ circ_0068367 are decreased in PBMCs. 88,89 Several circRNAs can be used as diagnostic markers for SLE.
Indeed, the combination of anti-dsDNA, hsa_circ_0082688 and hsa_circ_0082689 can distinguish patients with SLE from healthy controls with high sensitivity, specificity and accuracy. 88 Similarly, hsa_circ_0000479 can be used to distinguish SLE from rheumatoid arthritis (RA). 86 Correspondingly, the combination of anti-dsDNA and hsa_circ_0000479-can differentiate SLE from ankylosing spondylitis (AS), RA and healthy controls. 90 circRNA expression is also correlated with the severity of SLE.
For example, circIBTK expression is negatively correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score as well as the levels of complement C3 and anti-dsDNA in patients with SLE. 91 circIBTK can also bind to miR-29b to regulate DNA demethylation and the AKT signalling pathway, suggesting both miR-29 and circIBTK as potential biomarkers and therapeutic targets for SLE. 91 Moreover, the levels of hsa_circ_0075699 and hsa_circ_0021372 in whole blood are correlated with complement C3 and C4 levels in children with SLE, while the level of hsa_ circ_0057762 is positively correlated with the SLEDAI-2000 score. 92 In contrast, the expression of circPTPN22, has_circ_0049220, and hsa_circ_0049224 is inversely correlated with the SLEDAI score. 93,94 An association between hsa_circ_0000479 and hsa_circ_0082688 with the levels of complement C3 and anti-dsDNA, respectively, has also been reported.
In addition to SLE, the lesion of discoid lupus erythematosus significantly and differentially expresses at least 161 circRNAs that are associated with the immune response, inflammatory reaction, and T cell co-stimulation, as evidenced by gene ontology (GO) analysis. 95

| Atopic dermatitis
As another common chronic inflammatory dermatosis, AD is linked to genetic predisposition, immune system disorder and epidermal barrier disruption. 96 Although the exact role of circRNAs in AD is not fully understood, their expression is significantly altered in AD lesions compared with non-lesional and healthy skin. For example,

| Lichen planus
Lichen planus is an inflammatory mucocutaneous condition that affects the oral and genital mucous membranes in addition to the skin and typically develops in middle-aged adults, especially females. 97 Although lichen planus is considered a T cell-mediated autoimmune disease, 99 its pathogenesis remains nebulous. A recent study identified abnormal expression of 135 circRNAs in oral lichen planus (OLP); among them, chr6:31238920-31324013-, whose host gene is HLA-C (a regulator of the human immune system), is significantly highly expressed in OLP. 100 High expression of HLA-C on the cell surface can aggravate cytotoxic T cell responses. 101 Moreover, hsa_circ_0006867, which is derived from LRBA, is downregulated in OLP. 100 LRBA is involved in the secretion and membrane deposition of immune effector molecules. A lack of LRBA can cause defects in Tregs, resulting in immune disorders. 102 These results suggest that some circRNAs play a significant role in the pathogenesis of OLP.

| Severe acne
Acne, characterized by chronic inflammation of the pilosebaceous unit, 103 is estimated to affect 85% of individuals between the ages of 12 and 25, 104 making it the eighth most prevalent disease in the world. 105 Because acne negatively affects both the physical and mental health of patients, its pathogenesis has attracted attention.
Propionibacterium acnes, androgen, growth factors and inflammatory factors have all been proposed to play important roles in acne development. 104 Some circRNAs may also be involved in the onset of severe acne since lesional and non-lesional skin from patients with severe acne differentially expresses at least 538 circRNAs, including 271 upregulated and 267 downregulated circRNAs. 106 GO analyses and KEGG pathway enrichment showed that these circRNAs are related to metabolism, immune responses and inflammation. qPCR analysis also demonstrated significantly decreased expression of five circRNAs in severe acne. It is predicted that these five circRNAs regulate target gene expression by interacting with 213 miRNAs, highlighting circRNAs as potential biomarkers or drug targets of severe acne. 106 The role of circRNAs in inflammatory skin diseases other than psoriasis is shown in Table 1.

| CON CLUS I ON AND FUTURE PER S PEC TIVE S
circRNAs are an emerging research hotspot in the medical field owing to their great potential in the occurrence, development, diagnosis and treatment of several diseases. They also show potential as biomarkers for the diagnosis and assessment of disease severity or as effective therapeutic targets for many inflammatory dermatoses, including psoriasis, lupus erythematosus, AD, lichen planus and severe acne.
Although circRNAs are known to have multiple functions, such as miRNA sponge activity, 30

ACK N OWLED G EM ENTS
This review was supported by the National Nature Science Foundation of China (grant numbers 81472888 and 81602768).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.