The Role Of MicroRNA-1246in The Regulation Of B Cell Activation And The Pathogenesis Of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease which affects multiple organ systems and causes significant morbidity and mortality. One of the hallmarks of SLE is the production of anti-nuclear autoantibodies by uncontrolled overactivated B cells. The autoantibody-autoantigen immune complexes can deposit in different tissues and organs, leading to chronic inflammation and tissue damage in many parts of the body. It has been widely reported that complex interactions between genes, the environment, hormones, smoking, infections, drugs, and abnormalities in the adaptive immune system may contribute to the onset and progression of SLE. In recent years, several studies have shown that aberrant epigenetic mechanisms also play an important role in the pathogenesis of SLE.MicroRNAs (miRNAs), having a length of approximately19-25nucleotides, are one of the principal epigenetic regulatory mechanisms which have been identified as a large, novel sub-class of endogenous noncoding small RNAs (ncRNAs)[18]. miRNAs can regulate protein-coding genes post transcription by guiding a protein complex known as the RNA-induced silencing complex (RISC) to bind to the3’-untranslated region (3’-UTR) of target messenger RNAs (mRNAs). This inhibits protein translation and promotes mRNA degradation. Current estimates suggest that through this post-transcriptional gene silencing, miRNAs can regulate at least60%of human protein-coding genes. Recent studies have shown that miRNAs have a central role in the regulation, development, and function of immune system and could potentially serve as disease biomarkers and therapeutic targets. It has been reported that several miRNAs including miR-155, miR-146a, miR-326, miR-23b, miR-126, miR-142-3p/5p, miR-146a, miR-182, miR-150, and miR-124a modulate the pathogenesis of autoimmune diseases such as SLE, rheumatoid arthritis, and multiple sclerosis through their effects on T and B cell functions. Nevertheless, the mechanisms by which miRNA dysregulation contributes to the pathogenesis of autoimmune diseases such as SLE have not yet been completely investigated.At present, miR-1246has only been found in the ape and human genomes. In humans, its gene is located on chromosome two (2q31.1)^Some recent studies have reported an association between aberrant miR-1246expression and p53regulation, thereby explaining its role in the pathogenesis of cancer and Down syndrome. In response to DNA damage, p53induces miR-1246expression, which then reduces the level of DYRK1A, a Down syndrome-associated protein kinase. In addition, a p53-miR-1246-DYRK1A-NFAT pathway has been proposed in cancer pathogenesis. It is also reported that serum miR-1246has a strong potential to serve as a novel diagnostic and prognostic biomarker in Oesophageal squamous cell carcinoma. However, few studies have been done to identify the role of miR-1246in the pathogenesis of autoimmune diseases. Previous miRNA microarray studies by our group revealed that miR-1246was reduced to less than half in B cells of SLE patients compared to B cells of healthy controls.In this study, we confirm the expression patterns of miR-1246using real-time RT-PCR and investigate their involvement in SLE pathogenesis. We confirmed that abnormal activation of the Akt signaling pathway is associated with downregulation of P53expression which inhibits miR-1246expression. We discovered that miR-1246specifically targets EBF1mRNA by interacting with its3’UTR. Tansfection of miR-1246inhibitors into healthy B cells led to the upregulation of EBF1. Furthermore, inhibiting miR-1246expression in healthy B cells increased B cell function and enhanced IgG production. On the other hand, upregulation of miR-1246expression in over-activated B cells led to downregulation of EBF1levels, reduced B cell activity, and decreased IgG production. We therefore observed that through regulation of Ebfl expression, miR-1246can alter Akt phosphorylation levels in the BCR signal transduction pathway, which leads to regulation of p53protein expression levels and hence bring forth a further step in changing miR-1246expression levels. These studies and observations reveal that under-expression of miR-1246may be an important molecular mechanism that leads to B cell over-activation in SLE. We thus provide a theoretical framework towards the search of new and effective biological targets in SLE treatment. Part I The regulation of miR-1246expression by Akt signaling in B cells from SLE patientsSection I The expression levels of miR-1246, P53and Akt in B cells from SLE patientsObjective:To investigate whether there is abnormal miR-1246expression in B cells from SLE patients and the possible molecular mechanisms involved.1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients and healthy controls by density gradient centrifugation. B cells were isolated using microbeads and protocols provided by the manufacturer.2. Total RNA and protein were isolated with RNA isolation kits or protein isolation kits.3. Real-time polymerase chain reaction (Real-time PCR) was done*to determine miR-1246and P53mRNA expression levels.4. Akt protein and phosphorylation levels and P53protein levels were detected using western blot.Results:In comparison with healthy controls, miR-1246was significantly downregulated (p<0.001) in B cells from SLE patients. P53mRNA and protein levels were also significantly downregulated while Akt protein phosphorylation was markedly increased in B cells from SLE patients compared to controls (P<0.05or P<0.01). Akt protein phosphorylation levels were negatively correlated with P53protein expression levels (P<0.001) while P53mRNA and protein expression levels were positively correlated with miR-1246expression levels in B cells from SLE patients (P<0.05or P<0.001)Conclusion:Since p53protein expression is directly correlated with miR-1246expression, reduced miR-1246levels in B cells of SLE patients may be due to inhibition of P53protein expression by Akt phosphorylation. Section II Effects of Akt signaling pathway activation and inhibition on the expression of MIR-1246and P53Objective:To investigate whether activation or inhibition of Akt signaling affects MIR-1246and P53expression.Methods:1. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral venous blood of three healthy controls and three active SLE patients by density gradient centrifugation. B cells were isolated using microbeads and protocols provided by the manufacturer.2. Akt activator or the negative control were added to B cells from healthy controls incubated in1640culture medium. Akt inhibitor or the negative control were added to B cells from SLE patients incubated in1640culture medium.3. RNA and protein were isolated with RNA isolation kits or protein isolation kits.4. Real-time polymerase chain reaction (Real-time PCR) was done to determine the expression levels of miRNA and P53mRNA.5. Total Akt protein, Akt phosphorylation levels, and P53protein levels were detected by western blotting.Results:Compared to control-treated B cells from healthy controls, we observed significantly increased Akt protein phosphorylation levels, decreased P53mRNA and protein levels, and downregulated miR-1246expression in Akt-activated B cells from healthy controls (p<0.01). Compared to control-treated B cells from SLE patients, we observed significantly decreased Akt protein phosphorylation, increased P53mRNA and protein levels, and upregulated miR-1246expression in Akt-inhibited B cells from SLE patients (p<0.01).Conclusion:Akt activation can inhibit P53expression which results in impaired MIR-1246expression in B cells. On the other hand, Inhibition of Akt can enhance P53expression and hence up-regulate MIR-1246expression in B cells. Part Ⅱ Expression of miR-1246target genes in B cells of SLE patientsSection I Verification of miR-1246target genesObjective:To verify that Ebfl is the target gene of miR-1246using the reporter gene system.Methods:1. Bioinformatic algorithm softwares such as TargetScan, PicTar, and miRBase were used to find the potential target genes of miR-1246.2. A fragment sequence from the3’UTR of the target gene containing putative miRNA binding sites was amplified by human B cell genomic DNA PCR. The same procedure was used to generate reporter constructs with mutations in the3’UTR of the target gene. The3’UTR sequences were inserted into pMIR-REPORT luciferase miRNA Expression Reporter Vector (Ambion, USA) using Hind III and Spe I.3. We constructed a firefly luciferase reporter plasmid fused downstream to a segment of the Ebfl3’-UTR containing either the wild-type putative miR-1246binding sequence (EbWT-luciferase), or the miR-1246binding sequence containing three point mutations (CD EbflMut-luciferase).4. The constructs with negative control or mimic were then transiently co-transfected into Jurkat cells by electroporation.5. After48hours, firefly luciferase activity was measured using a luminometer and the Dual-Luciferase reporter assay system. Renilla luciferase was used as an internal control.Results:1. According to the bioinformatic softwares TargetScan and miRBase, Ebfl is the target gene of miR-1246.2. Co-transfection of EbflWT-luciferase and miR-1246mimic into Jurkat cells led to inhibition of Ebfl-luciferase activity (p<0.05), but failed to inhibit EbflMut-luciferase activity.Conclusion:Ebfl is the target gene of miR-1246. Through its effects on the3’-UTR, miR-1246can inhibit expression of its target gene Ebfl. Section II Expression of miR-1246target genes in SLE B cellsObjective:To investigate the expression levels of miR-1246target genes in B cells from SLE patients.Methods:1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of healthy donors and SLE patients by density gradient centrifugation. B cells were isolated using microbeads and protocols provided by the manufacturer.2. Total protein was extracted using protein isolation kits.3. Concentration of Ebfl protein was detected by western blot.Results:Expression of the Ebfl protein was significantly higher in B cells of SLE patients compared to healthy controls (P<0.01). In addition, Ebfl expression was negatively correlated with miR-1246expression levels in B cells from SLE patients (r=-0.82, P<0.001).Conclusion:Ebfl protein expression is upregulated and negatively correlated with miR-1246expression in B cells of SLE patients compared to healthy controls.Part III Abnormal miR-1246expression and autoimmunityObjective:To investigate the effects of changing miR-1246expression patterns on Ebfl expression, activity of the Akt signaling pathway, and autoimmune responses in B cells.Methods: 1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of three SLE patients and three healthy donors by density gradient centrifugation. B cells and CD4+T cells were isolated using microbeads and protocols provided by the manufacturer.2. miR-1246inhibitor or negative control were transfected into normal B cells by transient electroporation; miR-1246mimic or negative control were transfected into SLE B cells by transient electroporation.3. Concentrations of IgG in the supernatant were measured by the TB enzyme-linked immunosorbent assay (ELISA) method.4. Flow cytometric analyses were performed to determine the levels of CD40, CD80, and CD86proteins on B cell membranes.5. Total RNA and protein were isolated with RNA isolation kits or protein isolation kits.6. Real-time polymerase chain reaction (Real-time PCR) was done to determine the expression of miRNA,P53mRNA,CD40mRNA,CD80mRNA and CD86mRNA.7. Akt total protein and phosphorylation levels, including Ebfl and P53protein levels were detected by western blotting.Results:1. In comparison with B cells transfected with the negative control, the expression of miR-1246was decreased while the expression of Ebfl protein was significantly upregulated in B cells transfected with the miR-1246inhibitor (P<0.05). We also observed significantly increased CD40, CD80, and CD86mRNA and protein levels in miR-1246deficient cells, as well as increased Akt protein phosphorylation and higher IgG levels. Furthermore, P53mRNA and protein levels were significantly decreased (P<0.05or P<0.01)2. In comparison with B cells transfected with the negative control, expression of miR-1246was significantly increased while Ebfl expression protein was significantly downregulated in B cells transfected with the miR-1246mimic (P<0.05). We also observed significantly decreased CD40, CD80, and CD86mRNA and protein levels in cells with over-expressed miR-1246, as well as decreased Akt protein phosphorylation level and lower IgG levels. Furthermore, P53mRNA and protein levels were significantly increased (P<0.05or P<0.01)Conclusion:Inhibiting miR-1246expression in healthy B cells can increase Ebfl expression, enhance activity of the Akt signaling pathway, downregulate p53expression, and therefore promote B cell hyper-responsiveness. Enhancing miR-1246expression in B cells of SLE patients can decrease Ebfl expression, inhibit the Akt signaling pathway upregulate P53expression levels, inhibit antibody production, and therefore disable autoimmune responses.