CircPTPN22 modulates T-cell activation by sponging miR-4689 to regulate S1PR1 expression in patients with systemic lupus erythematosus.

BACKGROUND: Circular RNAs are involved in autoimmune disease pathogenesis. Our previous study indicated that circPTPN22 is involved in autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, but the underlying mechanisms remain unclear. METHODS: First, the expression of circPTPN22 was detected by real-time PCR and western blotting. After overexpression or knockdown of circPTPN22, the proliferation of Jurkat cells was detected by the CCK-8 assay, and the apoptosis of Jurkat cells was detected by flow cytometry. In addition, the relationship between circPTPN22-miR-4689-S1PR1 was confirmed by bioinformatic analyses, fluorescence in situ hybridization assays, RNA-binding protein immunoprecipitation, and dual luciferase reporter assays. RESULTS: We found that circPTPN22 expression was downregulated in the PBMCs of SLE patients compared to those of healthy controls. Overexpression of circPTPN22 increased proliferation and inhibited apoptosis of Jurkat T cells, whereas knockdown of circPTPN22 exerted the opposite effects. CircPTPN22 acts as a miR-4689 sponge, and S1PR1 is a direct target of miR-4689. Importantly, the circPTPN22/miR-4689/S1PR1 axis inhibited the secretion of TNF-α and IL-6 in Jurkat T cells. CONCLUSIONS: CircPTPN22 acts as a miR-4689 sponge to regulate T-cell activation by targeting S1PR1, providing a novel mechanism for the pathogenesis of SLE.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

RNA circles with minimized immunogenicity as potent PKR inhibitors.

Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.

A Previously Undiscovered Circular RNA, circTNFAIP3, and Its Role in Coronavirus Replication.

Circular RNAs (circRNAs) are a newly discovered class of noncoding RNAs (ncRNAs) present in various tissues and cells. However, the functions of most circRNAs have not been verified experimentally. Here, using deltacoronavirus as a model, differentially expressed circRNAs in cells with or without deltacoronavirus infection were analyzed by RNA sequencing to characterize the cellular responses to RNA virus infection. More than 57,000 circRNA candidates were detected, and seven significantly dysregulated circRNAs were quantitated by real-time reverse transcription-PCR. We discovered a previously unidentified circRNA derived from the TNFAIP3 gene, named circTNFAIP3, which is distributed and expressed widely in various tissues. RNA viruses, including deltacoronaviruses, rather than DNA viruses tend to activate the expression of endogenous circTNFAIP3. Overexpression of circTNFAIP3 promoted deltacoronavirus replication by reducing the apoptosis, while silencing of circTNFAIP3 inhibited deltacoronavirus replication by enhancing the apoptosis. In summary, our work provides useful circRNA-related information to facilitate investigation of the underlying mechanism of deltacoronavirus infection and identifies a novel circTNFAIP3 that promotes deltacoronavirus replication via regulating apoptosis. IMPORTANCE CircRNAs, a new class of ncRNAs, play important roles in cell growth, neural development, carcinogenesis, and anticarcinogenesis. Porcine deltacoronavirus is an emerging enteropathogenic coronavirus that causes diarrhea, but the role of host circRNAs in regulating its infection is unknown. Here, we performed expression profiling of circRNAs in mock- and deltacoronavirus- infected cells and identified the novel differentially expressed circular RNA circTNFAIP3. We demonstrate that circTNFAIP3 promotes deltacoronavirus replication by inhibiting apoptosis. Our findings first illustrate that circRNA can act as an apoptosis negative regulator during RNA virus infection and help to explore the underlying mechanism of deltacoronavirus infection.

Circular RNA circSamd4a Regulates Antiviral Immunity in Teleost Fish by Upregulating STING through Sponging miR-29a-3p.

Circular RNAs (circRNAs) are a subgroup of endogenous noncoding RNA that is covalently closed rings and widely expressed. In recent years, there is accumulating evidence indicating that circRNAs are a class of important regulators, which play an important role in various biological processes. However, the biological functions and regulation mechanism of circRNAs in lower vertebrates are little known. In this study, we discovered a circRNA Samd4a (circSamd4a) that is related to the antiviral immune response of teleost fish. It can act as a key regulator of the host's antiviral response and play a key role in inhibiting Sininiperca chuatsi rhabdovirus replication. Further studies have shown that circSamd4a may act as a competing endogenous RNA, which can enhance the STING-mediated NF-κB/IRF3 signaling pathway by adsorbing miR-29a-3p, thereby enhancing the antiviral immune response. Therefore, circSamd4a plays an active regulatory role in the antiviral immune response of bony fish. Our research results provide a strong foundation for circular RNA to play a regulatory role in the antiviral immune response of teleost fish.

The circular RNA circBCL2L1 regulates innate immune responses via microRNA-mediated downregulation of TRAF6 in teleost fish.

Growing numbers of studies have shown that circular RNAs (circRNAs) can function as regulatory factors to regulate the innate immune response, cell proliferation, cell migration, and other important processes in mammals. However, the function and regulatory mechanism of circRNAs in lower vertebrates are still unclear. Here, we discovered a novel circRNA derived from the gene encoding Bcl-2-like protein 1 (BCL2L1) gene, named circBCL2L1, which was related to the innate immune responses in teleost fish. Results indicated that circBCL2L1 played essential roles in host antiviral immunity and antibacterial immunity. Our study also identified a microRNA, miR-30c-3-3p, which could inhibit the innate immune response by targeting inflammatory mediator TRAF6. And TRAF6 is a key signal transduction factor in innate immune response mediated by TLRs. Moreover, we also found that the antiviral and antibacterial effects inhibited by miR-30c-3-3p could be reversed with the expression of circBCL2L1. Our data revealed that circBCL2L1 functioned as a competing endogenous RNA (ceRNA) of TRAF6 by competing for binding with miR-30c-3-3p, leading to activation of the NF-κB/IRF3 inflammatory pathway and then enhancing the innate immune responses. Our results suggest that circRNAs can play an important role in the innate immune response of teleost fish.

Circular RNA hsa_circ_0001598 promotes programmed death-ligand-1-mediated immune escape and trastuzumab resistance via sponging miR-1184 in breast cancer cells.

Approximately 25% of breast cancer (BC) patients are HER2-positive. Trastuzumab is used as a targeted therapy drug to treat HER2-positive BC patients; however, the drug resistance remains a big challenge. Circular RNAs (circRNAs) are reported to be involved in drug resistance, but the role of circ_0001598 has never been studied in BC. First, we identified the expression of circ_0001598 by RT-qPCR in BC. The gain-of-function and loss-of-function studies were applied to study the functional roles of circ_0001598 and its target gene. We observed upregulation of circ_0001598 in BC tissues, especially in trastuzumab-resistant BC samples. We further identified that miR-1184 is a functional target of circ_0001598. Moreover, it was found that programmed death-ligand 1 (PD-L1) was a direct target of miR-1184. The oncogenic effects of circ_0001598 in promoting BC cell growth, trastuzumab-resistance, PD-L1 expression, and escaping of CD8 T cell killing were abolished after the restoration of miR-1184. In conclusion, we demonstrate that circ_0001598/miR-1184/PD-L1 signaling plays a crucial role in the regulation of BC progression and trastuzumab-resistance phonotypes, which suggests that circ_0001598 may be a molecular target to treat HER2-positive BC patients.

Multiple Sclerosis: circRNA Profile Defined Reveals Links to B-Cell Function.

BACKGROUND AND OBJECTIVES: To investigate the total circular RNA (circRNA) profile in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls (HCs). METHODS: Hybridization microarray was used to define the circRNA profile in peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with RRMS (10 in relapse and 10 in remission) and 10 HCs. We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-PCR with an independent cohort of 47 patients with RRMS (19 in relapse and 28 in remission) and 27 HCs. RESULTS: Microarray analysis revealed 914 transcripts to be differentially expressed between patients with RRMS in relapse and HCs (p < 0.05). We validated 3 circRNAs from 5 showing highest levels of differential expression in the RRMS relapse vs HC group: hsa_circRNA_101348, hsa_circRNA_102611, and hsa_circRNA_104361. Their expression was significantly increased during relapse in RRMS (p = 0.0002, FC = 2.9; p = 0.01, FC = 1.6; and p = 0.001, FC = 1.5, respectively) and in patients showing gadolinium enhancement on brain MRI (hsa_circRNA_101348, p = 0.0039, FC = 2.4; hsa_circRNA_104361, p = 0.029, FC = 1.7). Bioinformatic analysis revealed 15 microRNAs interacting with these circRNAs in a complementary manner and led to the discovery and validation of 3 protein-coding RNAs upregulated in patients with RRMS during relapse. Two of these, AK2 and IKZF3, have previously been implicated in B-cell function. DISCUSSION: circRNAs display a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNA in the known disturbed B-cell activity in RRMS and thus represent a novel biomarker for monitoring relapse activity.

Up-regulation of circPVT1 in T cell acute lymphoblastic leukemia promoted cell proliferation via miR-30e/DLL4 induced activating NOTCH signaling.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer with dismal prognosis. Recent studies disclosed that circPVT1 played an oncogene role in various cancers. But its role in T-ALL is still unclear. In this study, we found the expression levels of circPVT1 in bone marrows and cell lines of T-ALL were significantly up regulated and knock-down of circPVT1 in T-ALL cell lines could inhibit the cell proliferation and increase the cell apoptosis. Further analysis showed that circPVT1 could bind directly to miR-30e and contributed to the activate the Notch signaling by regulating miR-30e/DLL4 pathway. The levels of circPVT1 were obviously related to cumulative relapse rate and 5-year survival rate. In conclusion, our study reveals that circPVT1 participates in the progression of T-ALL through the miR-30e/DLL4 pathway and might represent a potential therapeutic target for T-ALL treatment.

circFAT1 Promotes Cancer Stemness and Immune Evasion by Promoting STAT3 Activation.

Cancer stemness and immune evasion are closely associated, and play critical roles in tumor development and resistance to immunotherapy. However, little is known about the underlying molecular mechanisms that coordinate this association. Here, it is reported that elevated circular RNA FAT1 (circFAT1) in squamous cell carcinoma (SCC) unifies and regulates the positive association between cancer stemness and immune evasion by promoting STAT3 activation. circFAT1 knockdown (KD) reduces tumorsphere formation of SCC cells in vitro and tumor growth in vivo. Bioinformatic analysis reveals that circFAT1 KD impairs the cancer stemness signature and activates tumor cell-intrinsic immunity. Mechanistically, circFAT1 binding to STAT3 in the cytoplasm prevents STAT3 dephosphorylation by SHP1 and promotes STAT3 activation, resulting in inhibition of STAT1-mediated transcription. Moreover, circFAT1 KD significantly enhances PD1 blockade immunotherapy by promoting CD8+ cell infiltration into tumor microenvironment. Taken together, the results demonstrate that circFAT1 is an important regulator of cancer stemness and antitumor immunity.

Identification and characterization of circRNAs in the liver of blunt snout bream (Megalobrama amblycephala) infected with Aeromonas hydrophila.

Circular RNAs (circRNAs), a class of non-coding RNAs, play an important role in regulating various biological processes. In the present study, circRNAs from the Megalobrama amblycephala liver were identified at five different time points post Aeromonas hydrophila using RNA-seq technology. A total of 250 circRNAs were identified, of which 106 were differentially expressed (DE) in ten pairwise comparisons. GO and KEGG analyses showed that the parental genes of DE circRNAs were enriched in phagocytosis, complement and coagulation cascades, and Fc gamma R-mediated phagocytosis pathways. According to ceRNA hypothesis, the interaction network of circRNAs, miRNAs and mRNAs was constructed. Moreover, WGCNA was conducted, and five specific modules significantly related to bacterial infection were identified. All the above results reveal the important role of circRNAs in immune response, which enriches the information of circRNAs in teleost, and helps to understand the immune response mechanism of M. amblycephala to A. hydrophila.

Circular RNA circDtx1 regulates IRF3-mediated antiviral immune responses through suppression of miR-15a-5p-dependent TRIF downregulation in teleost fish.

Circular RNAs (circRNAs) represent a class of widespread and diverse covalently closed circular endogenous RNAs that exert crucial functions in regulating gene expression in mammals. However, the function and regulation mechanism of circRNAs in lower vertebrates are still unknown. Here, we discovered a novel circRNA derived from Deltex E3 ubiquitin ligase 1 (Dtx1) gene, namely, circDtx1, which was related to the antiviral responses in teleost fish. Results indicated that circDtx1 played essential roles in host antiviral immunity and inhibition of SCRV replication. Our study also found a microRNA miR-15a-5p, which could inhibit antiviral immune response and promote viral replication by targeting TRIF. Moreover, we also found that the antiviral effect inhibited by miR-15a-5p could be reversed with the circDtx1. In mechanism, our data revealed that circDtx1 was a competing endogenous RNA (ceRNA) of TRIF by sponging miR-15a-5p, leading to activation of the NF-κB/IRF3 pathway, and then enhancing the innate antiviral responses. Our results indicated that circRNAs played a regulatory role in immune responses in teleost fish.

Systematic Identification and Analysis of Circular RNAs of Japanese Flounder (Paralichthys olivaceus) in Response to Vibrio anguillarum Infection.

Circular RNA (circRNA) is a new class of non-coding RNA that is structured into a closed loop without polyadenylation. Recent studies showed that circRNAs are involved in the host immune response to pathogen infection. Japanese flounder (Paralichthys olivaceus), an important economical marine fish cultured in north Asia, is affected by Vibrio anguillarum, a pathogenic bacterium that can infect a large number of fish. In this study, we systematically explored the circRNAs in the spleen of V. anguillarum-infected flounder at different infection time points. A total of 6581 circRNAs were identified, 148 of which showed differential expression patterns after V. anguillarum infection and were named DEcirs. Most of the DEcirs were strongly time-specific. The parental genes of the DEcirs were identified and functionally classified into diverse pathways, including immune-related pathways. Among the immune-related DEcirs, seven were predicted to sponge 18 targeted miRNAs that were differentially expressed during V. anguillarum infection (named DETmiRs). Further analysis showed that the DEcirs and their corresponding DETmiRs intertwined into complicated immune related networks. These results indicate that in flounder, circRNAs are regulated by V. anguillarum and form interactive networks with mRNAs and miRNAs that likely play important roles in the immune defense against pathogen infection.

Integrative analysis of circRNA/miRNA/mRNA regulatory network reveals the potential immune function of circRNAs in the Bombyx mori fat body.

Bombyx mori nucleopolyhedrosis virus (BmNPV) is one of the greatest threats to sustainable development of the sericulture industry. Circular RNA (circRNA), a type of non-coding RNA, has been shown to play important roles in gene expression regulation, immune response, and diseases. The fat body is a tissue with both metabolic and immune functions. To explore the potential immune function of circRNAs, we analyzed differentially expressed (DE)circRNAs, microRNAs(miRNAs), and mRNAs in the B. mori fat body in response to BmNPV infection using high-throughput RNA sequencing. A total of 77 DEcircRNAs, 32 DEmiRNAs, and 730 DEmRNAs that are associated with BmNPV infection were identified. We constructed a DEcircRNA/DEmiRNA/DEmRNA and DEcircRNA/DEmiRNA/BmNPV gene regulatory network and validated the differential expression of circ_0001432 and its corresponding miRNA (miR-2774c and miR-3406-5p) and mRNA (778467 and 101745232) in the network. Tissue-specific expression of circ_0001432 and its expression at different time points were also examined. KEGG pathway analysis of DEmRNAs, target genes of DEmiRNAs, and host genes of DEcircRNAs in the network showed that these genes were enriched in several metabolic pathways and signaling pathways, which could play important roles in insect immune responses. Our results suggest that circRNA could be involved in immune responses of the B. mori fat body and help in understanding the molecular mechanisms underlying silkworm-pathogen interactions.

The emerging role and significance of circular RNAs in viral infections and antiviral immune responses: possible implication as theranostic agents.

Circular RNAs (circRNAs) are ubiquitously expressed, covalently closed rings, produced by pre-mRNA splicing in a reversed order during post-transcriptional processing. Circularity endows 3'-5'-linked circRNAs with stability and resistance to exonucleolytic degradation which raises the question whether circRNAs may be relevant as potential therapeutic targets or agents. High stability in biological systems is the most remarkable property and a major criterion for why circRNAs could be exploited for a range of RNA-centred medical applications. Even though various biological roles and regulatory functions of circRNAs have been reported, their in-depth study is challenging because of their circular structure and sequence-overlap with linear mRNA counterparts. Moreover, little is known about their role in viral infections and in antiviral immune responses. We believe that an in-depth and detailed understanding of circRNA mediated viral protein regulations will increase our knowledge of the biology of these novel molecules. In this review, we aimed to provide a comprehensive basis and overview on the biogenesis, significance and regulatory roles of circRNAs in the context of antiviral immune responses and viral infections including hepatitis C virus infection, hepatitis B virus infection, hepatitis delta virus infection, influenza A virus infection, Epstein-Barr virus infection, kaposi's sarcoma herpesvirus infection, human cytomegalovirus infection, herpes simplex virus infection, human immunodeficiency virus infection, porcine epidemic diarrhoea virus infection, ORF virus infection, avian leukosis virus infection, simian vacuolating virus 40 infection, transmissible gastroenteritis coronavirus infection, and bovine viral diarrhoea virus infection. We have also discussed the critical regulatory role of circRNAs in provoking antiviral immunity, providing evidence for implications as therapeutic agents and as diagnostic markers.

Viral non-coding RNAs: Stealth strategies in the tug-of-war between humans and herpesviruses.

Oncogenic DNA viruses establish lifelong infections in humans, and they cause cancers, often in immunocompromised patients, despite anti-viral immune surveillance targeted against viral antigens. High-throughput sequencing techniques allowed the field to identify novel viral non-coding RNAs (ncRNAs). ncRNAs are ideal factors for DNA viruses to exploit; they are non-immunogenic to T cells, thus viral ncRNAs can manipulate host cells without evoking adaptive immune responses. Viral ncRNAs may still trigger the host innate immune response, but many viruses encode decoys/inhibitors to counter-act and evade recognition. In addition, ncRNAs can be secreted to the extracellular space and influence adjacent cells to create a pro-viral microenvironment. In this review, we present recent progress in understanding interactions between oncoviruses and ncRNAs including small and long ncRNAs, microRNAs, and recently identified viral circular RNAs. In addition, potential clinical applications for ncRNA will be discussed. Extracellular ncRNAs are suggested to be diagnostic and prognostic biomarkers and, with the realization of the importance of viral ncRNAs in tumorigenesis, approaches to target critical viral ncRNAs are emerging. Further understanding of viral utilization of ncRNAs will advance anti-viral therapeutics beyond conventional medication and vaccination.

The role of circular RNAs in viral infection and related diseases.

Circular RNAs (circRNAs) are a class of non-coding RNAs with a special covalently closed circular structure, which is formed by precursor mRNA (pre-mRNA) through "back-splicing". CircRNAs are more stable than linear RNAs because they are resistant to exoribonucleases. Viral infections often cause abnormal expression of circRNAs, which could serve as novel biomarkers for the diagnosis of viral infections by detecting specific circRNAs in cells, body fluids, or tissues. CircRNAs also play a critical role in regulating host immune response and virus replication. Here, we reviewed the production and function of circRNAs, mainly focusing on their regulation on virus infection, to provide novel insights into the potential role of circRNAs as diagnostic marker or treatment targets for viral infection.

Emerging roles of circular RNAs in innate immunity.

The proper function of the innate immune system depends on an intricate network of regulation that promotes effective responses to pathogens while avoiding autoimmunity. Circular RNAs (circRNAs), a class of RNAs that lack 5' and 3' ends, have emerged as key actors in these networks. Recent studies have demonstrated that endogenous circRNAs in eukaryotes regulate the activation of innate immune proteins and cells through diverse modes of action. Some DNA viruses also encode circRNAs, and foreign circRNAs have been found to stimulate an innate immune response. This review summarizes recent investigations that reveal the critical roles that circRNAs play in innate immunity and points to future areas of study in this emerging field.

Aberrant Non-Coding RNA Expression in Patients with Systemic Lupus Erythematosus: Consequences for Immune Dysfunctions and Tissue Damage.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease with heterogeneous clinical manifestations. A diverse innate and adaptive immune dysregulation is involved in the immunopathogenesis of SLE. The dysregulation of immune-related cells may derive from the intricate interactions among genetic, epigenetic, environmental, and immunological factors. Of these contributing factors, non-coding RNAs (ncRNAs), including microRNAs (miRNAs, miRs), and long non-coding RNAs (lncRNAs) play critical roles in the post-transcriptional mRNA expression of cytokines, chemokines, and growth factors, which are essential for immune modulation. In the present review, we emphasize the roles of ncRNA expression in the immune-related cells and cell-free plasma, urine, and tissues contributing to the immunopathogenesis and tissue damage in SLE. In addition, the circular RNAs (circRNA) and their post-translational regulation of protein synthesis in SLE are also briefly described. We wish these critical reviews would be useful in the search for biomarkers/biosignatures and novel therapeutic strategies for SLE patients in the future.

Circular RNAs in Immune Response and Viral Infection.

Circular RNAs (circRNAs) are a diverse class of RNAs with varying sizes, cellular abundance, and biological functions. Investigations from the past decade have revealed that circRNAs are ubiquitously found in eukaryotes and have defined the different biological roles of circRNAs to illuminate this previously unrecognized class of molecules. In the context of the immune system, immune responses and immune-related diseases alter circRNA expression. More recently, several oncogenic double-stranded DNA viruses have been found to encode circRNAs. In this review, we summarize the current understanding of circRNAs and their emerging functions in immune regulation and autoimmune disorders, and discuss the identification and potential roles of viral circRNAs during infections. Finally, we present promising areas for future investigations in the nascent field of circRNAs.