Circular RNA circLOC101928570 Suppress Systemic Lupus Erythematosus Progression Via Targeting the miR-150/c-myb Axis

Background: Accumulating evidence supports the implication of circRNAs in systemic lupus erythematosus (SLE). however, little is known about their the detailed mechanisms and the roles of circRNAs in the pathogenesis of SLE. Methods: Quantitative real time-PCR (qRT-PCR) was used to determine the levels of circLOC101928570 and miR-150 in peripheral blood mononuclear cells (PBMCs) of SLE. Overexpression and knockdown experiments were conducted to assess the effects of circLOC101928570. Fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP), luciferase reporter assays, western blot, ow cytometry analysis and enzyme-linked immunosorbent assay (ELISA) were used to investigate the molecular mechanisms underlying the function of circLOC101928570. Results: The results showed that the level of circLOC101928570 was signicantly down-regulated in SLE and correlated with systemic lupus erythematosus disease activity index (SLEDAI). Functionally, circLOC101928570 acted as a miR-150 sponge to relieve the repressive effect on its target c-myb, which modulates the activation of immune inammatory responses. CircLOC101928570 knockdown enhanced apoptosis. Moreover, circLOC101928570 promote the transcriptional level of IL2RA through directly regulate miR-150/c-myb axis. Conclusion: Overall, our ndings demonstrated that circLOC101928570 played a critical role in SLE. The down-expression of circLOC101928570 suppressed SLE progression through miR-150/c-myb/IL2RA axis. Our ndings identied that circLOC101928570 serve as a potential biomarker for the diagnosis and therapy of SLE.

In this study, we aimed to analyze circRNA pro les expressed in PBMCs of SLE patients, investigate whether circLOC101928570 differentially expressed and closely related to the disease activities of SLE patients. We focused on the effect of circLOC101928570 competitively bind to miR-150 and regulate the expression of c-myb, which might regulate the transcription of IL2RA and eventually protected against diseasees progression. This study may provide a promising biomarker for the prevention, diagnose and treatment of the SLE.

Patients samples
There are 62 SLE patients and healthy volunteers were recruited from the First A liated Hospital of Army Medical University between 2017 and 2020. The SLE patients included ful lled at least four of the American College of Rheumatology (ACR) 1997 revised criteria of SLE (Hochberg, 1997). Demographic, clinical, and laboratory characteristics of each subject were recorded, and disease activity was evaluated with SLEDAI (Bombardier et al. 1992). The information of SLE patients can be found in (Table 1, Additional le 1: Table 1). All participants were Han Chinese. Sequence used was (5'-CCGGAATTCCGGGAAAGCGTCACTTGGGGAAAA-3'). PLKO.1-puro (Addgene plasmid # 8453) was used to design short hairpin RNA , they were transfected by Lipofectamine 3000 (Invitrogen, United States) into cells. The transfection process lasted 48 hours.
Luciferase reporter assay 293T cells in 24-well plates were cotransfected with miR-150-3p/5p mimic, inhibitor, and negative control, luciferase reporter vectors (SV40-Luc-MCS) with wild type or mutant circLOC101928570 were designed and constructed by Genechem (Shanghai, China). The IL2RA 3' UTR sequences containing two wild-type c-myb predicted binding sites were inserted into the region directly downstream of a T7 promoter-driven re y luciferase cassette in a psiCHECKTM-2 vector (Promega, United States). All constructs were veri ed by sequencing. 293T cells were seeded into 24-well plates and were co-transfected with a mixture of 1ug of luciferase reporter plasmid and PCDH, plasmid pCDH-MYB, shNC, shMYB. After 48h, luciferase activity was measured using Dual-Luciferase® Reporter Assay System (Promega, United States). The relative re y luciferase activity was normalized to Renilla luciferase activity. All experiments were performed in triplicate.

Short hairpin RNA
To stably knock down circLOC101928570 expression, Jurkat cells were cultured and infected with lentivirus carrying shRNA targeting circLOC101928570 and a negative control vector, PLKO.1-puro was used to design short hairpin RNA, the restriction site were AgeI (R3552S), EcoRI (R3101T), after 1300bp has a single restriction site KpnI (R0142M). For the lentivirus package, HEK-293T cells were transfected with the core plasmid PLKO.1-shRNA, with the psAX2 packaging plasmid and pMD2G envelope plasmid for 48 h to obtain the lentivirus supernatant. The shRNA sequences used was showed in (Additional le 3: Table 3). All constructs were veri ed by sequence analysis. with no homology to any other human genes.

Apoptosis
Double staining of Annexin V and 7-aminoactinomycin-D (7-AAD) was carried out using a PE Annexin Enzyme-linked immunosorbent assay (ELISA) Concentrations of IL-4 and IFN-γ in Peripheral blood serum supernatants were analyzed by Human IL-4 instant ELISA Kit (eBioScience, United States) and Human IFN gamma Platinum ELISA Kit (eBioScience, United States) following the manufacturer's instructions, respectively. The concentrations were calculated according to their corresponding stand curves.

Prediction of ceRNAs for circLOC101928570
Mutually targeted method was applied to predict putative ceRNAs for circRNA. To predict ceRNAs for circLOC101928570, we use software circMir1.0 identi ed circLOC101928570 targeting miRNAs.
Pull-down assay Biotinylated circLOC101928570 probe was speci cally designed and synthesized for binding to the junction site of circLOC101928570. The biotin-coupled RNA complex was pulled down by incubating the cell lysates with Pierce™Streptavidin Magnetic Beads (Thermo Fisher Scienti c, United States) following the manufacturer's instructions. The enrichment of miRNAs in the capture fractions was evaluated by qRT-PCR analysis. Probe sequences used were listed in (Additional le 4: Table 4).

RNA-binding protein immunoprecipitation (RIP)
RIP assay was performed by using a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, United States), according to the manufacturer's instructions. Brie y, PBMCs were harvested and lysed in RIP lysis buffer on ice for 30 min. After centrifugation, the supernatant was incubated with 30μl of Protein G agarose beads (Roche, United States) and antibodies. After overnight incubation, the immune complexes were centrifuged then washed six times with washing buffer. The beads bound proteins were further analyzed using western blotting. The immunoprecipitated RNA was applied to qRT-PCR analysis.

Western blot analysis
Complete proteome from PBMCs was extracted after lysing in RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Sigma-Aldrich, United States) and then separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis. The samples were electro transferred into polyvinylidene uoride membrane (Millipore, United States). Post blocking with 5% fat free milk, the membranes were treated with prepared primary antibodies: anti-c-myb (Abcam, England), rabbit IL2Rα antibody (CST, United States), rabbit GAPDH antibody (CST, United States) was used as control. Membranes were washed, followed by treating with secondary antibody anti-rabbit antibody (CST, United States). The signal of the blot was examined by Pierce ECL Western Blotting Substrate (Thermo Fisher Scienti c, United States) with ChemiDocTM Touch Imaging System (Bio-Rad, United States). The integrated density values were calculated using Quantity One software (Bio-Rad, United States).

Flow cytometry analysis
The ow cytometric analysis was performed on freshly isolated PBMCs as a previous study showed that CD25 expression is affected by freezing-thaw procedures. PBMCs were stained with uorochromeconjugated antibodies to identify blood CD4+ and CD8+ T cell subsets by ow cytometry. The staining procedure was conducted blinded to genotype and performed simultaneously for each pair. Prior to staining, FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the PBMCs to prevent non-speci c binding. We used the following monoclonal antibodies speci c for: CD3 . Quantitative values are expressed as means±standard deviation (SD) of at least three independent repetitions. Statistical differences among groups were tested with unpaired two-tailed student's t test. A P-value less than 0.05 was chosen to indicate statistical signi cance.

Results
Identi cation and characterization of circLOC101928570, a circRNA that speci cally showed a lower expression level in SLE To identify the essential circRNAs that contribute to the progression of SLE, we analyzed the circRNA RNA-seq data to identify circRNAs (Additional le 5: Table 5) with signi cant differences (|fold change|>2, FDR<0.05) between healthy controls and SLE patients (Miao et al. 2019). We selected and identi ed 4 circRNAs signi cantly related with SLE (Fig. 1A). We applied 62 patient samples and 62 healthy samples for further analysis. We then focused on circLOC101928570, which was expressed at low level in PBMCs of SLE patients, for further study (Fig. 1B). Patients expressed low level of circLOC101928570 and had a signi cantly higher the SLEDAI score (Fig. 1C), higher complement C3 level (Fig. 1D). To assess the diagnostic value of circLOC101928570 for SLE, receiver operating characteristic (ROC) curve analysis was performed to determine the relative circLOC101928570 expression between the 62 patients and 62 healthy controls (Fig. 1E). The area under the curve (AUC) was 0.8985 and the 95% con dence interval (95%CI) was 0.8441−0.9530. We next evaluated the circular structure of circLOC101928570, which was aroused from exon 3 of the LOC101928570 gene (chr6: 77271340-77273307), sanger sequencing validated the backspliced junction of circLOC101928570, as showing in (Fig. 1F). There are two CPG islands existed in the transcriptional core region of circLOC101928570 by using the software (http://www.ebi.ac.uk/emboss/cpgplot/), as showing in (Fig. 1G). Then we used the exonuclease RNase R to examine the stability of the circLOC101928570, circLOC101928570 showed a strong resistance to digestion by RNase R, whereas the linear RNA of LOC101928570 and β-Actin were highly degraded (Fig.  1H). FISH result showed that circLOC101928570 is predominantly localized in the cytoplasm (Fig. 1I). Together, these data suggest that circLOC101928570 was signi cantly low expressed in SLE patients and correlated with SLE.
CircLOC101928570 acted as a sponge for miR-150 To explore the possible mechanism of functional circLOC101928570, we identi ed its ceRNAs with circMir1.0 software (http://www.bioinf.com.cn/). The result showed that circLOC101928570 has two targets with miR-150 ( Fig. 2A). The miRNAs were extracted after pull-down assay, and the level of 10 candidate miRNAs was detected by real-time PCR. MiR-150-3p/5p were abundantly pulled down by circLOC101928570 in PBMC cells (Fig. 2B). To determine whether circLOC101928570 functions as a miRNAs sponge, We next preformed AGO2 immunoprecipitation to determine whether circLOC101928570 served as a platform for AGO2 and miR-150. The result showed that circLOC101928570 does bind to AGO2, QRT-PCR results further supported this observation (Fig. 2C). To con rm circLOC101928570 could be regulated by miR-150-3p/5p, we constructed luciferase reporters containing wild type and mutated putative binding sites of circLOC101928570 transcripts respectively (Fig. 2D), then co-transfected with miR-150-3p/5p mimcs or inhibitors into 293T cells. Luciferase reporter assays showed that the luciferase activities of circLOC101928570 wild type reporter were signi cantly reduced when transfected with miR-150-3p/5p mimics compared with control reporter or mutated luciferase reporter (Fig. 2E). Whereas, miR-150-3p/5p inhibitor signi cantly increased the luciferase signal of wild-type circLOC101928570 reporter (Fig. 2F). QRT-PCR further con rmed that circLOC101928570 knockdown could increase the miR-150 level and circLOC101928570 overexpression had an opposite role in Jurkat cell lines (Fig. 2G). However, miR-150 can't signi cantly in uence circLOC101928570 level (Fig. 2H). Moreover, RNA FISH assay revealed that circLOC101928570 and miR-150 were co-localized in cytoplasm (Fig. 2I). These results showed that circLOC101928570 can bind to miR-150.
CircLOC101928570 decreased the c-myb expression by sponging miR-150 In order to explore the function of miR-150 in SLE, we then detected the expression of miR-150 using qRT-PCR in PBMCs obtained from 36 patients with SLE and 25 healthy controls. The result showed that the expression of miR-150 was upregulated in SLE (Fig. 3A). MiR-150 expression was also contrarily correlated with complement C3 level (Fig. 3B). There was strong positively correlated between miR-150 expression and the SLEDAI score in patients with SLE (Fig. 3C). To assess the diagnostic value of miR-150 in SLE, we also performed ROC curve analysis with the relative miR-150 expression in the 36 patients and 25 healthy controls (Fig. 3D) performed two short hairpin RNAs (shRNA-circLOC101928570#1 and shRNA-circLOC101928570#2). Jurkat cells with stable circLOC101928570 knockdown with lentiviral shRNA and circLOC101928570 overexpression plasmid with lentiviral were constructed. We found that shRNA-circLOC101928570 could successfully knockdown circLOC101928570 expression but had no effect on LOC101928570 mRNA expression in jurkat cells (Fig. 3E). Similarly, circLOC101928570 was successfully overexpressed in jurkat cells, while LOC101928570 mRNA expression had no obvious change (Fig. 3F). To explore whether circLOC101928570 could regulate the c-myb via competitively binding with miR-150, we transfected miR-150 inhibitor and circLOC101928570 overexpression plasmids into jurkat cells. CircLOC101928570 overexpression increased the expression of c-myb, while transfected miR-150 mimics signi cantly attenuated the circLOC101928570-induced increased expression of c-myb (Fig. 3G). Downregulation of circLOC101928570 resulted in decreased expression of c-myb. Furthermore, transfected miR-150 inhibitor promoted the decreased expression of c-myb (Fig. 3H). These data demonstrated that circLOC101928570 regulates the c-myb by competitively binding of miR-150 to mediate the immune in ammatory response in SLE.

CircLOC101928570 knockdown enhanced apoptosis
As a proto-oncogene, c-myb plays a crucial role in the process of cell development, differentiation and apoptosis. Down-regulated expression of c-myb will cause cell apoptosis (Malaterre et al., 2007;Oh and Reddy, 1999;Liu, 2004). Our study has shown that circLOC101928570 decreased the c-myb expression by sponging miR-150. To further explore the function of circLOC101928570, we examined the cell apoptosis in stable Jurkat cells, and found that the percentage of cells in apoptosis was signi cantly increased in the shRNA-circLOC101928570#1 group compared with shRNA-NC group ( Fig. 3I and 3J). The proportion of cells that were in apoptosis was lower in the circLOC101928570 overexpression group than that of NC group ( Fig. 3I and 3K). These ndings suggested that circLOC101928570 negatively regulated early apoptosis of Jurkat cells. Identifying the factors contributing to the enhanced apoptosis of SLE T cells will deepen our understanding of SLE pathogenesis.
C-myb transcriptionally regulating IL2RA expression by binding to IL2RA To further explore the pathogenesis of circLOC101928570 involvement in SLE. Next, through the prediction of transcription factor c-myb target genes by software (http://cistrome.org/db/#/), result showed that IL2RA might bind c-myb in Th1 and Th2 CD4+ T lymphocytes ( Fig. 4A and 4B). CD4+T lymphocytes are an important factor in the pathogenesis of SLE, mainly manifested by the immune imbalance of CD4+T lymphocytes, and the differentiation of CD4+T lymphocytes is regulated by IL-2(He et al. 2016). However, the speci c mechanism remains to be clari ed. Normal serum IL-2 level is an important condition to maintain the normal function of CD4+T lymphocytes, B cells and NK cells. As a transcription factor, c-myb may regulate target gene expression in transcriptional level, thereby exerting biological functions. We analyzed the potential binding DNA sequence loop of c-myb and found two theoretical binding sites in the top 2000nt of the promoter domain of IL2RA gene (http://jaspar.genereg.net) (Fig. 4C). Hence, we speculated that c-myb may regulate IL2RA expression at the transcriptional level. To con rm the supposition, a luciferase plasmid with the top 2000nt of the promoter domain of IL2RA gene (psicheck2-WT) and a luciferase plasmid with mutant sequences in both two binding sites of the top 2000nt of the promoter domain (psicheck2-Mutant) were generated (Fig. 4D). In addition, we performed two short hairpin RNAs (shRNA-MYB#1 and shRNA-MYB#2), MYB overexpression plasmid was constructed with PCDH vector. We found that shRNA-MYB could successfully knockdown MYB expression and MYB was successfully overexpressed in 293T cells (Fig. 4E  and 4F). Luciferase reporter assays demonstrated that c-myb enhanced the luciferase activity of psicheck2-WT in a dose dependent manner, but not that of psicheck2-Mutant ( Fig. 4G and 4H), suggesting that c-myb enhanced IL2RA expression by directly binding to the promoter domain of IL2RA.
These data demonstrated that c-myb may suppress SLE progression by positively regulating IL2RA expression in the transcriptional level. To explore whether circLOC101928570 could regulate the expression of IL2RA, we transfected circLOC101928570 overexpression plasmid, circLOC101928570-NC, shRNA-circLOC101928570#1, shRNA-NC into jurkat cells. Upregulated circLOC101928570 increased the expression of IL2RA (Fig. 4I). Downregulation of circLOC101928570 resulted in decreased expression of IL2RA (Fig. 4J). These data indicated function of circLOC101928570 participating in regulating IL-2RA expression.

CircLOC101928570 suppresses IL2RA expressions in CD4+T cells subsets of SLE
We analyzed the cell cytokine expressions in the supernatant of PBMCs from different 55 SLE and 33 healthy groups by ELISA. IL-4 expression was increased in SLE groups compared with healthy groups (Fig. 5A). There was no signi cant difference in the IFN-γ expression in SLE patients compared with healthy groups (Fig. 5B). IL-4/IFN-γ level in SLE was higher than healthy groups (Fig. 5C). The IL-4/IFN-γ ratio of SLE was negatively correlation with the expression of circLOC101928570 (Fig. 5D). Meanwhile, the Treg/Th17 ratios negatively correlated with the expression of circLOC101928570 (Fig. 5E). We analyzed the Treg and Th17 percentages in PBMCs from different SLE and healthy groups by ow cytometry (Fig. 5F and 5G). By a linear regression analysis, the ratio of Th1/Th2 and Treg/Th17 ratios were correlated with the expression of circLOC101928570. Next, We analyzed the expression of IL2RA on Th1, Th2, Tc1 and Tc2 in the CD4+ T cell populations between SLE patients and healthy controls by ow cytometry. The results demonstrated that the expression of IL2RA on Th1, Th2, Tc1 and Tc2 in SLE patients was lower than healthy groups (Fig. 5H and 5I). We analyzed the percentages IL2RA of Th1, Th2, Tc1 and Tc2 cells in PBMCs from different SLE and healthy groups by ow cytometry (Fig. 5J and 5K). In this study, we screened out a downregulated circRNA named circLOC101928570 according to the results of RNA-seq and qRT-PCR analysis. We con rmed that circLOC101928570 expression was downregulated in SLE patients compare with healthy controls, and the expression of circLOC101928570 was correlated with disease activity of SLE. Furthermore, we investigated the function and mechanism of circLOC101928570. Bioinformatics analysis showed that circLOC101928570 binds to miR-150-3p/5p. Luciferase reporter and RIP assays con rmed the direct interaction between circLOC101928570 and miR-150, suggesting the circLOC101928570 functions as a "miRNA sponge" of miR-150. Previous studies have demonstrated the pathogenic role of miR-150 in SLE. MiR-150 was identi ed to be with positively correlated with chronicity scores and the expression of pro brotic proteins in lupus nephritis patients. of early apoptosis of Jurkat cells. Next, we identi ed that c-myb is interacted with IL2RA by bioinformatics analysis and Dual luciferase reporter assay, which suggesting that circRNAs is implicated in numerous aspects of post-transcriptional of mRNA. Moreover, we found that circLOC101928570 expression is negative correlation with IL-4/IFN-γand Treg/Th17 ratios in SLE patients. The expression of IL2RA on Th1, Th2, Tc1 and Tc2 of CD4+ T cell populations in SLE patients was also signi cantly lower than healthy groups. Which may show that circLOC101928570 may affect the cytokine expressions and the differentiation of T cell in SLE.
IL2RA is a subunit of the high a nity receptor for interleukin-2 (IL2). The gene locus of IL2RA has been ascertained as risk factor for a diverse series of autoimmune diseases including SLE. The IL-2 pathway is critical for the maintenance of immune homeostasis. IL-2 signaling plays a role in activation-induced cell death and is vital to regulatory T cell homeostasis (Carr et al. 2009;Huang et al. 2012). In our study, we found that the expression of IL2RA on Th1, Th2, Tc1 and Tc2 of CD4+ T cell populations in SLE patients was also signi cantly lower than healthy groups. C-myb transcriptionally regulating IL2RA expression by binding to IL2RA. These data indicated circLOC101928570 may in uence IL2RA expression in CD4+T cells subsets of SLE.   Scale bar: 10µm. IgG: immunoglobulin G. All data are presented as the means±SD (n=3). *P < 0.1, **P < 0.01, ***P < 0.001. control shRNA (NC-shRNA). J, K The percentage of cells of apoptosis was signi cantly increased in the shRNA-circLOC101928570#1 group, signi cantly decreased in the circLOC101928570 group. Results are represented as mean ± SD (n=3). ***P < 0.001.   Results are represented as mean ± SD (n=3). NS: no signi cance. ***p < 0.001.