The Role Of MicroRNA-29b And MicroRNA-132in The Regulation Of CD4~+T Cells Activation And Pathogenesis Of Systemic Lupus Erythematosus

Part I The expression profiling and target gene prediction of miRNAs in CD4+T cells from SLE patientsObjective:To identify the differentially expressed miRNAs in CD4+T cells from patients with SLE, and predict their target gene and function.Methods:We use high-throughput qPCR method to detect the expression levels of1080miRNAs in CD4+T cells from9SLE patients and9normal controls, and apply TargetScan/miRanda/PicTar software and go-pathway analysis to predict the target gene and function of differentially expressed miRNAs.Results:29differentially expressed miRNAs were detected in CD4+T cells of SLE patients than those in normal controls. The16upregulated miRNAs were miR-1302,-320a,-126,-3200,-107,-29b,-1260,-1913,-451,-98,-3125,-132,-744,-21,-606,-3190-5p and13downregulated miRNAs were miR-3926,-4311,-7-2*,-1253,-633,-1279,-3201,-558,-620,-664,-609,-3148,-488*. In the predicted target genes of the29miRNAs,221genes might play pivotal roles in the pathogenesis of SLE. spl, a transcription factor regulating DNMT1expression, may be a potential target gene of miR-29b; while MeCP2may be a potential target gene of miR-132.Conclusions:Aberrant miRNA expressions might play an important role in the pathogenesis of SLE. And spl and MeCP2may be the potential target genes of miR-29b and miR-132, respectively. Part Ⅱ miR-29b contributes to DNA hypomethylation of CD4+T cells in SLE by targeting sp1/DNMT1Section Ⅰ The expression signature of miR-29b in CD4+T cells of patients with SLE Objective:To further verify the aberrant expression of miR-29b in CD4+T cells of SLE patients.Methods:We used real-time quantitative PCR to detect the miR-29b expression levels in CD4+T cells from36SLE patients and28normal controls, and analyzed the correlations between sp1and DNMT1protein levels.Results:miR-29b levels were significantly increased in lupus CD4+T cells, as compared to that in normal controls. Significantly negative correlations were observed between miR-29b levels and spl protein levels, and also between miR-29b levels and DNMT1protein levels.Conclusions:The miR-29b upregulation might play an important role in onset of SLE by affecting sp1and DNMT1expression. Section Ⅱ The validation of miR-29b affecting DNMT1expression by targeting sp1Objective:To verify the regulation of spl on DNMT1expression and regulation of miR-29b on DNMT1expression by targeting sp1.Methods:We designed and synthsized sp1-siRNA and negative control in vitro and transiently transfected them into Jurkat cells or primary CD4+T cells using nucleofector Ⅱ machine, respectively. After48h transfection, we collected the cells and dectected mRNA and protein levels of sp1and DNMT1by RT-PCR and western-blot. With the same method, we designed and synthsized miR-29b mimics and negative control in vitro and transiently transfected them into Jurkat cells or primary CD4+T cells using nucleofector Ⅱ machine, respectively. After48h transfection, we collected the cells and dectected mRNA and protein levels of spl and DNMT1by RT-PCR and western-blot.Results:The mRNA and protein levels of spl and DNMT1in sp1-siRNA transfected Jurkat cells were significantly lower than those in the control group (P<0.005and P<0.01), while mRNA and protein levels of sp1and DNMT1in miR-29b mimics transfected Jurkat cells were also significantly reduced than those in the control group(P<0.005and P<0.01). In spl-siRNA or miR-29b mimics transfected primary CD4+T cells, the expression levels of sp1and DNMT1mRNA and protein were also significantly decreased than those in the control group, repectively (all P<0.05).Conclusions:sp1can regulate DNMT1expression, and miR-29b may suppress spl protein levels by binding to and degrading mRNA of sp1. Section Ⅲ Overexpression of miR-29b induced CD4+T cell autoreactivity in normal controlsObjective:To investigate the effect of miR-29b overexpression on levels of global DNA methylation and autoimmunity related genes expression in CD4+T cell from normal controls.Methods:We designed and synthsized miR-29b mimics and negative control in vitro and transiently transfected them into primary CD4+T cells of normal controls using nucleofector Ⅱ machine. The global DNA methylation levels were evaluated by the MethyflashTM DNA Methylation Quantification Kit. CD11a and CD70promoter methylation levels were detected by bisulfate modification and HRM-PCR. CDlla and CD70mRNA and protein levels were determined by RT-qPCR and flow cytometry, respectively.Results:Compared with the negative control, the methylation levels of global DNA and CD11a、CD70promoter region were significantly reduced (all P<0.05), while mRNA and protein levels of CDlla and CD70were significantly elevated (both P<0.05for CD11a, P<0.005and P<0.05for CD70; respectively)in miR-29b mimics transfected group.Conclusions:The miR-29b overexpression can cause DNA hypomethylation and thereby downregulation of autoimmunity related genes and increase autoreactivity of CD4+T cells. Section Ⅳ Inhibition of miR-29b reduced CD4+T cell autoreactivity in SLE patientsObjective:To investigate the effect of downregulating miR-29b expression on levels of global DNA methylation and autoimmunity related genes expression in CD4+T cell from SLE patients.Methods:We designed and synthsized miR-29b inhibitor and negative control in vitro and transiently transfected them into primary CD4+T cells of SLE patients using nucleofector Ⅱ machine. The global DNA methylation levels were evaluated by the MethyflashTM DNA Methylation Quantification Kit. CD11a and CD70promoter methylation levels were detected by bisulfate modification and HRM-PCR. CD11a and CD70mRNA and protein levels were determined by RT-qPCR and flow cytometry, respectively.Results:Compared with the negative control, the mRNA (both P<0.05)and protein levels of spl and DNMT1were significantly elevated,and the methylation levels of global DNA and CD11a、CD70promoter region were significantly increased (all P<0.05), and also mRNA and protein levels of CDlla and CD70were significantly decreased (both P<0.05for CDlla, both P<0.05for CD70; respectively) in miR-29b inhibitor transfected group.Conclusions:Inhibition of miR-29b expression can reverse DNA hypomethylation by upregulating sp1and DNMT1levels and thereby downregulation of autoimmunity related genes and reduce autoreactivity of CD4+T cells in SLE patients. Part Ⅲ miR-132contributes to overexpression of methylation-sensitive genes in SLE by targeting MeCP2Section Ⅰ The expression signature of miR-132in CD4+T cells from patients with SLEObjective:To further verify the aberrant expression of miR-132in CD4+T cells of SLE patients. Methods:We used real-time quantitative PCR to detect the miR-132expression levels in CD4+T cells from36SLE patients and28normal controls, and analyzed the correlation between miR-132levels and MeCP2protein levels.Results:miR-132levels were significantly upregulated in lupus CD4+T cells, as compared to that in normal controls. Significantly negative correlation was observed between miR-132levels and MeCP2protein levels.Conclusions:The miR-132upregulation might play an important role in onset of SLE by affecting MeCP2expression. Section II The validation of miR-132targeting MeCP2Objective:To verify that MeCP2is the target gene of miR-132.Methods:We designed and synthsized miR-132mimics and negative control in vitro and transiently transfected them into Jurkat cells or primary CD4+T cells using nucleofector Ⅱ machine, respectively. After48h transfection, we collected the cells and detected mRNA and protein levels of MeCP2by RT-qPCR and western-blot. We further verify the relationship between miR-132and MeCP2using luciferase reporter gene system. A fragment from the3’-UTR of target gene MeCP2containing putative miR-132binding sites was amplified by PCR and then cloned into psicheck-2expression vector to construct MeCP2wild type luciferase recombinant vector, while construct MeCP2mutant luciferase recombinant vector using site-directed mutagenesis kit. Then the recombinant vector was transiently co-transfected into Jurkat cells with miR-132mimics or negative control by liposome transfection. After48h transfection, firely luciferase activity was measured using the Dual-Luciferase reporter assay system. Renilla luciferase was used as internal control.Results:The MeCP2mRNA levels have no significant change in miR-132transfected Jurkat cells and primary CD4+T cells than those in the control group (both P>0.05), while the protein levels of MeCP2were significantly decreased in both miR-132transfected group than those in the control (both P<0.05). Co-transfection of miR-132mimics and recombinant vector can inhibit the MeCP2wild type vector luciferase activity (P<0.05), but failed to inhibit MeCP2mutant vector luciferase activity (F>0.05).Conclusions:MeCP2are the corresponding target gene of miR-132. miR-132can repress the mRNA translation by binding to3’-UTR region of MeCP2. Section Ⅲ Overexpression of miR-132induced CD4+T cell autoreactivity in normal controlsObjective:To investigate the effect of miR-132overexpression on levels of autoimmunity related genes expression in CD4+T cell from normal controls.Methods:We designed and synthsized miR-132mimics and negative control in vitro and transiently transfected them into primary CD4+T cells of normal controls using nucleofector Ⅱ machine. The binding levels of MeCP2protein in promoter region of CD11a and CD70were evaluated using Chromatin immunnoprecipitation (ChIP). CDlla and CD70promoter methylation levels were detected by bisulfate modification and HRM-PCR. CDlla and CD70mRNA and protein levels were determined by RT-qPCR and flow cytometry, respectively.Results:Compared with the negative control, the combination levels of MeCP2protein and CD11, CD70promoter region were significantly decreased (P<0.005and P<0.05), and the methylation levels of CDlla, CD70promoter region have no significant change (both P>0.05), while mRNA and protein levels of CDlla and CD70were significantly elevated (both P<0.05for CDlla and CD70, respectively) in transfected group.Conclusions:The miR-132overexpression can decline the expression silencing of MeCP2on CD11and CD70gene and thereby upregulate autoimmunity related genes expression and increase autoreactivity of CD4+T cells. Section Ⅳ Inhibition of miR-132reduced CD4+T cell autoreactivity in SLE patientsObjective:To investigate the effect of downregulating miR-132expression on levels of autoimmunity related genes expression in CD4+T cell from SLE patients.Methods:We designed and synthsized miR-132inhibitor and negative control in vitro and transiently transfected them into primary CD4+T cells of SLE patients using nucleofector Ⅱ machine. The binding levels of MeCP2protein in promoter region of CD11a and CD70were evaluated using Chromatin immunnoprecipitation (ChIP). CDlla and CD70promoter methylation levels were detected by bisulfate modification and HRM-PCR. CDlla and CD70mRNA and protein levels were determined by RT-qPCR and flow cytometry, respectively.Results:Compared with the negative control, the MeCP2protein levels were significantly increased (P<0.05), the combination levels of MeCP2protein and CD11, CD70promoter region were significantly increased (both P<0.05), but the methylation levels of CDlla, CD70promoter region have no significant change (both P>0.05), while mRNA and protein levels of CDlla and CD70were significantly reduced (both P<0.05for CDlla and CD70, respectively) in transfected group.Conclusions:Inhibition of miR-132expression can reverse CD11a and CD70overexpression in some extent by upregulating MeCP2levels and reduce autoreactivity of CD4+T cells in SLE patients.