Background
Systemic lupus erythematosus (SLE) is a chronic and incurable autoimmune disease, which involves multiple organs, including skin, kidneys, and central nervous system [
1,
2]. In recent years many studies have been dedicated to elucidating the pathogenesis of SLE, which is still unknown. A very significant pathophysiological feature of SLE is the dysfunction of T cells, B cells, and dendritic cells (DC) [
3‐
6], and research on the dysfunction of immune cells through SLE progression is a hot topic.
Circular RNAs (circRNAs) are noncoding RNAs (ncRNAs) with a covalently closed loop structure without 5′ cap and a 3′ polyadenylated tail. They are highly stable and widely exist in eukaryotic cells. They do not usually encode protein, but can occur in any genomic region, regulating gene expression in eukaryotes. Recently circRNAs were found to be enriched with functional microRNA (miRNA) binding sites and could act as miRNA sponges to regulate gene expression [
7,
8]. Furthermore, more and more studies have demonstrated that circRNAs are expressed abnormally in many human diseases and might play a significant role in the pathogenesis and diagnosis of these diseases [
9‐
13].
DNA methylation is abnormal in T cells and B cells from patients with SLE. Besides global hypomethylation on lupus T and B cells, the gene-specific and site-specific methylation has been identified to be responsible for SLE. Aberrant DNA methylation plays an important role in the initiation and development of SLE and provides an insight into the related diagnosis biomarkers and therapeutic options in SLE [
14,
15]. Some studies showed that the PI3K/AKT signaling pathway played a role in the differentiation of peripheral B cell and T cell homeostasis [
16‐
19]. Many researchers also found there is abnormal activation of the PI3K/AKT signaling pathway in SLE and they thought this might participate in the pathogenesis of SLE. For example, in MRL-lpr lupus models, CD4+ T cells show higher levels of AKT activation than in wild-type mice [
20]. Anomalous activity of AKT kinases has been documented in peripheral blood B cells and T cells from patients with SLE [
21,
22]. Rapamycin has been successfully used to target the AKT/mTOR axis for treatment of patients with SLE [
3].
In this study, we characterized a new circRNA, hsa_circ_0077179, which was derived from the IBTK gene locus, termed circIBTK, and which was downregulated in patients with SLE. We further estimated the value of circIBTK as a biomarker in SLE and studied the role of circIBTK in the DNA hypomethylation and abnormal activation of the AKT signaling pathway.
Methods
Patients and controls
This research was approved by the Institutional Research Ethics Committee of Shanghai General Hospital and abided by the ethical guidelines of the Declaration of Helsinki. Informed consent was obtained from all the patients involved in this study. The diagnosis of SLE was established based on the 1997 revised American Rheumatism Association criteria and all the patients were diagnosed with SLE for the first time or without treatment with glucocorticoid or immunosuppressive agents for one month. Clinical data used in this research on 42 patients and 35 age-matched and gender-matched healthy controls from Shanghai, China are summarized in Additional file
1: Table S1.
Cell isolation and culture
Whole blood (10 ml) was collected in EDTA collection tubes from each subject, and human peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Biosciences) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum at 37 °C with 5% CO2 for 24 h before transfection.
CircRNA sequencing analysis
Total RNA was extracted from PBMCs using Trizol (Invitrogen, Carlsbad, CA, USA) according to the instructions of the manufacturer. Before sequencing, RNAs were digested with Rnase R (Epicentre Technologies, Madison, WI, USA) to remove the linear RNAs and enrich the circular configuration. The sequencing analysis was performed on Illumina HiSeq2000 Platform (Illumina Inc., San Diego, CA, USA).
Nuclear-cytoplasmic fractionation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using the DNA/RNA Isolation Kit (DP422, Tiangen Biotech, Beijing, China) according to the instructions supplied by the manufacturer. The nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Total RNA from the nuclear and cytoplasmic fractions was isolated with Trizol (Invitrogen) according to the instructions provided by the manufacturer. After reverse transcription, complementary DNA (cDNA) was amplified by using SYBR-Green Premix (Takara, Otsu, Japan). MiR-29b expression levels were detected using the Hairpin-itTM Quantitation PCR Kit (GenePharma, Shanghai, China). The expression of miR-29b and nuclear circIBTK were normalized to the expression of U6 and the expression of IBTK and cytoplasmic circIBTK were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data were analyzed using the delta cycle threshold (Ct) method. Primers for circIBTK were designed by Genechem (Shanghai, China). The primers for IBTK were forward, 5’CTTACATGTCTGCTGCTTTTGG-3′; reverse, 5′- GAGACACATAAGCAATTCACTGC-3′. The primers for GAPDH were forward, 5′- GAAGATGGTGATGGGATTTC-3′; reverse, 5’-GAAGGTGAAGGTCGGAGT-3′. The primers for U6 were forward, 5’-TCGCTTCGGCAGCACATA-3′; reverse, 5’-TTTGCGTGTCATCCTTGC-3′.
Transfection and luciferase reporter assay
MiR-29b mimics, miR-29b inhibitor and NC oligonucleotides were obtained from GenePharma. CircIBTK expression plasmids were designed by Genechem. SiRNA for circIBTK: sequence, 5’-GGAAUUUCCUUGUCAUAAAUG-3′, anti-sequence, 5’-UUUAUGACAAGGAAAUUCCUU-3′. Oligonucleotides were transfected by Hiperfect transfection reagent (Qiagen, Valencia, CA, USA) and plasmids were transfected by Lipofectamine 3000 (Invitrogen) into cells. All cells were incubated for 48 h afterwards. For circIBTK and miR-29b luciferase reporter assay, the circIBTK sequences containing wild-type miR-29b predicted binding sites were inserted into the region directly downstream of a cytomegalovirus (CMV) promoter-driven firefly luciferase cassette in a pCDNA3.1 vector. For PTEN 3’ UTR and miR-29b luciferase reporter assay, the PTEN 3’ UTR sequences containing two wild-type miR-29b predicted binding sites were inserted into the region directly downstream of a CMV promoter-driven firefly luciferase cassette in a pCDNA3.1 vector. Mutant reporter plasmids were prepared using Mutagenesis Kit (Stratagene, La Jolla, CA, USA). All constructs were verified by sequencing. PBMCs were seeded into 96-well plates and were co-transfected with a mixture of 30 ng of firefly luciferase reporter, 5 ng of pRL-TK Renilla luciferase reporter, and miRNA mimics or inhibitor. After 48 h of incubation, the firefly and Renilla luciferase activities were quantified using the Dual Luciferase Assay System (Promega, Madison, WI, USA).
Western blot analysis
Western blot analysis was carried out using standard procedures. Cells were lysed using radioimmunoprecipitation (RIPA) lysis buffer (Boster, Wuhan, China). Protein concentrations were detected using bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Antibodies used in the assays were β-actin (number 3700; Cell Signaling Technology, Danvers, MA, USA), AKT (pan) (number 4691; Cell Signaling Technology), phospho-AKT (Ser473) (number 4060; Cell Signaling Technology), PTEN (number 9552; Cell Signaling Technology).
DNA extraction and global methylation analysis
Genomic DNA was extracted from PBMCs using the DNA/RNA Isolation Kit (Tiangen Biotech) according to the instructions provided by the manufacturer. Global DNA methylation status in DNA samples was detected using the Methylflash Methylated DNA Quantification Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer’s recommended protocol.
Statistical analysis
Statistical analysis was performed using the SPSS program (version 18.0; SPSS, Chicago, IL, USA). The statistical significance of differences between two groups was tested using Student’s t test or the chi square (χ2) test. Multiple comparisons were performed using one-way analysis of variance (ANOVA) followed by the Newman–Keuls test. Spearman’s analysis was used to test correlation. P < 0.05 was considered as statistically significant.
Discussion
Recently, more and more research has focused on the function of circRNAs in many human diseases, while the expression profile and function of circRNAs in SLE remain unclear. In this study, we identified many circRNAs in SLE and found that some of these were differentially expressed in PBMCs from patients with SLE compared with healthy controls, which suggested that these RNAs might be regulated and exert a potential function.
CircIBTK, which was derived from the IBTK gene locus, was downregulated in patients with SLE. Bruton’s tyrosine kinase (BTK) is a member of the Tec family of non-receptor protein tyrosine kinases and a downstream signaling molecule of the B cell antigen receptor (BCR) signaling pathway, involved in the development, activation, and survival of B cells. Some studies have proved that BTK plays a significant role in the initiation and development of SLE. For example, transgenic mice over expressing BTK specifically in B cells produce antinuclear antibody and develop lupus-like symptoms [
26]. Furthermore, when BTK inhibitor was used to treat the lupus mice, it alleviated damage in the kidney, and the production of autoantibody [
27‐
29]. In humans, higher BTK expression in B cells from peripheral blood is associated with lupus nephritis [
30]. Inhibitor of Bruton’s tyrosine kinase (IBTK) is an inhibitor of BTK that can bind to the PH domain of BTK to downregulate BTK kinase activity, BTK-mediated calcium mobilization, and nuclear factor kappa B-driven transcription [
31]. We showed that the mRNA level of IBTK was downregulated in PBMCs from patients with SLE and this indicated that IBTK might be regulated or exert a potential function in SLE, which needed further exploration.
CircRNAs can act as potential biomarkers for disease and we showed that circIBTK is a marker of SLE. In this study, circIBTK was downregulated in SLE and the expression of circIBTK was strongly inversely correlated with the SLEDAI score. This revealed that the expression of circIBTK could help estimate the activity of SLE. Furthermore, circIBTK expression was notably increased when patients received efficacious treatment. So, circIBTK expression might indicate the effectiveness of treatment for SLE. What is more, the ROC curve also indicated that circIBTK might act as a potential diagnostic biomarker of SLE. We revealed the important role of circIBTK in SLE in this study.
In this study, we found that circIBTK might function as a miR-29b sponge. And we used luciferase reporter assays to verify this prediction. In patients with SLE compared to healthy controls, miR-29b levels were proved to be upregulated in CD4+ T cells [
23]. We also proved that miR-29b levels were upregulated in PBMCs from patients with SLE compared to healthy controls. Like circIBTK, miR-29b also had the potential function of helping diagnose and estimate the activity and effectiveness of treatment of SLE. According to some studies, miR-29b could induce DNA demethylation via various ways [
23‐
25]. Our results showed that miR-29b could induce DNA demethylation in SLE and miR-29b expression was inversely correlated with DNA methylation in SLE. What is more, expression of PTEN was decreased in SLE and the level of PTEN expression was inversely correlated with disease activity [
32]. Anomalous activity of AKT kinases has been documented in peripheral blood B cells and T cells from patients with SLE [
21,
22], and this may participate in the progress of SLE. We also proved that miR-29b could activate the AKT signaling pathway by suppressing PTEN expression by directly targeting PTEN. These results indicated that miR-29b could play a significant role in the pathogenesis of SLE, and might be a useful therapeutic target in SLE. Our data also demonstrated that circIBTK could inhibit DNA demethylation and activation of the AKT signaling pathway via the regulation of miR-29b in SLE. As DNA demethylation and activation of the AKT signaling pathway are critical for the initiation and development of SLE, and circIBTK and miR-29b could regulate both DNA demethylation and activation of the AKT signaling pathway, this explains why circIBTK and miR-29 correlated with clinicopathological variables in patients with SLE and act as biomarkers of SLE.
Conclusions
In conclusion, circIBTK and miR-29b were abnormally expressed in PBMCs from patients with SLE and could regulate DNA methylation and activation of the AKT signaling pathway in PBMCs in SLE. Our study explained the important role of circIBTK and miR-29 in SLE progression and suggested that circIBTK and miR-29 could act as biomarkers and therapeutic targets for SLE.
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