Hsa_circ_0060450 Negatively Regulates Type I Interferon-Induced Inflammation by Serving as miR-199a-5p Sponge in Type 1 Diabetes Mellitus

Circular RNAs (circRNAs) constitute a class of covalently circular non-coding RNA molecules formed by 5′ and 3′ end back-splicing. The rapid development of bioinformatics and large-scale sequencing has led to the identification of functional circRNAs. Despite an overall upward trend, studies focusing on the roles of circRNAs in immune diseases remain relatively scarce. In the present study, we obtained a differential circRNA expression profile based on microarray analysis of peripheral blood mononuclear cells (PBMCs) in children with type 1 diabetes mellitus (T1DM). We characterized one differentially expressed circRNA back-spliced from the MYB Proto-Oncogene Like 2 (MYBL2) gene in patients with T1DM, termed as hsa_circ_0060450. Subsequent assays revealed that hsa_circ_0060450 can serve as the sponge of miR-199a-5p, release its target gene, Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), encoded by the tyrosine-protein phosphatase non-receptor type 11 gene (PTPN11), and further suppress the JAK-STAT signaling pathway triggered by type I interferon (IFN-I) to inhibit macrophage-mediated inflammation, which indicates the important roles of circRNAs in T1DM and represents a promising therapeutic molecule in the treatment of T1DM.


INTRODUCTION
Type 1 diabetes (T1DM) is an autoimmune disease, also known as insulin-dependent diabetes mellitus, owing to the complete absence of insulin secretion. During the pathogenesis of T1DM, insulin-producing pancreatic β cells are attacked by the immune system, resulting in destruction of islet function and decreased insulin secretion (1,2). Immune tolerance mediated by Treg cells is outweighed by immune activation involving autoreactive CD8 + T cells, Th1 cells, and B cells (3). Furthermore, the expansion of IL-17-secreting Th17 cells also contributes to pancreatic inflammation in patients with T1DM (4,5). In addition to adaptive immune cells, innate immune cells are involved in the onset of T1DM (6)(7)(8). Emerging evidence suggests that the absence of Toll-like receptor signaling in innate immune cells, caused by TIR-domain-containing adapter-inducing interferonβ (TRIF) deficiency, has a protective effect against diabetes in the non-obese diabetic (NOD) mouse via alteration of the gut microbiota and reduced immune cell activation (9).
Macrophages, known as inflammatory mediator-secreting immune cells, play a critical role in insulitis, and support autoimmune T cells to aggravate the infiltration of inflammatory cells during T1DM (10,11). Macrophage migration inhibitory factor (Mif) knockout (KO) mice were found to express lower levels of costimulatory molecules in the streptozocin-induced diabetic mouse model and Toll-like receptors on the surface of their macrophages, leading to functional deficit of macrophages and impaired T cell activation, thereby alleviating pancreatic injury (12). Moreover, Unanue et al. have showed that isletresident macrophages are in a persistent inflammatory state prior to signs of β cell pathology and their depletion significantly reduced the onset of diabetes (13,14).
In addition to inflammation caused by bacterial infection, viral infection and subsequent IFN-I production are linked to T1DM pathogenesis (15)(16)(17). Viral infections can lead to apoptosis of pancreatic β cells and pancreas damage (18). Some viruses, such as coxsackievirus B1, parainfluenza, and enteroviruses initiated a devastating immune response in pancreatic β cells and other surrounding cells, resulting in the occurrence of fulminant T1DM through the induction of IFN-I and inflammation (19,20). In addition, 2 independent longitudinal studies have showed the expression of IFN-I-induced genes in PBMCs isolated from children at risk for T1DM preceded onset of autoimmunity (21,22). Moreover, some studies have confirmed that abrogation of IFN-I function significantly ameliorated the incidence of diabetes in NOD mice (23,24). Marro et al. also found that macrophages promoted autoreactive CD8 + T cell infiltration into islets in the Rip-LCMV-GP T1DM model via IFN-I signaling, and abrogation of IFN-I signaling on macrophages significantly limited the onset of T1DM (25). Circular RNAs (circRNAs) are a class of covalently circular non-coding RNA molecules (26,27). Existing studies have shown circRNAs are generated mainly through three distinct mechanisms: exon skipping, RNA binding protein (RBP)driven circularization, and intron pairing-driven circularization, which bring the 5 ′ and 3 ′ termini of mRNA into spatial proximity and induce back-splicing. The length of most human exonic circRNAs is <1,500 nucleotides (nt), and the median length is 500 nt (28). Compared with their homologous linear transcripts, circRNAs are not easily degraded by RNase R (29), and highly conserved between tissues, developmental stages, and cell types (30 (32). Substantial evidence has been published describing the competitive endogenous RNA (ceRNA) mechanism, which is the most widely studied mechanism in circRNA research. Through adsorption of microRNAs, circRNA reduces the quantity of free microRNAs in the cytoplasm and releases target genes of microRNAs, leading to occurrence or inhibition of certain diseases. Notably, despite an overall upward trend, circRNA studies focusing on immune diseases are relatively scarce. In the present study, we obtained a differential circRNA expression profile based on microarray analysis of PBMCs in children with T1DM. We characterized an upregulated circRNA, backspliced from MYB Proto-Oncogene Like 2 (MYBL2) in patients with T1DM, termed as hsa_circ_0060450. Subsequent assays revealed that hsa_circ_0060450 can serve as the sponge of miR-199a-5p, releasing its target Src homology 2 (SH2)containing protein tyrosine phosphatase 2 (SHP2) encoded by tyrosine-protein phosphatase non-receptor type 11 gene (PTPN11) in macrophages, and further suppress the JAK-STAT signaling pathway triggered by IFN-I to inhibit macrophagemediated inflammation.

Human Normal and T1DM Samples
Peripheral blood samples of T1DM patients and healthy controls were collected from the Children's Hospital of Fudan University, Shanghai, China. The follow-up PBMCs were separated by Ficoll-Hypaque (GE Healthcare) within 24 h after collection. Human samples were obtained with informed consents from their parents, and the study was approved by the Research Ethics Board of the Children's Hospital of Fudan University.

Microarray Analysis
PBMCs of T1DM patients and healthy controls were separated and performed SBC Human (4 * 180K) ceRNA microarray analysis (Shanghai Biotechnology Corporation, China). In brief, total RNA was extracted and purified using miRNeasy Mini Kit (QIAGEN, GmBH, Germany), followed by amplification and label using Low Input Quick Amp Labeling Kit, One-Color (Agilent technologies, Santa Clara, CA, USA). Each slide was hybridized with Cy3-labeled cRNA using Gene Expression Hybridization Kit (Agilent technologies, Santa Clara, CA, USA) in Hybridization Oven. After hybridization, slides were washed in staining dishes with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, USA), and were then scanned by Agilent Microarray Scanner (Agilent technologies, Santa Clara, CA, USA). Finally, data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, USA), and raw data were normalized by Quantile algorithm. Fold change > 2.0 and P < 0.05 was the criterion to screen differentially expressed circRNAs between T1DM patients and healthy controls. Microarray data have been deposited in the Gene Expression Omnibus (GEO) under accession numbers GSE133225 (submitted in other draft).

RNA Extraction and Reverse Transcription Quantitative PCR (RT-qPCR)
Total RNA was extracted using TRIzol (Invitrogen, USA) followed by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Japan), according to the manufacturer's instructions. Real-time quantitative PCR reaction was performed with SYBR R Premix Ex Taq TM II (Takara, Japan) using the Roche 480 Real Time PCR System. We used βactin as internal control and calculated fold change via the 2 − CT method. The primer pairs used for qPCR are listed in Supplementary Table 2.

Nuclear and Cytoplasmic Separation
Nuclear and cytoplasmic separation assay was conducted using the Nuclear and Cytoplasmic Extraction Kit (CWBiotech, Beijing, China) according to the manufacturer's protocol. Briefly, 1 × 10 7 cells were centrifuged at 3 × 10 3 rpm for 5 min, resuspended in 500 µl of ribonuclease inhibitor added-Nc-buffer A, and incubated on ice for 20 min. Then, 27.5 µl of Nc-Buffer B was added, and incubated on ice for 1 min. The mixtures were then centrifuged at 12,000 g, 4 • C for 15 min. The supernatants (cytoplasmic component) were mixed with TRIzol reagent for RNA extraction. Next, the nuclear pellets were resuspended with 250 µl of ribonuclease inhibitor added-Nc-buffer C, and then mixed with TRIzol reagent for RNA extraction. RNA extraction and RT-qPCR were performed as previously described.

Dual-Luciferase Reporter Assay
The wild type (WT) and mutant hsa_circ_0060450 fragments and SHP2 3 ′ -UTR containing corresponding miRNAs binding sites were fused into Renilla luciferase gene (hRluc) included psicheck2 vector (Promega, Madison, WI, USA) using the Xho I and Not I restriction sites. The fused recombinant vectors were verified by sanger sequencing. The PCR primers for WT and mutant hsa_circ_0060450 fragments and SHP2 fragments are listed in Supplementary Table 2.

RNase R Treatment
A total of 5 µg of RNA extracted from cells using TRIzol reagent was incubated at 37 • C for 30 min with RNase R (20 U/µl, Lucigen, USA) treatment according to the manufacturer's instructions; the control group was treated under RNase R-free conditions. The remaining RNA was then purified with phenol/chloroform mixture (5:1, Sigma-Aldrich, USA). Subsequent RNA extraction and RT-qPCR was performed as previously described.

Actinomycin D Treatment
THP1 cells were seeded into a 24-well plate at a density of 120,000 cells/well with PMA (50 ng/ml) stimulation for 48 h. Then, total RNA was harvested at 0, 3, 6, 12, and 24 h, with or without actinomycin D (ActD) treatment (1 µg/ml). Subsequent RNA extraction and RT-qPCR was performed as previously described.

Statistical Analysis
Results from three independent, replicate experiments were obtained to confirm the reproducibility of each experiment. Bars represent the means ± SEM. The two-tailed Student's t-tests was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons of multiple groups. A p-value of < 0.05 was considered to indicate statistically significant data. Statistical analyses were performed with SPSS v. 19.0 software and visualized data were obtained with Graphpad prism 7.0 software.

Hsa_circ_0060450 Is Upregulated in PBMCs of T1DM Patients
In a pilot study, we obtained the differential circRNA expression profile based on microarray analysis of PBMCs in children with T1DM and age-matched healthy controls. We identified 15 upregulated and 17 downregulated circRNAs between T1DM patients and healthy controls (n = 3, fold change > 2, and p < 0.05) ( Figure 1A). Next, we verified the expressions of the top 10 circRNAs (Figure 1B) with the highest differential expression fold in a larger population of clinical samples of PBMCs collected from 20 T1DM children and 20 healthy controls by RT-qPCR, and the results showed hsa_circ_0060450 was detectable and significantly upregulated in PBMCs of T1DM patients than controls (Figure 1C). In addition, through ROC curve analysis, we found that the expression of hsa_circ_0060450 in PBMCs could discriminate normal subjects and T1DM patients ( Figure 1D). Using RT-PCR with divergent primers and DNA sequencing, we confirmed the back-splicing site sequences of hsa_circ_0060450 ( Figure 1E). The full-length sequences of hsa_circ_0060450 were listed in Supplementary Figure 1. After treatment with RNase R, hsa_circ_0060450 did not degrade readily compared with its cognate linear transcript MYBL2 (Figure 1F). Actinomycin D assay also revealed that hsa_circ_0060450 is more stable than its linear transcript MYBL2, with a half-life exceeding 24 h (Figure 1G). Furthermore, to investigate the roles of hsa_circ_0060450 in T1DM pathogenesis, we separated human PBMCs to detect the expression of hsa_circ_0060450 in T cells, B cells and monocytes, respectively, and found the expression of hsa_circ_0060450 is higher in monocytes compared with T cells and B cells (Figure 1H). Given that monocytes can transform into macrophages to participate in pancreas inflammation, we next mainly explored the function of hsa_circ_0060450 in macrophages. Subsequently, we performed nuclear and cytoplasmic separation experiments in THP1derived macrophages, and found that ∼40% of hsa_circ_0060450 is located in the cytoplasm, with the remaining proportion in the nucleus (Figure 1I).
Hsa_circ_0060450 Negatively Regulates IFN-I-Induced Inflammation Through the JAK-STAT1/3 Pathway To further explore whether hsa_circ_0060450 plays a role in IFN-I-induced inflammation, we designed two small interfering RNAs (siRNAs) to silence hsa_circ_0060450 expression in THP1-derived macrophages (Figure 2A). Notably, the two siRNAs were targeted specifically to the back-splicing site of hsa_circ_0060450, ensuring that only hsa_circ_0060450 was knocked down and its cognate linear transcript MYBL2 was not targeted. RT-qPCR assay showed that hsa_circ_0060450 silencing resulted in significant upregulation of the expressions of IFIT1, IFIH1, CXCL10, and iNOS under IFN-I stimulation ( Figure 2B). We then performed Western blotting assay to investigate the effect of hsa_circ_0060450 on the JAK-STAT1/3 pathway activated by IFN-I. Experimental results showed that hsa_circ_0060450 silencing promoted STAT1 and STAT3 protein phosphorylation after 15 min and 30 min of IFN-I stimulation ( Figure 2C). This was indicative of the inhibitory action of hsa_circ_0060450 on IFN-I-induced inflammation. Collectively, these results suggest that hsa_circ_0060450 negatively regulates IFN-I-induced inflammation through the JAK-STAT1/3 pathway.
Hsa_circ_0060450 Serves as a Sponge of miR-199a-5p to Inhibit Inflammation Induced by IFN-I Recent evidence suggests that exonic circRNAs within the cytoplasm can serve as a "sponge" of microRNAs. Therefore, we determined to investigate whether hsa_circ_0060450 adsorbs microRNAs to exert its function. We first selected several microRNAs that have been studied for their pro-inflammatory effects, and among these microRNAs, miR-199a-5p, miR-133a-3p and miR-133b were predicted to have binding sites to hsa_circ_0060450 by RNA22 database (https://cm.jefferson. edu/rna22/Interactive/) ( Figure 3A). Next, we performed luciferase assay by constructing hsa_circ_0060450 fragmentcontaining psicheck2 recombinant vectors ( Figure 3B). Results showed that the luciferase activity of the group co-transfected with miR-199a-5p mimics and psicheck2 recombinant vector containing hsa_circ_0060450 fragment, as well as that of the other two groups co-transfected with miR-133a or miR-133b mimics and psicheck2 recombinant vector containing hsa_circ_0060450 fragment, was significantly reduced (Figure 3C). We also performed luciferase assay to confirm the target sequence of miR-199a-5p, miR-133a or miR-133b in hsa_circ_0060450 by constructing mutant hsa_circ_0060450 fragment-containing psicheck2 recombinant vectors ( Figure 3A). Results showed that the luciferase activity of the group co-transfected with miR-199a-5p, miR-133a or miR-133b mimics and corresponding hsa_circ_0060450-mutant vector was not changed compared with the NC group ( Figure 3D and Supplementary Figure 2). Furthermore, we investigated the pro-inflammatory effects of these three microRNAs in THP1-derived macrophages. The Western blotting results suggested that miR-199a-5p overexpression promoted STAT1 and STAT3 protein phosphorylation ( Figure 3E); however, the overexpression of miR-133a and miR-133b inhibited STAT1 and STAT3 protein phosphorylation after IFN-I stimulation (Supplementary Figures 3A,B). Of note, RT-qPCR assay indicated that miR-199a-5p overexpression significantly upregulated the expressions of IFIT1, IFIH1, Frontiers in Immunology | www.frontiersin.org   (Figure 3F), consistent with the pro-inflammatory features of miR-199a as reported previously (33,34), and miR-133a or miR-133b overexpression significantly reduced the expressions of IFIT1, IFIH1, CXCL10, and CCL5 under the stimulation of IFN-I (Supplementary Figure 3C). To summarize, hsa_circ_0060450 may serve as a sponge of miR-199a-5p to inhibit inflammation induced by IFN-I.

miR-199a-5p Promotes IFN-I-Induced Inflammation by Targeting SHP2
MicroRNAs can bind to the 3 ′ UTR of their targets to suppress translation or accelerate mRNA degradation (35,36). miR-199a-5p may act as a pro-inflammatory factor by binding to its target to promote JAK-STAT1/3 pathway activation. We then selected five typical negative regulators of JAK-STAT pathway through a survey of the literature: SHP2, SOCS3, SHP1, SOCS1, and PTP1B (37). RT-qPCR assay demonstrated that the overexpression of miR-199a-5p reduced the expression of SHP2 at mRNA level, but not that of the other four negative regulators ( Figure 4A). Western blotting assay also showed that overexpression of miR-199a-5p reduced the expression of SHP2 at protein level ( Figure 4B). SHP2 was predicted to contain three miR-199a-5p binding sites according to the starBase database (http:// starbase.sysu.edu.cn/index.php) (Figure 4C), and subsequent luciferase assay showed that the luciferase activity of the group co-transfected with miR-199a-5p mimics and part SHP2 sequence-containing psicheck2 vector was significantly reduced ( Figure 4D). These findings suggest that SHP2 is a target of miR-199a-5p.

DISCUSSION
In this study, we obtained a differential circRNA expression profile based on microarray analysis of PBMCs in children with T1DM. We then characterized one circRNA, back-spliced from MYBL2, termed as hsa_circ_0060450, which exhibited upregulated expression in patients with T1DM. Subsequent assays revealed that hsa_circ_0060450 serves as a "sponge" of miR-199a-5p to inhibit IFN-I-induced inflammation ( Figure 5F). miR-199a-5p was reported to exert various biological functions, and existing studies have indicated the potential roles of miR-199a-5p in the pathogenesis of diabetes. Lin et al. demonstrated that miR-199a-5p is upregulated in pancreatic β cells in response to high glucose and promotes apoptosis and ROS generation in type 2 diabetes (38). In addition, a bioinformatics analysis showed that mmu-miR-199a-5p is closely related to inflammatory response, and insulin signal pathway in the liver of diabetic mice (39). SHP2 is a widely expressed SH2 domain-containing protein tyrosine phosphatase (40). Although sufficient evidence has shown that SHP2 is involved in tumorigenesis, its roles in immune regulation have also been gradually elucidated. Shuai et al.   (44). Despite these existing studies, the present study is the first, to our knowledge, to investigate the role of hsa_circ_0060450-miR-199a-5p-SHP2 axis in IFN-I-induced inflammation in T1DM. Unexpectedly, even if showing an upward trend in the PBMCs of T1DM patients, hsa_circ_0060450 did not prevent the occurrence of pancreatic inflammation and T1DM. Possibly, we consider that hsa_circ_0060450 is produced reactively during pancreatic inflammation, but the amount produced in the body is not sufficient to inhibit macrophage inflammation. In addition, there may be other circRNAs or other types of molecules in T1DM patients that antagonistically inhibit anti-inflammatory effect of hsa_circ_0060450. Therefore, it is necessary to administer a larger dose of exogenous hsa_circ_0060450 to achieve a certain effect of alleviating inflammation and treating T1DM.
Generally, circRNAs are classified into three types: exon circular RNA (ecircRNA), circular intronic RNA (ciRNA), and exon-intron circRNA (EIciRNA) (45). EcircRNA mainly exists in the cytoplasm, serving as a "sponge" of microRNAs, while the other two circRNAs are confined to the nucleus owing to the presence of introns (46)(47)(48). Zhang et al. demonstrated that intron-derived circular RNA ci-ankrd52 exists in the nucleus and cis-regulates transcription of its parental gene ANKRD52 via the catalytic effect of RNA Pol II (47). Li et al. subsequently found that intron-exon circRNA circEIF3J and circPAIP2 form an RNA-protein complex with U1SNP and RNA Pol II through RNA-RNA interaction to cis-regulate the transcription of their parental genes (46). The parental gene of hsa_circ_0060450, MYBL2, is a transcription factor of the MYB family, and was first studied to be a physiological regulator of cell cycle progression, cell differentiation and cell survival, but recently it was frequently found deregulation in numerous cancer entities and significantly drove cancer initiation and progression (49). Moreover, a new non-synonymous SNP R687C in exon 14 of MYBL2 was identified in a genome scan study in Ashkenazi patients with type 2 diabetes (50), suggesting a potential role of MYBL2 in diabetes. In the present study, we found MYBL2 showed an upregulated trend in T1DM patients compared with healthy controls (Supplementary Figure 4). In addition, we discovered that 40% of hsa_circ_0060450 is present in the cytoplasm, with the remaining proportion in the nucleus, and in the current study we mainly focused on the role of cytoplasmic hsa_circ_0060450; however, it remains to be elucidated whether hsa_circ_0060450 in the nucleus also functions in T1DM through the interaction with MYBL2.
In summary, in this study, we obtained a differential circRNA expression profile, and characterized a differentially expressed circRNA, hsa_circ_0060450. Subsequent experiments showed that hsa_circ_0060450 can adsorb miR-199a-5p, releasing its target SHP2, by which hsa_circ_0060450 inhibits macrophage-mediated inflammation through the suppression of JAK-STAT1/3 signaling pathway triggered by type I interferon. Overall, this study indicates the important role of hsa_circ_0060450 in T1DM and implies a promising therapeutic molecule in the treatment of T1DM in the future.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Research Ethics Board of the Children's Hospital of Fudan University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
LY, XH, CZ, CS, SH, WX, YG, QL, FL, and WL designed, carried out experiments, and analyzed data. LY and YZ wrote the manuscript. YZ and JF planned, designed, supervised, and coordinated the overall research efforts. All authors contributed to the article and approved the submitted version.