The Role Of MicroRNA-142-3p/5p In The Regulation Of T Cell Activation And The Pathogenesis Of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by uncontrolled T cell overactivation that triggers inflammation and tissue damage in many parts of the body. Although the molecular mechanisms that regulate the onset and progression of SLE remain unclear, it has been widely reported that multiple epigenetic mechanisms contribute to its pathogenesis, in addition to various genetic factors.MicroRNAs (miRNAs) are one of the principal epigenetic regulatory mechanisms. They are endogenous21-25nucleotide non-coding RNA molecules that are potent negative modulators of genes involved in several cellular processes. MiRNAs can regulate the expression of target genes by binding to the3'-untranslated region (3'-UTR) of target messenger RNAs (mRNAs), leading to their degradation or translational repression. To date, approximately one thousand miRNAs have been identified, which are predicted to regulate at least one-third of protein coding transcripts in the mammalian genome. Recent studies have shown that miRNA regulate the function of both the innate and the adaptive immune system, are involved in various immune pathways, and could potentially serve as disease biomarkers and therapeutic targets. Altered miRNA expression has been reported in human autoimmune diseases including SLE, rheumatoid arthritis and multiple sclerosis. However, how miRNA dysregulation contributes to the pathogenesis of autoimmune diseases such as SLE has not been thoroughly investigated.Has-mir-142is a T cell-specific miRNA known to play a role in regulating T-cell development. The has-mir-142locus produces two transcripts:miR-142-5p which is expressed from the5'arm of the locus and miR-142-3p expressed from the3'arm. Previous miRNA microarray studies by our group revealed that miR-142-3p and miR-142-5p were reduced to less than half in SLE CD4+T cells compared with healthy control CD4+T cells. In this study, we have confirmed the expression patterns of miR-142-3p and miR-142-5p using real-time RT-PCR and investigated their involvement in SLE pathogenesis. We discovered that miR-142-3p specifically targets CD84mRNAs which are members of the signaling lymphocytic activation molecule (SLAM) family; and that miR-142-5p specifically targets SAP (SLAM-associated protein) mRNA by interacting with its3'UTR. Tansfection of miR-142-3p/-5p inhibitors into healthy CD4+T cells led to the upregulation of CD84and SAP. Furthermore, inhibiting miR-142-3p/5p expression in healthy CD4+T cells increased T cell function and promoted IgG production in co-cultured B cells. On the other hand, upregulated miR-142-3p/-5p expression in SLE CD4+T cells led to restored CD84and SAP levels, reduced T cell activity, and decreased IgG production. Furthermore, we observed that the decrease in miR-142-3p/5p expression may be mediated by the histone modifications and DNA methylation within the regulatory region500bp upstream of the miR-142precursor sequence. Together, these results provide novel insights into the mechanisms by which miRNA dysregulation contributes to the pathogenesis of SLE.Part Ⅰ Abnormal miR-142-3p/5p expression in CD4+T cells of SLE patients and validation of its target genesSection I Abnormal miR-142-3p/5p expression in SLE CD4+T cellsObjective:To investigate miR-142-3p/5p expression in SLE CD4+T cells.Methods:1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients and healthy donors by density gradient centrifugation. CD4+T cells were isolated using microbeads and protocols provided by the manufacturer.2. Total RNA were isolated with RNA isolation kits.3. cDNAs were synthesized using the miScript Reverse Transcription Kit4. Altered microRNA were confirmed by real-time polymerase chain reaction (Real-time PCR).Results:Both miR-142-3p and miR-142-5p were significantly downregulated (p<0.001, p<0.001) in CD4+T cells from SLE patients.Conclusion:miR-142-3p/5p were significantly downregulated in SLE CD4+T cells. Section II The validation of miR-142-3p/5p target genesObjective:To verify CD84and SAP are the target genes of miR-142-3p and miR-142-5p respectively by using reporter gene systerm.Methods:1. Use of bioinformatic algorithm software, such as miRBase, TargetScan, PicTar, to find potential target genes of miR-142-3p/5p;2. A fragment sequence from the3'UTR of the target gene containing putative miRNA binding sites was amplified by PCR from human CD4+T cell genomic DNA. The same procedure was used to generate reporter constructs with mutations in the3'UTR of the target gene.3'UTR sequences were inserted into pMIR-REPORT luciferase miRNA Expression Reporter Vector (Ambion, USA) using Spe I and Hind III.3. We constructed a firefly luciferase reporter plasmid fused downstream to a segment of the CD843'-UTR containing either the wild-type putative miR-142-3p binding sequence (CD84WT-luciferase), or the miR-142-3p binding sequence containing three point mutations (CD84Mut-luciferase). Then again constructed a firefly luciferase reporter plasmid fused downstream to a segment of the SAP3'-UTR containing either the wild-type putative miR-142-5p binding sequence (SAPWT-luciferase), or the miR-142-5p binding sequence having two point mutations (SAP Mut-luciferase). 4. The constructs were then co-transfected into Jurkat cells with mimic or negative control by electroporation.5After48hours firefly luciferase activity was measured using the Dual-Luciferase reporter assay system and luminometer. Renilla luciferase was used as an internal control.Results:1. According to the bioinformatic software TargetScan and miRBase, SLE-associated SLAM (signaling lymphocyte activation molecule) family member CD84is a predicted target of miR-142-3p, and SLAM-associated protein (SAP) is a predicted target of miR-142-5p.2. Co-transfection of miR-142-3p mimic and CD84WT-luciferase in Jurkat cells can inhibit the expression of CD84WT-luciferase activity (p<0.05), but failed to inhibit CD84Mut-luciferase activity.3. Co-transfection of miR-142-5p mimic and SAPWT-luciferase in Jurkat cells inhibited the expression of SAPWT-luciferase activity (p<0.05), but failed to inhibit SAPMut-luciferase activity.Conclusion:CD84and SAP are the corresponding target genes of miR-142-3p and miR-142-5p respectively. miR-142-3p/5p can repress mRNA translation of target genes by binding to their3'-UTR. Section Ⅲ The effect of changing miR-142-3p/5p expression patterns on the expression of target genes in CD4+T cellsObjective:To investigate the effect of changing miR-142-3p/5p expression patterns on the expression of target genes in CD4+T cells.Methods:1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients and healthy donors by density gradient centrifugation. CD4+T cells were isolated using microbeads and protocols provided by the manufacturer.2. miR-142-3p inhibitor, miR-142-5p inhibitor or negative controls were transfected into normal CD4+T cells by transient electroporation; miR-142-3p mimic, miR-142-5p mimic or negative controls were transfected into SLE CD4+T cells by transient electroporation.3. Total RNA and protein were isolated with RNA isolation kits or protein isolation kits.4.Altered microRNAs were confirmed by real-time polymerase chain reaction (Real-time PCR).5. CD84and SAP protein levels were detected using western blot.Results:1. In comparison with CD4+T cells transfected with the negative control, the expression of miR-142-3p was decreased in CD4+T cells transfected with miR-142-3p inhibitor (P<0.05). The expression of CD84protein, but not mRNA, was significantly upregulated in CD4+T cells transfected with miR-142-3p inhibitor (P<0.05).2. The expression of miR-142-5p was decreased and the expression of SAP protein was significantly upregulated in CD4+T cells transfected with miR-142-5p inhibitor (P<0.05).3. The expression of miR-142-3p was upregulated (P<0.05) and the expression of CD84protein was significantly decreased (P<0.05) in CD4+T cells transfected with miR-142-3p mimic.4. The expression of miR-142-5p was upregulated (P<0.05) and the expression of SAP protein was significantly decreased (P<0.05) in CD4+T cells transfected with miR-142-5p mimic.Conclusion:Downregulation of miR-142-3p/5p expression can upregulate expression of CD84/SAP in CD4+T cells; overexpression of miR-142-3D/5p can inhibit expression of CD84/SAP in SLE CD4+T cells Section IV Expression of miR-142-3p/5p target genes in SLE CD4+T cellsObjective:To investigate expression of miR-142-3p/5p target genes in SLE CD4+T cellsMethods:1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients and healthy donors by density gradient centrifugation. CD4+T cells were isolated using microbeads and protocols provided by the manufacturer.2. Total protein extracted using protein isolation kits.3. CD84and SAP protein levels were detected by western blot.Results:1. Expression of the CD84protein was significantly higher in CD4+T cells of SLE patients compared to healthy controls (P<0.01) and was negatively correlated with miR-142-3p expression level in SLE CD4+T cells (r=-0.621,P=0.003)2. Expression of the SAP protein was significantly higher in CD4+T cells of SLE patients compared to healthy controls (P<0.01) and was negatively correlated with miR-142-5p expression level in SLE CD4+T cells (r=-0.708, P=0.001)Conclusion:The CD84and SAP protein expressions were significantly higher in CD4+T cells of SLE patients compared to controls and were negatively correlated with miR-142-3p/5p expression level in SLE CD4+T cells. Part II Abnormal miR-142-3p/5p expression and autoimmunitySection I Inducing T cell autoreactivity by inhibiting miR-142-3p/5p expressionObjective:To investigate the effect of inhibiting miR-142-3p/5p expression on autoreactivity in CD4+T cells.Methods:1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4+T cells and B cell were isolated using microbeads and protocols provided by the manufacturer.2. CD4+T cells were transfected with a negative control, the miR-142-3p inhibitor, the miR-142-5p inhibitor, or both inhibitors.3. Flow cytometric analyses were performed to determine the levels of CD40L and ICOS proteins on CD4+T cell membranes.4. Concentrations of IL4, IL10and IL21in the supernatant were assessed by ELISA.5. CD4+T cell proliferation was measured with a cell proliferation enzyme-linked immunosorbent assay (ELISA) bromodeoxyuridine (colorimetric) kit.6. Conjugate frequencies of CD4+T cell and B cell were detected by flow cytometry. 7. concentrations of IgG in the supernatant were measured by ELISA.Results:Compared to control-transfected CD4+T cells, we observed significantly increased IL4, IL10and IL21protein levels in miR-142-3p and/or-5p deficient cells, as well as increased CD40L and ICOS protein expression and higher cell proliferation. Furthermore, cells collected from these three groups showed higher CD4+CD19+conjugates than the controls, and significantly higher rates of IgG synthesis.Conclusion:Inhibiting miR-142-3p/5p expression in healthy CD4+T cells increases CD4+T cell function and promotes B cell hyper-responsiveness. Section Ⅱ Inhibiting autoreactivity of T cells by overexpression of miR-142-3p/5pObjective:To investigate whether overexpression of miR-142-3p/5p can inhibit autoreactivity of CD4+T cells from SLE patients.Methods:1. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients by density gradient centrifugation. CD4+T cells and B cell were isolated using microbeads and protocols provided by the manufacturer.2. CD4+T cells were transfected with the negative control, the miR-142-3p mimic, the miR-142-5p mimic, or both mimics.3. Flow cytometric analyses were then performed to determine the levels of CD40L and ICOS proteins on CD4+T cell membranes.4. Concentrations of IL4, IL10and IL21in the supernatant were assessed by ELISA.5. CD4+T cell proliferation was measured with a cell proliferation enzyme-linked immunosorbent assay (ELISA) bromodeoxyuridine (colorimetric) kit.6. Conjugate frequencies of CD4+T and B cells were detected by flow cytometry.7. concentrations of IgG in the supernatant were measured by ELISA.Results:Compared to control-transfected CD4+T cells, we observed significantly decreased IL4, IL10and IL21protein levels in SLE CD4+T cells transfected with the mir-142-3p and/or mir-142-5p mimic, as well as reduced CD40L and ICOS protein expression and decreased cell proliferation. Furthermore, cells collected from these three groups exhibited lower levels of CD4+CD19+conjugates than the controls, and also had significantly decreased IgG production. Conclusion:Overexpression of miR-142-3p/5p in SLE CD4+T cells decreases CD4+T cell function and inhibits antibody production. Part III Abnormal histone modification patterns and DNA methylation status in the MIR-142regulatory region of SLE CD4+T cellsSection I Abnormal histone modification patterns in the MIR-142regulatory region of SLE CD4+T cellsObjective:To explore histone methylation status in the MIR-142regulatory region of SLE CD4+T cells.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients and healthy donors by density gradient centrifugation. CD4+T cells were isolated using microbeads and protocols provided by the manufacturer. ChIP and real-time PCR experiments were carried out to examine H3K4trimethylation (H3K4me3) and H3K27trimethylation (H3K27me3) binding levels of MIR-142regulatory region.Results:Compared to healthy controls, SLE CD4+T cells showed enriched levels of H3K27me3(ChIP-1:4.17±1.32vs1.61±0.59, ChIP-2:4.30±1.19vs1.59±0.56, ChIP-3:4.21±1.16vs1.54±0.60; P<0.001), while H3K4me3levels were almost equal to those of matched controls (P=0.716).Conclusion:The downregulation of miR-142-3p/5p is associated with increased H3K27me3enrichment in the MIR-142regulatory region in SLE CD4+T cells. Section II DNA methylation status in the MIR-142regulatory region in SLE CD4+T cellsObjective:To explore DNA methylation status in the MIR-142regulatory region of SLE CD4+T cells.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of SLE patients and healthy donors by density gradient centrifugation. CD4+T cells were isolated using microbeads and protocols provided by the manufacturer. Bisulfite sequencing was performed to determine the methylation status of CpG pairs within the MIR-142regulatory regionResults:Compared to healthy controls, the average methylation status of the11CpG pairs in the area (-409bp--9bp) was similar in SLE patients; however, the3CpG pairs closest to the transcription start site (-15bp～-9bp) were significantly hypermethylated. Conclution:The downregulation of miR-142-3p/5p is associated with the hypermethylation of CpG pairs in the MIR-142regulatory region in SLE CD4+T cells.