The Role Of MicroRNA-126 In The Regulation Of T Cell DNA Methylation And The Pathogenesis Of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic and potentially fatal autoimmune disorder characterized by T lymphocyte autoreactivity and the production of autoantibodies that cause widespread tissue damage. Although the mechanisms that initiate these manifestations remain unclear, it has been widely reported that epigenetic factors play a central role in the onset and progression of SLE.DNA methylation is an epigenetic regulator of several biological processes, including embryonic development, gene transcription, X chromosome inactivation, genomic imprinting and chromatin modification. DNA methylation involves the addition of a methyl group to the pyrimidinyl ring of a cytosine, primarily within CpG pairs, and is catalyzed by DNA methyltransferases (DNMTs). Methylation of CpG islands in promoter regulatory regions is associated with transcriptional inactivation of the corresponding gene, while demethylation of the same region leads to transcriptional activation. Treating murine T cells with DNA demethylating drugs can induce lupus-like autoreactivity in vitro, and lupus-like symptoms if the cells are injected back into host mice. T cells from patients with active lupus have genome-wide decreases in deoxymethylcytosin and gene-specific hypomethylation. These methylation deficits correlate with upregulated expression of autoimmune-related genes such as CD11a (ITGAL), perforin (PRF1), CD70 (TNFSF7) and CD40 ligand (TNFSF5). Overexpression of these genes results in T cells autoreactivity, spontaneous monocyte/macrophage killing, B cells immunoglobulin overproduction. Recent studies have demonstrated an association between DNA hypomethylation in SLE and a decrease in the enzymatic activity of DNMTs, especially DNMT1, suggesting a mechanism by which T cell DNA becomes hypomethylated. However, the molecular mechanism of T cell DNA hypomethylation in SLE is unclear.MicroRNAs (miRNAs) are endogenous 21-25 nucleotide non-coding RNA molecules that regulate the expression of target genes by specifically binding to and interfering with their messenger RNAs (mRNAs). Recent studies have shown that miRNAs are involved in various immune responses and are associated with autoimmune disease, and could potentially serve as diagnostic biomarkers or therapeutic targets. In the present study, we compared miRNA expression profiles from CD4+T cells of SLE patients with those of healthy controls by microarray.The upregulation of one of these genes, miR-126, was confirmed by stem-loop RT-PCR and its involvement in SLE was studied. Therefore, we propose the pathogenesis of SLE as follows:overexpression of miR-126 can decrease methylation level at regulatory sequence of autoimmunity-related genes in T cell, results in upregulated expression of the genes, then leads to spontaneous monocyte/macrophage killing and overstimulation of IgG synthesis, finally causes SLE. To prove the hypothesis, we used the following approach:firstly, to validate the target gene of miR-126 and examine the effect of upregulated and downregulated expression of miR-126 on normal or SLE CD4+T cells; Secondly, to investigate the pathogenesis of miR-126 regulates DNA methyloation in SLE CD4+T cells. These studies would further reveal the mechanisms of demethylation of T cell genes in lupus, and provide a theoretical basis for more effective therapy of SLE.Objective:To predict the target genes of miR-126 according to the bioinformatic algorithm software and verify its role on target genes by using reporter gene systerm.Methods:Firefly luciferase expression vector was constructed to verify the relationship between miR-126 and DNMT1. Constructed a firefly luciferase reporter plasmid fused downstream to a segment of the Dnmtl 3'-UTR containing either the wild-type putative miR-126 binding sequence (Dnmt1WT-luciferase), or the miR-126 binding sequence containing two point mutations (Dnmt1Mut-luciferase). The constructs were then co-transfected into Jurkat cells with pSilencer-mir-126 or negative control construct pSilencer4.1CMV-negative by electroporation. After 48 hours firefly luciferase activity was measured using the Dual-Luciferase reporter assay system and luminometer. Renilla luciferase was used as internal control.Results:The SLE-associated methyltransferase enzyme DNMT1 is a predicted target of miR-126, according to the bioinformatic algorithm software, such as miRBase, TargetScan, PicTar. Co-transfection of pSilencer-mir-126 and Dnmt1WT-luciferase in Jurkat cells can inhibit the expression of Dnmt1 WT-luciferase activity (p< 0.05), but failed to inhibit Dnmt1Mut-luciferase activity.Conclusion:The prediction of the bioinformatic algorithm software suggests that DNMT1 is the corresponding target gene of miR-126 which can repress the mRNA translation by binding to the 3'-UTR of DNMT1. Objective:To investigate the effect of overexpression of miR-126 on expression and methylation status of autoimmunity related genes and autoreactivity in normal CD4+T cells.Methods:miR-126 expressed plasmid (psilencer4.1-CMV-miR-126) and control plasmid (psilencer4.1-CMV-negative) were constructed, then transfected into normal human CD4+T cells by transient electroporation. Real-time RT-PCR was used to detect the expression of miR-126, DNMT1, CD11a and CD70 mRNA. CD11a and CD70 protein levels were measured by flow cytometry. DNMT1 protein level was assessed by Western Blotting. Detection of IgG antibody productions were performed following the protocol of kits. The methylation status of CD11a (ITGAL) and CD70 (TNSFN7) promoter regulatory elements were detected by sodium bisulfite sequencing.Results:Compared with CD4+T cells transfected control plasmid,the expression of miR-126 CD11a and CD70 mRNA was upregulated in CD4+T cells transfected with psilencer4.1-CMV-miR-126 (P<0.05). The expression of DNMT1 protein, but not mRNA, was significantly decreased in CD4+T cells transfected with psilencer4.1-CMV-miR-126 (P<0.05). Increased IgG production (P<0.01) and enhanced CDlla and CD70 expression by flow cytometric were also found in CD4+T cells transfected with psilencer4.1-CMV-miR-126 (P<0.05). The average methylation levels of CpG pairs in ITGAL promoter sequence (-1263 to-1111) and TNSFN7 promoter sequence (-517 to-343) was significantly reduced (P<0.05, P<0.05) in CD4+T cells transfected with psilencer4. 1-CMV-miR-126.Conclusion:Overexpression of miR-126 can upregulate expression of autoimmunity related genes, and increase autoreactivity in CD4+T cells.Objective:To investigate the effect of downregulating miR-126 expression on expression and methylation status of autoimmunity related genes, and autoreactivity in SLE CD4+T cells.Methods:miR-126-inhibitor was transfected into SLE CD4+T cells by transient electroporation. Real-time RT-PCR was used to detect the expression of miR-126, DNMT1 and autoimmunity related genes CD11a and CD70 mRNA. CD11a and CD70 protein levels were measured by flow cytometry. DNMT1 protein level was assessed by Western Blotting. Production of IgG antibody were performed following the protocol of kits. The average methylation status of ITGAL and TNSFN7 promoter regulatory elements were detected by the sodium bisulfite sequencing.Results:Compared with control group, the expression of miR-126, CD11a and CD70 mRNA were significantly downregulated in SLE CD4+ T cells transfected with miR-126-inhibitor (P<0.05). The expression of DNMT1 protein, but not mRNA, was obviously increased in CD4+T cells transfected with miR-126-inhibitor (P<0.05). Percent of CD11a staining cells and CD70 were decreased in CD4+T cells transfected with miR-126-inhibitor (P<0.05). Reduced IgG production was also found in SLE CD4+T cells transfected with miR-126-inhibitor (P<0.01). The average methylation status of CpG pairs at ITGAL promoter sequence (-1263 to-1111) and TNSFN7 promoter sequence (-517 to-343) was significantly increased (P<0.05, P<0.05) in SLE CD4+T cells transfected with miR-126-inhibitor.Conclusion:Downregulation of miR-126 expression can inhibit expression of autoimmunity related genes, and reduce autoreactivity in SLE CD4+T cells.Objective:To explore the mechanisms by which miR-126 is upregulated in SLE CD4+T cells.Methods:we analyzed the expression and the DNA methylation status within promoter region of EGFL7 by Real-time PCR and bisulfite genomic sequencing analysis.Results:EGFL7 mRNA expression was upregulated in SLE CD4+T cells compared with healthy controls (p<0.05), and positively correlated with miR-126 expression in SLE CD4+T cells (R=0.538, p=0.015). The average methylation level of EGFL7 in SLE CD4+T cells was lower than in healthy controls (p<0.05).Conclution:The upregulation of miR-126 and its host gene EGFL7 in SLE CD4+T cells is associated with the hypomethylation of the EGFL7 promoter.