Behçet’s disease (BD) is a rare, chronic, multisystemic ailment characterized by inflammation and autoimmunity. It can cause ocular lesions, recurring oral and vaginal ulcers, skin rashes and lesions, and arthritis, and may also involve the nervous system, intestines, and blood vessels (1). Epidemiologists have found that the incidence of BD varies widely around the globe, from 0.1 per 100,000 people in Hawaii to 664 per 100,000 people in northern Jordan (2). BD is associated with several environmental factors, for instance infections and the microbiome. Viral infection has long been hypothesized to be one of the main etiological factors of BD. Infectious agents such as hemorrhagic streptococcus or differences in the composition of the saliva or intestinal microbiome trigger congenital inflammation, which is subsequently maintained by an adaptive immune response (3–5). BD has recently been classified as a form of MHC-I-opathy, and the latest research shows that the most potent genetic susceptibility factor is HLA-B51. The variable endoplasmic reticulum aminopeptidase 1 (ERAP1) haplotype Hap10 participates in the pathogenesis of BD by generating restricted HLA-B51 peptides (6–8). An epistatic interaction between HLA-B51 and ERAP1 may affect the tuning of microbial and/or endogenous peptides, disrupting the homeostasis of regulatory T cells (Tregs) and the amplification of type 1 T helper (Th1) and Th17 effector cells. Genetic differences in the expression of cytokine genes may affect their susceptibility and impact on their functionality (9). To prevent severe and potentially life-threatening consequences such as neurologic complications, the occlusion of large blood vessels, and damage to the eyes, it is crucial to detect BD in its early stages (10). Unfortunately, no specific diagnostic laboratory test exists and diagnosis relies on clinical criteria (11). Rapid diagnosis, disease activity surveillance, and therapy for BD can be greatly improved through the discovery of new and therapeutic targets and sensitive biomarkers.

Non-coding RNAs (ncRNAs) derive from larger regions of the genome and can carry out biological functions at the RNA level but lack the ability to code for proteins (12). Through base complementary pairing, ncRNAs bind to target genes directly or indirectly and control the transcriptional translation of those genes (13, 14). Small endogenous RNAs called microRNAs (miRNAs), a subcategory of ncRNAs, control gene expression (15). More than 60% of genes that encode protein may be controlled by miRNAs (16, Figure 1 ). MiRNAs play crucial roles in immune reaction and inflammation, among other physiological and pathological processes (17–20). MiRNAs can regulate autoimmune disease BD by regulating immune cells. Type 1 T helper (Th1) cell/Th17 cell amplification and regulatory T cells (Tregs) damage has been found to be one of the main pathogenetic mechanisms of BD (21, 22); miRNAs can also participate in the pathogenesis of BD by regulating Th1 and Th17 cells, and Tregs. For example, the Tregs/Th17 cell imbalance could be caused by highly expressed miRNA-19b-3p, probably by inhibiting the expression of cluster of differentiation 46 (CD46) (23). Elevated levels of inflammatory cytokines such as interleukin-1 (IL-1) and IL-17 in BD have been found to be associated with the aberrant expression of miRNAs, such as miRNA-155, which regulates the Th17 immune response by targeting Ets-1 in BD, and miR-155 and IL-17 expression are significantly increased in CD4⁺ T cells from patients with active BD. Functional variants of miR-196a2 confer the risk of BD by regulating the expression of the miR-196a gene and regulating the production of pro-inflammatory IL-1β (24, 25). The upregulation of miR-3591-3p and downregulation of miR-638, miR-155, and miR-4488 have been linked to the etiology of BD (26–28). The role of miRNAs in BD is being investigated, and the underlying mechanism between miRNAs and BD must be clarified. Furthermore, some miRNAs may serve as potential biomarkers of illness and/or molecular tools to develop new approaches for the treatment of BD.

Research on miRNAs is critical to understanding BD. As a result, in this review we explore the function, mechanisms, and potential future applications in therapeutic research of miRNAs in BD.

MiR-155 is one of the first miRNAs identified in humans, mice, and chickens. It is located on chromosome 21and its sequence is conserved (36). MiR-155 was originally defined as a B-cell integration cluster (BIC) gene that regulates adaptive and innate immunity and is encoded by the host gene MIRHG155 (37). MiR-155 promotes dendritic cell maturation, migration to lymph nodes, T-cell activation, and cytokine release (38). Several reports have indicated that miR-155 expression is increased within a range of activated immune cells, highlighting the significant role of miR-155 in the immune response (39–41). MiR-155 is involved in the generation of cytokines by dendritic cells (DCs) induced by defective autophagy (42–44). MiR-155 may contribute to the onset of BD by regulating autophagy (45). According to one study (46), miR-155 expression is reduced in patients with active BD. As a direct target of miR-155, the transforming advancement factor-activated kinase 1 binding protein 2 (TAB2) is upregulated in the DCs of individuals with active BD and can be inhibited by miR-155 expression. TAB2 is involved in the miR-155-mediated effects on autophagy. TAB2 can interact with beclin-1 or ATG13 to control autophagy, and, in turn, affects the generation of TNF-α, IL-1, and IL-6 by DCs, which participate in the progression of BD. One study showed that P62/SQSTM1, which acts as an autophagic substrate, is decomposed during autophagy (47). The study revealed that, despite the existence of many autophagosomes in BD cells, autophagy degradation was inhibited because the levels of protein expression of P62 in the DCs of patients with active BD were much higher than those of patients without active BD and those of healthy subjects (HSs). In summary, certain triggers in genetically susceptible individuals stimulate autophagosome formation. Therefore, the defective control of the succedent degradation of autophagy products result in local stimulation of pro-inflammatory cytokine release. MiR-155 may serve as a potential prognostic and diagnostic biomarker in BD (47). Numerous autoimmune illnesses, including rheumatoid arthritis (RA), overexpress miR-155 and are associated with a release of proinflammatory cytokines. MiR-155 expression levels were found to be significantly increased in patients with AS and in an in vivo model of psoriasis. Studies have shown that its upregulation leads to the increased expression of IL-21 and STAT3 as characteristic of Th-17 lymphocytes, leading to worsening inflammatory conditions in patients with AS. Through the PTEN signaling pathway, highly expressed miR-155 encourages the proliferation of psoriatic cells and inhibits apoptosis, and could be a potential therapeutic option for the treatment of psoriasis progression (48–50). Thus, the investigation of miR-155 is vital for comprehending the pathogenesis of BD. Manipulating miR-155 levels may also provide a novel approach to treating BD.

MiR-181b belongs to the miR-181 family, of which the other members are miR-181a, miR-181c, and miR-181d (51). Based on its ability to limit the activity of activation-induced cytidine deaminases, miR-181b was identified as a regulator of the initial antibody repertoire of B cells (52). Numerous tumors and non-tumor pathologies (cardiovascular pathologies, metabolic disorders, and neurologic or infectious diseases) have been associated with levels of miR-181b (53–56). The miR-181 family, as we will describe, controls several important biological processes, for instance cell apoptosis, division, autophagy, mitochondrial properties, and immunological response (57–59). MiR-181b, the most abundant member of the miR-181 family expressed in endothelial cells (EC), has been shown to reduce EC activation by specifically targeting protein 3 in vivo and in vitro. The main site of immune-mediated damage is the endothelium (51). A key characteristic of BD is the EC damage caused by it. Growing evidence has shown that TNF-α plays an increasingly important role in the immune pathogenesis of BD, and the serum level of TNF-α is higher in patients with BD (60). The overexpression of miR-181 in BD may be a marker of endothelial injury in this disease. In one investigation, the results showed that blood levels of TNF-α, high-sensitivity C-reactive protein (hs-CRP), e-selectin, IL-6, and vascular cell adhesion molecule 1 (VCAM-1) were significantly increased in patients with BD, together with higher levels of serum miR-181b expression compared with controls (61). Importin-3 and a set of enriched NF-κB sensitive genes, including E-selectin and adhesion molecules VCAM-1, were suppressed by the overexpression of miR-181b in ECs both in vivo and in vitro (51). Conversely, Iliopoulos et al. (62) discovered that miR-181b specifically targets CYLD in ER-Src cells, increasing NF-κB activity and maintaining the inflammatory response. Simultaneously, Wang et al. (63) discovered that overexpression of miR-181b causes upregulated p65, a component of the NF-κB signaling pathway. This demonstrated that miR-181b targets the NF-κB signaling pathway and functions as a pro-inflammatory agent (64, 65). This miRNA-targeted therapy may be a way to modulate the inflammatory response in BD (61). However, the specific function of miR-181b in the pathological process of BD and its potential use as a diagnostic and predicted marker of the disease remains unknown, and therefore further study is needed.

MiRNA-23b belongs to the miR-23b/27b/24-1 cluster that is encoded by the chromosomal region 9q22.32, which also generates miR-23b, a pleiotropic regulator of numerous developmental processes and clinical conditions (66, 67). Pleiotropic miR-23b plays a significant role in the control of cellular immunological responses, cell development, and cell physiological functioning (68–70). miR-23b is closely related to the appearance and progression of many diseases, including cancer and autoimmune diseases (71, 72). According to numerous studies, miR-23b modulates the pathogenesis of autoimmune diseases by targeting a variety of protein molecules, including chemokine ligand 7 (CCL-7), inhibitory-κB kinase alpha (IKK-α), the transforming advancement factor-activated kinase 1 binding protein 2 (TAB2) and the transforming advancement factor-activated kinase 1 binding protein 3 (TAB3) (73). One miRNA that is linked to the differentiation of Tregs is miR-23b. Lower levels of Tregs in BD are consistent with the lower expression of miR-23b (74). According to a recent study, miR-23b expression may be downregulated in patients with BD, which may contribute to the activation of the Notch system and of Th1/Th17 cells, and CD4 T cells transfected with the miR-23b inhibitor markedly promoted both IL-17-expressing and IFN-γ-expressing T cells. The abnormal increase in immune microenvironment stimulation and the loss of immunomodulatory effects due to the decline of miR-23b can jointly lead to the overactivation of the Notch pathway and the expansion of inflammatory cells, which may be related to the development of BD (75). Furthermore, miR-23b is considered a biomarker of RA and is negatively correlated with inflammation in RA (76). Studies have shown that inflammatory cytokines such as IL-17 promote the development inflammatory autoimmune diseases, such as systemic lupus erythematosus (SLE), whereas the suppression of miR-23b decreases the expression of the inflammatory factor IL-17 (77). Similar to RA, the inhibition of miR-23b interferes with the onset of SLE by blocking IKK-α, TAB2, and TAB3 expression. It has been predicted that the increased expression of miR-23b will aid in the treatment of SLE (73). Thus, manipulating miR-23b levels may also provide a novel approach to treating BD.

MiRNA-21 is located within the vacuolar membrane protein 1 (VMP1) gene on chromosome 17 (17q23.2) and is recognized as one of the most abundant and highly conserved miRNAs (78). MiR-21 expression increases in many mammalian cells and regulates various biological functions such as cell differentiation, proliferation, migration, and apoptosis (79). It also regulates many signaling pathways, and can contribute to angiogenesis, endothelial cell migration, and differentiation (80, 81). The upregulation or downregulation of miR-21 has been shown to be involved in the pathogenesis of immune diseases. Higher levels of CD4 T cells producing IL-17 were detected in BD patients (82). The differentiation of CD4 T cells in response to Th17, Th2, or Th1 may be influenced by the expressions of miR-146b, miR-21, miR-326, and their potential target transcripts (83). Serum IL-17 levels were also found to be clearly upregulated in patients with BD (84). The expression of the cytokine IL-17 has been shown to be lower in mice treated with the miR-21 antibody. RhoB and programmed cell death 4 (PDCD4) are the main functional targets of MiR-21. The expression of PDCD4 expression increases during apoptosis (85). MiR-21 exerts an antiangiogenic effect by targeting RhoB expression in endothelial cells (86). In BD mice models caused by herpes simplex virus (HSV), it was found that RhoB mRNA expression was consistently upregulated after the suppression of miR-21. According to Lu et al. (87), RhoB, PD-1, and PDCD were negatively interrelated with miR-21 expression, whereas toll-like receptor 4 (TLR4) expression was positively correlated with that of miR-21. TLR plays a crucial role in the context of the innate immune response. The inflammatory progression of BD can be explained by the accumulation of overproduced inflammatory cells and the delay of inflammatory cell apoptosis. The level of miR-21 expression has been linked to symptoms similar to those of BD-like mouse models caused by HSV. MiR-21 suppression improved BD-like inflammation symptoms by controlling cytokine (IL-6 and IL-17) expression and TLR4 levels. MiR-21 antagomir can be used as an immunomodulator to control BD-like mouse models caused by HSV (88). In addition, miR-21 is dysregulated in other immune disorders. For example, it is overexpressed in AS and psoriasis. In psoriasis, the inhibition of miR-21 by locked nucleic acid (LNA)-modified anti-miR-21 drugs reduced disease pathology in mouse xenotransplant models of patient-derived psoriatic skin. A prospective therapeutic strategy for the management of psoriasis involves targeting miR-21 (89–91). These studies will offer a potential treatment approach for individuals with BD. As immune modulators, miR-21 antagomir may be helpful for treating BD, brought on by HSV infection. The expression level of miR-21 is higher in BD patients with severe eye involvement, ROC curve analysis shows that miR-21 has a high sensitivity to the diagnosis of BD, and miR-21 can be used as a potential biomarker for the diagnosis of BD patients (83).

Hsa-microRNA-196a2 (miR-196a2), originally identified by Lagos-Quintana et al. (92), features a nucleotide change from C to T and is situated in the 3′-untranslated location of its precursor miR-196a2 (93). As a defined miRNA polymorphism, miR-196a2 is significantly associated with the risk of cancer (94, 95). Single-nucleotide polymorphisms (SNPs) are one of the most significant genetic variations in the human genome. SNPs are DNA changes that are distributed throughout human chromosomes. They are genetic variants based on single-nucleotide mutations (96). Genetic SNPs in miRNA genes may have a significant impact on how they are processed, expressed, or matured, and may lead to sensitivity to or the development of a number of diseases (97, 98). Hoffman et al. (99) demonstrated that miR-196a2 rs11614913 exerts biological effects on target gene production and also affects the transcript level of mature miR-196a. The miRNA-196a2 polymorphism is also associated with BD. Qi et al. (24) reported a higher risk of BD is conferred by the rs11614913 TT genotype in uveitis. MiR-196a2 may be a common inducer of distinct phenotypes of inflammatory bowel disease (IBD) and BD. It has been reported that a functional variant of miR-196a2 increases the risk of BD in uveitis by controlling the expression of the miR-196a gene. The current findings demonstrate that miR-196a2 expression is reduced in patients with active intestinal BD. Downregulated miR-196a2 may be participated in intestinal BD pathogenesis by targeting Bach1/HO-1. Lower expression of miR-196a2 downregulates HO-1 gene expression and enhances the expression of the pro-inflammatory cytokines IL-17, IFN-γ, TNF-α, and IL-1β in monocytes, which may be related to the pathogenesis of intestinal BD (100). There is also research that shows a functional variant of miR-196a2 confers risk for BD, but not for Vogt–Koyanagi–Harada (VKH) syndrome or acute anterior uveitis (AAU), by modulating the expression of the miR-196a gene and regulating the production of pro-inflammatory IL-1β and monocyte chemoattractant protein-1 (MCP-1) (24).

Other miRNAs also play a significant role in BD. Research shows that patients with BD have impaired regulation of the inflammatory state, which could be caused by abnormal T-cell homeostasis. The upregulation of the Th17 and Th1 pathways and the reduction in the suppressive regulation of Treg cells may play vital roles in this scenario. A study by De Santis et al. found that the increased expression of miR-106b-25 may affect Tregs by altering the TGF-β signaling pathway (101). The differentiation of CD4 T cells into Th17, Th2, or Th1 responses may be influenced by the expression of miR-146b and its potential target genes (102). MiR-326 plays a key role in regulating Th17 differentiation and thus contributes to the pathogenesis of BD. The association between the lack of vitamin D and the pathogenesis of BD is generally known. A higher expression level of the miR-326 gene inhibits the expression of VDR. MiR-326 mediates Th17 differentiation by inhibiting Ets-1 translation and is a negative regulator of Th17 differentiation. Thus, miR-326 can be considered a therapeutic target (103). MiR-326 shows high sensitivity and specificity in the prediction of ocular involvement and uveitis in patients with BD. Measuring the expression rate of miR-326 can be used as a biomarker to predict severe ocular involvement and uveitis (83).

The Notch signaling pathway is a highly conserved pathway in mammals and is essential for immune cell homeostasis and differentiation (104). It is also intimately connected to the transmission of immunological signals. The Notch signaling pathway has no significant pro-inflammatory or anti-inflammatory effects, although its effects are greatly determined by the type of immune cell and the cellular environment (105). A previous study reported that the enhanced activation of the Notch signaling pathway may be strongly related to the etiology and pathogenesis of BD. Hes and Hey proteins are transcriptional repressors that negatively control downstream target gene expression (106). Hes1 is the most representative target gene in the Notch signaling pathway, and Hes1 mRNA expression is upregulated in the peripheral blood mononuclear cells (PBMCs) of patients with BD, indicating increased activation of the Notch signaling pathway, which may result in increased STAT3 phosphorylation and, in turn, activation of the Th17 response. This pathway may play a role in BD lesions. Several miRNAs have been shown to regulate the Notch signaling pathway. The activation of the Notch signaling pathway in BD may be related to the reduced expression of miR-23b (75). In patients with BD, the reduced expression of miR-23b may help to activate and amplify the function of Th17 and Th1 cells and the Notch signaling pathway. The Th17 response can be preferentially suppressed by blocking the Notch signaling pathway. Studies have shown that members of the NF-κB family control miR-23b transcription, and that this control is based on the Act-1/IL-17 signaling pathway, which implies that there is a vicious cycle among miR-23b, the Notch signaling pathway, and IL-17 (73).

The AKT/mammalian target of rapamycin (mTOR) signaling pathway regulates many cellular processes, including cellular proliferation, metabolism, and viability. The PI3K/AKT/mTOR signaling pathway is generally present in all tissues and cells (107). It plays a significant role in proliferation, cell autophagy, apoptosis cell cycle, and differentiation. One of the most well-known signaling mechanisms controlling autophagy is the Akt/mTOR signaling pathway (106, 108). In DCs, miR-155 can downregulate the phosphorylation of Akt and mTOR. There are reports indicating that, following the transfection of miR-155 mimics, the p-Akt/Akt and p-mTOR/mTOR ratios in the DCs treated with LPS increased markedly (45). MiR-155 may induce the Akt/mTOR signaling pathway to initiate autophagy and may also participate in the development of BD (27, 109). Patients with active BD have activated autophagy in their DC, and TAB2 mRNA can not only translate proteins to keep TLR4 signaling activated [the interaction of which with the gut microbiota may be involved in the development of BD (110)] but also compete with miR-155 to enhance the expression of the LPS-stimulated SOCS1 protein to prevent extensive immune responses (111). The regulation of autophagy by the AKT/mTOR pathway may be related to the pathogenesis of BD.

The NF-κB signaling pathway is a key regulator of cell survival, immunity, inflammation, carcinogenesis, and organogenesis (112). Once activated, the NF-κB signaling pathway regulates a number of transcriptional outcomes, driving the production of proteins essential for cell survival, cell cycle progression, immunological control, and NF-κB regulators, establishing a negative feedback loop (113). The NF-κB signaling pathway is crucial to the pathogenesis of BD. Numerous autoimmune disorders, such as SLE, RA, and IBD, have been linked to an imbalanced macrophage polarization (114). Studies have shown that BD serum promotes macrophage polarization toward the pro-inflammatory phenotype of M1 macrophage through NF-κB signaling. Thus, enhanced phagocytosis and M1 macrophage polarization may play a role in the inflammation induced by BD through their promotion of Th1 and Th17 differentiation (115). In a recent study, the phosphorylation of p65 and degradation of IκBα, and also the upregulation of TNF-α in macrophages treated with BD serum, demonstrate the activation of the NF-κB signaling pathway, leading to its amplification. Inducing NF-κB activation in BD can result in a higher profile of inflammatory cytokines, which in turn creates an environment polarized by M1 (38). Other studies have shown that NF-κB regulates genes involved in inflammatory responses and genes associated with inhibiting apoptosis, leading to resistance to apoptosis in the T-cell subpopulations of BD and participation in the pathogenesis of BD. The apoptotic refractoriness of activated T cells in BD is what leads to a prolonged inflammatory response. Key antiapoptotic proteins, the short isoform of Bcl-xL and cellular FLIP (cFLIP), were significantly upregulated in activated T cells in BD. The apoptosis-refractory properties of activated T cells in BD are what trigger the inflammatory response (116). Overall, NF-κB is crucial to the pathogenesis of BD, and inhibiting the NF-κB signaling pathway or using miRNA-targeted therapy may be effective ways to reduce the inflammatory response in BD (65).