Emerging Role of Non-Coding RNAs in Regulation of T-Lymphocyte Function

T-lymphocytes (T cells) play a major role in adaptive immunity and current immune checkpoint inhibitor-based cancer treatments. The regulation of their function is complex, and in addition to cytokines, receptors and transcription factors, several non-coding RNAs (ncRNAs) have been shown to affect differentiation and function of T cells. Among these non-coding RNAs, certain small microRNAs (miRNAs) including miR-15a/16-1, miR-125b-5p, miR-99a-5p, miR-128-3p, let-7 family, miR-210, miR-182-5p, miR-181, miR-155 and miR-10a have been well recognized. Meanwhile, IFNG-AS1, lnc-ITSN1-2, lncRNA-CD160, NEAT1, MEG3, GAS5, NKILA, lnc-EGFR and PVT1 are among long non-coding RNAs (lncRNAs) that efficiently influence the function of T cells. Recent studies have underscored the effects of a number of circular RNAs, namely circ_0001806, hsa_circ_0045272, hsa_circ_0012919, hsa_circ_0005519 and circHIPK3 in the modulation of T-cell apoptosis, differentiation and secretion of cytokines. This review summarizes the latest news and regulatory roles of these ncRNAs on the function of T cells, with widespread implications on the pathophysiology of autoimmune disorders and cancer.

INTRODUCTION T-lymphocytes (T-cells) play a central role in adaptive immunity and are involved in the pathogenesis of immune-related disorders and cancer, thus several therapeutic strategies have been developed to stimulate their effector functions (1). During the process of maturation in the thymus, T cells express T cell receptors (TCR) on their surface. Moreover, they can express either CD8 or CD4 glycoproteins, thus being categorized as glycoprotein on their surface and are called CD8+ (cytotoxic) or CD4+ cells (helper) T cells (2). Based on the distinctive cytokine profiles, T helper (Th) cells can be categorized to Th1, Th2, Th9, Th17, Th22, regulatory T cells (Tregs), and follicular helper T cells (Tfhs) subtypes (3). Each cell type can be recognized by epigenetic and genetic signatures. For instance, Treg cells are described by over-expression of the FOXP3 transcription factor (4) Demethylation of the intronic conserved non-coding sequence 2 is required for maintenance of FOXP3 expression and regulation of stability of Tregs upon re-exposure to cytokines (5). In Tregs, this intronic sequence acts a sensor for IL-2 and STAT5 (5). The expression of a number of transcription factors has been shown to be altered in CD8+ T cells during clearing an Bacterial or Viral infection (6). Notably, it is possible to predict the potential of these cells to make memory cells based on gene signatures (6). For instance, expressions of Bcl-2 and Cdh-1 have been shown to be surged in the memory subset of CD8+ T cells (6). In addition, chromatin configurations have been found to influence the function of T cells (6). Non-coding RNAs (ncRNAs) carry a regulatory function in several biological processes including implications in immune checkpoint inhibitor treatment (7). Recent studies have highlighted the impact of different classes of non-coding RNAs in T cell functions. In this review, we highlight the function of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in regulation of T cells. These three classes of ncRNAs have regulatory effects on expression of mRNA coding genes. In fact, lncRNAs and circRNAs can sequester miRNAs and decrease availability of miRNAs. Since miRNAs can inhibit expression of target mRNAs, the sequestering effects of circRNAs and lncRNAs on miRNAs release miRNA targets from inhibitory effects of miRNAs (8).

miRNAs AND T CELL REGULATION
miRNAs are about 22 nucleotides in length and regulate expression of their target transcripts mostly through binding to their 3' UTR (9,10). These small molecules are generated in the forms of precursors by RNA polymerases II and III. The mature miRNA is generated through a series of cleavage events in the nucleus and cytoplasm (9). Given their various regulatory functions, miRNAs are important players in the regulation of several physiologic and pathophysiologic processes (11). As for the regulation of T-cell differentiation, several examples of important miRNAs have been reported. For instance, miR-15/ 16 hampers T cell cycle, their survival and differentiation to memory T cells. Experiments in miR-15/16 deficient T cells have shown that these miRNAs directly inhibit the expression of a broad network of genes contributing in the regulation of cell cycle progression, survival, and development of memory cells (12). Another study has shown miR-15a/16-1 silencing in CD4+ T cells leads to the production of higher levels of IL-22, while upregulation of miR-15a/16-1 results in down-regulation of the IL-22 expression through suppression of the aryl hydrocarbon receptor. miR-15a/16-1 silenced CD4+ T cells were superior to wild-type CD4+ T cells in terms of tissue repair capacity because of their higher capability in production of IL-22. Furthermore, IL-22 has been shown to decrease miR-15a/16-1 levels through activation of phosphorylated STAT3-c-myc signaling (13).
A high throughput miRNA profiling in human peripheral gd T cells of healthy subjects has led to identification of 14 differentially expressed miRNAs between ab and gd T cells.
Let-7 family miRNAs are also involved in the regulation of T cells functions. In vivo experiments demonstrated that, let-7g-5p may attenuate the frequency of Th17 cells of rheumatoid arthritis (RA) and block Th17 differentiation (18). Let-7f-5p inhibits Th17 differentiation through targeting STAT3. This miRNA has been found to be downregulated in CD4+ T cells of patients with multiple sclerosis (MS) (19). Finally, let-7d-3p regulates the expression of IL-17 in CD4 + T cells by targeting AKT1 and modulation of AKT1/mTOR signaling pathway (20). miR-210 is another miRNA whose deletion enhances T cell differentiation and Th17 polarization under hypoxic situation through modulation of HIF-1a expression (21). Expression of this miRNA has also been found to be over-expressed in both psoriasis patients and psoriasis animal models where it stimulates Th17 and Th1 cell differentiation but suppresses Th2 differentiation via inhibiting expressions of STAT6 and LYN. Ablation of miR-210 in animals and intradermal injection of miR-210 antagonist has reversed the immune imbalance and blocked the development of psoriasis-like inflammatory response in an animal model of psoriasis. TGF-b and IL-23 have been shown to increase the expression of miR-210 through the induction of HIF-1a, and subsequent recruitment of P300 and enhancement of histone H3 acetylation in miR-210 promoter (22). miR-181c has been shown to enhance Th17 differentiation and promote autoimmunity through targeting Smad7 and modulating TGF-b pathway and IL-2 expression (13). Overexpression of miR-181c has suppressed activation of T cell, impaired cytoskeleton arrangement in T cells by targeting BRK1 (23). Meanwhile, miR-181a has been reported to restrict IFN-g production by targeting Id2 so regulating IFN-g-mediated CD8+ T cell responses mediated by (24). This miRNA also promotes expression of TGF-b and IL-10 and inhibits function of Tregs through modulating the PI3K/Akt pathway (25). Figure 1 illustrates the role of various ncRNAs in regulating the differentiation of T cells via the PI3K/Akt/mTOR and MAPK/ERK signaling pathways. Table 1 summarizes the impact of miRNAs on regulation of function of T cells.

LncRNAs AND T CELL REGULATION
LncRNAs are typically longer than 200 nucleotides and may also be several kilobases long (69). They exert diverse effects on chromatin structure, transcription of genes and posttranscriptional regulation of gene expression (70). These effects are exerted through both chromatin-based mechanisms and the interaction with other types of transcripts. Moreover, by serving as decoy, scaffold, and enhancers, lncRNAs influence genes expressions though various mechanisms (71). Several lncRNAs have been found to influence the function of T cells. For instance, IFNG-AS1 is up-regulated in the intestinal tissue of patients with active inflammatory bowel disease (IBD). Specific overexpression of IFNG-AS1 in T cells has led to significant enhancement of inflammatory cytokines, while attenuation of production of anti-inflammatory cytokines. Media from IFNG-AS1-overexpressing T cells has induced a potent inflammatory response in primary human peripheral blood mononuclear cells (PBMCs) (72). Lnc-ITSN1-2 is another lncRNA that affect T cells differentiation. This lncRNA has been shown to increased proliferation and activation of CD4+ T Cells and promote their differentiation to Th1/Th17 through targeting miR-125a and upregulating IL-23R (73).
The regulatory role of NEAT1 on T cells functions has been validated in different contexts, including sepsis, primary Sjögren's syndrome, RA and hepatocellular carcinoma (HCC) (74,75). Downregulation of NEAT1 has restricted immune response in mouse model of sepsis and induced T cell apoptosis through modulating miR-125/MCEMP1 axis (76). This lncRNA has been shown to promote expression of CXCL8 and TNF-a and activate MAPK signaling pathway. NEAT1 expression has been up-regulated in CD4+ and CD8+ T cells of patients with primary Sjögren's syndrome (34). Similarly, this lncRNA has been found to be up-regulated in peripheral blood mononuclear cells of RA patients. Its silencing has led to inhibited differentiation of Th17 cells from CD4+ T cells by downregulating STAT3 through modulating its ubiquitination (77). Finally, NEAT1 has been found to be upregulated in PBMCs of HCC patients parallel with up-regulation of Tim-3. NEAT1 silencing has blocked apoptosis of CD8+T cells and increased their cytolysis function. Further, NEAT1 has been shown to exert such effects through miR-155/Tim-3 pathway. Taken together, NEAT1 has been suggested as an i m p o r ta n t t a r g e t f o r e n h a n c i n g t h e e ffi c i e n c y o f immunotherapy (78).
MALAT1 is another lncRNA with prominent role in the regulation of T cell function. This lncRNA regulates Th1/Th2 ratio by sponging miR-155 and modulating expression of CTLA4 (79). On the other hand, MEG3 has been found to enhance proportion of Th17 cells and regulate Treg/Th17 ratio by sponging miR-17 and upregulating RORgt (80). Moreover, this lncRNA decreases proliferation of CD4+T cell and inhibits Th1 and Th17 differentiation by absorbing miR-23a and modulating expression of TIGIT (81). Figure 2 represents the role of various ncRNAs in regulating the JAK2/STAT3 and NF-kB signaling pathways in the regulation of function of T cells. Table 2 summarizes the impact of lncRNAs on T cell function.

CircRNAs AND T CELL REGULATION
CircRNAs are another group of ncRNAs that can be occasionally translated into proteins. The enclosed structure of circRNAs has endowed them a certain resistance to RNases and thus increases the stability in different body compartments FIGURE 1 | A schematic representation of the role of various non-coding RNAs in modulating the differentiation of T cells via the PI3K/Akt/mTOR and MAPK/ERK signaling cascades. a) The MAPK/ERK pathway can be triggered via several growth factors including PDGF, EGF, NGF, and insulin. Upon receptor dimerization, activation of its tyrosine kinase module could be triggered, subsequently recruiting Grb2 and SOSto the phosphorylated domain, thus creating the Grb2-SOS complex. Furthermore, the GTP binding protein RAS interacts with the Grb-2-SOS complex that in turn leads to the activation of RAS. Activated GTP-bound RAS plays an effective role in upregulating the phosphorylation of MEK1/2 (MAPKK), which then phosphorylates ERK1/2 (MAPK). Eventually, ERK is transferred to the nucleus where it triggers the activation of various target genes involved in a variety of cellular processes (26)(27)(28)(29). b) The PI3K/Akt/mTOR signaling is activated by a subset of growth factors such as PI3KCI, which phosphorylates PIP2 to PIP3. PIP3 has an important role in recruiting AKT which gets activated through double phosphorylation (via PDK1 and mTORC2). In addition, activated AKT suppresses TSC2 through phosphorylation. Inactive TSC1/2 complex is able to bind RHEB, which eventually triggers the activation of mTORC1. The mTORC1 has a significant impact on many downstream proteins, such as S6K1/2 and 4EBP1 (30,31). Besides, exposure to IL-17 results in receptor-mediated activation of Src, MAPKs, and PI3K/Akt signaling cascades (32). Moreover, subsequent to JAK activation, CRKL is phosphorylated by TYK2 that could result in CRKL complexation with STAT5. STATs in turn interacts with individual mediators of the PI3K/AKT signaling cascade (33). Accumulating finding has demonstrated that miR-let-7d-3p via directly suppressing AKT1 could regulate expression level of IL-17 in CD4+ T cells through the AKT1/mTOR signaling pathway (20). In addition, another research has authenticated that overexpression of lncRNA NEAT1 could promote the expression levels of CXCL8 and TNF-a in Sjögren's syndrome via positively regulating MAPK signaling (34). Green arrows indicate upregulation of target genes modulated via ncRNAs (lncRNAs, and miRNAs), red arrows depict inhibition by these ncRNAs. All the information regarding the role of these ncRNAs in modulating T call differentiation can be seen in and.       (108). A genome wide transcriptome profiling of circRNAs has revealed that the median length of circRNAs is about 530 nt (109). Four categories of circRNA shave been identified: exonic circRNAs, circRNAs from introns, exon-intron circRNAs and intergenic circRNAs (110). The impact of this group of transcripts on T cell functions has been discovered during the recent decade. Several studies have shown that circRNAs can bind with miRNAs, thus decreasing bioavailability of miRNAs and releasing miRNA targets from their inhibitory effects. This kind of interactions between circRNAs and miRNAs has critical biological impacts. A high-throughput microarray study found down-regulation of circ_0001806 in patients with cryptococcal meningitis as compared to healthy controls. Circ_0001806 silencing has increased the intensity of fungal infection in the animal models and decreased their survival. Circ_0001806 has been suggested to regulate molecular cascades associated with the host antimicrobial response in T cells. Functionally, circ_0001806 has been shown to increase ADM level, decrease cell apoptosis and reverse G1/S arrest in T cells through sequestering miR-126. Thus, circRNA-1806/miR-126 cascade has an essential role in the regulation of cell cycle and apoptosis in T cells (111). Another high throughput circRNA profiling in patients with systemic lupus erythematosus (SLE) has led to identification of 127 differentially expressed circRNAs in T cells of these patients. Among them, circRNA hsa_circ_0045272 has been reported to be the down-regulated. Functional studies have shown that hsa_circ_0045272 silencing increases early apoptosis of Jurkat cells and enhances production of IL-2 by activated Jurkat cells. Binding of this circRNA with hsa-miR-6127 has been validated through functional studies (112). Hsa_circ_0012919 has been reported to be up-regulated in CD4+ T cells of SLE patients in two independent studies. In a microarray study of circRNAs signature in these patients, hsa_circ_0012919 has been among differentially expressed circRNAs between SLE patients and healthy subjects.
Expression of this circRNA has been associated with SLE features. Down-regulation of hsa_circ_0012919 has enhanced expression of DNMT1, decreased CD70 and CD11a levels and inverted the DNA hypomethylation of these genes in CD4+ T cells of SLE. Hsa_circ_0012919 has been found to regulate expressions of KLF13 and RANTES through sequestering miR-125a (113). This circRNA has also been found to increase the expression of MDA5 in CD4+ T cells through downregulating miR-125a-3p (114). Hsa_circ_0005519 is another circRNA influencing T cell function. This circRNA has been found to be up-regulated in CD4+ T cells of asthmatic patients. Expression of this circRNA has been inversely correlated with hsa-let-7a-5p levels. Expression of hsa_circ_0005519 in CD4+ T cells has been correlated with fraction of exhaled nitric oxide and eosinophil ratio in the circulation of these patients. Hsa_circ_0005519 has been predicted to sequester hsa-let-7a-5p and release IL-13/IL-6 from its inhibitory effect (115). Being up-regulated circRNA in nasal mucosa of patients with allergic rhinitis, circHIPK3 has been found to promote differentiation of CD4+ T cells to Th2 by targeting miR-495 and increasing expression of GATA-3 (116). Figure 3 illustrates the role of different ncRNAs in Th2-cell differentiation through modulating the IL-4-STAT6-GATA3 axis. Table 3 shows the impact of circRNAs on T cell function.

SUMMARY
Numerous miRNAs, lncRNAs and circRNAs have been found to influence activity, survival or differentiation of T cells under physiologic or pathologic conditions. These molecules can affect pathophysiology of autoimmune conditions such as MS, SLE, RA, IBD and asthma via this route. Moreover, several of these non-coding RNAs influence immune evasion of cancer cells and their response to immunotherapeutic modalities. Notably, both lncRNAs and circRNAs can serve as sponges for miRNAs. Through this molecular mechanism, lncRNAs and circRNAs bind with certain miRNAs to decrease their bioavailability. Thus, circRNAs and lncRNAs can indirectly affect expression of miRNAs targets. Circ_0001806/miR-126, hsa_circ_0045272/hsa-miR-6127, hsa_circ_0012919/miR-125, hsa_circ_0005519/hsa-let-7a-5p, circHIPK3/miR-495 are examples of circRNA/miRNA axes regulating T cell functions. In addition, lnc-ITSN1-2/miR-125a, NEAT1/miR-125, NEAT1/ miR-155, MALAT1/miR-155, MEG3/miR-17, MEG3/miR-23a, Gm15575/miR-686 are examples of lncRNA/miRNA pairs in this regard. These examples not only indicate the intricate network between these classes of transcripts, but also provide clues to find the most important modules in the regulation of T cell functions. Contribution of miR-125, miR-155, MEG3 and NEAT1 in more than one of these molecular axes suggests their significance in the regulation of T cell function. Most notably, all of these four non-coding RNAs have essential roles in cancer development or suppression (118)(119)(120), further highlighting the intercalation of cancer-related and immune-related molecular pathways.
Consistent with the important roles of lncRNAs, circRNAs and miRNAs in the regulation of function of T cells and their impact on differentiation of different classes of T cells, therapeutic targeting of these ncRNAs represent an efficient tool for management of disorders related with abnormal function of T cells. Forced up-regulation or silencing of these transcript can affect signaling pathways that modulate T cell responses, thus alleviating tissue damage caused by abnormal T cell responses. Moreover, assessment of ncRNAs signature is a probable strategy for prediction of course of T cell-related disorders.
Taken together, the significant impact of non-coding RNAs on differentiation, survival, cytokine production and activity of T cells potentiates these molecules as important targets for treatment of various disorders, particularly cancer. Moreover, non-coding RNAs participate in the pathogenesis of autoimmune disorders via affecting epigenetic regulation of genes with crucial roles in the regulation of effector T cells as well as Tregs (121). Thus, identification of the role of these transcripts in the regulation of T cell functions can provide new modalities for treatment of this kind of disorders as well. High throughput sequencing method and assessment of the competing endogenous RNA network through bioinformatics methods is an efficient strategy in identification of appropriate targets for therapeutic interventions.

FUTURE PERSPECTIVES
High throughput sequencing strategies and identification of differential expressions of lncRNAs, circRNAs, miRNAs FIGURE 2 | A schematic illustration of the role of various noncoding-RNAs in modulating the JAK2/STAT3 and NF-kB signaling pathway as major regulators of T cell function. a) In JAK/STAT pathway, JAKs bind to the receptor, and upon multimerization, upregulation of JAK proteins is mediated via transphosphorylation. Consequently, JAKs have a significant part in STATs phosphorylation. After dimerization of STATs, they translocate to the nucleus, where they either activate or suppress several target genes. This cascade is remarkably involved in the control of immune responses. Dysregulation of JAK-STAT signaling is associated with different immune disorders (82,83). Besides, REG3A, acts as a key molecule for overexpression of the JAK2/STAT3 pathway which effectively contributes to triggering inflammation (84). b) The NF-kB canonical or classical signaling pathway is initiated from the cell surface receptor of pro-inflammatory cytokines and PAMPs containing TNFR, TLR and T/B cell receptor. The activation of IKK complex is triggered via binding of ligand molecules to transfer the signal across the cell surface. This complex generally comprises heterodimer of IKKa and IKKb catalytic subunits and an IKKg regulatory subunit. The released NF-kB dimers (most generally the p50-P65 dimer) could be translocated to the nucleus, and bind to DNA to trigger activation of the down-stream gene transcription (85)(86)(87). In addition, NF-kB signaling cascade could be regulated via TNFAIP3 through deubiquitinating TNFR1-RIP1, IL-1R/TLR4-TRAF6, and NOD2-RIP2 pathways (88). Moreover, canonical NF-kB cascade could be activated by various members of the TNFRSF including GITR, TNFR2, 4-1BB, and DR3 but not OX40 in Treg cells and modulates induction of Foxp3, markers of Th2/Th17 response (89). Mounting studies have revealed that multiple ncRNAs (lncRNAs and miRNAs) have an effective role in as major regulators of T cell function through regulating the JAK/STAT and NF-kB cascades. As an illustration, recent research has detected that downregulation of miR-128-3p could notably reduce the inflammation response of rheumatoid arthritis via attenuating the activity of NF-kB pathway and promote expression of TNFAIP3 (35). Another study has figured out that reducing the expression of lncRNA NEAT1 could lead to suppression of Th17/CD4+ T cell differentiation via downregulating STAT3 expression in rheumatoid arthritis patients (77). Furthermore, upregulation of DQ786243 could play a remarkable role in elevating the expression level of miR-146a through modulating Foxp3, and thereby suppressing the NF-kB signaling cascade in oral lichen planus disease (90). Green arrows indicate upregulation of target genes modulated via ncRNAs (lncRNAs, and miRNAs), red arrows depict inhibition by these ncRNAs. All the information regarding the role of these ncRNAs in modulating T call differentiation can be seen in Tables 1, 2.     (117). Growing evidence has confirmed that the interactions between CircHIPK3, LncGAS5, and miR-495 could play a crucial role in the modulation of Th2 differentiation in allergic rhinitis (116). Green arrows indicate upregulation of target genes by ncRNAs (lncRNA, and circRNA), red arrows depict inhibitory effects of by these ncRNAs.