MicroRNA-142-3p/5p对CD4~+T细胞活化的调控及其在系统性红斑狼疮发病中的作用
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摘要
系统性红斑狼疮(Systemic Lupus Erythematosus, SLE)是一种严重危害人类健康的自身免疫性疾病。其特点是T细胞过度激活,过度辅助自身B细胞产生大量的自身抗体,造成体内多器官的炎症反应和组织损伤。目前虽然导致SLE的发生、发展的分子机制尚未明确,但近年来的研究表明,除了遗传因素外,表观遗传机制在SLE的发病机制中起着重要作用。
MicroRNAs (miRNAs)为表观遗传调控机制之一。miRNAs是一类长度约21-25个核苷酸的内源性非编码RNA分子,被认为是细胞生命活动中相关基因的潜在负调节子。miRNA可通过与靶mRNA的3’端非翻译区、编码区或5’端非翻译区结合进而抑制翻译或引发mRNA降解,阻遏基因转录后的翻译过程。miRNA是生物体自身的一套正常的调控机制,直接调节哺乳动物的基因组中30%以上蛋白编码基因的转录。最近的研究表明,miRNA参与调节人体各种免疫过程,可以作为多种疾病诊断的生物学标志物或治疗靶点。在人类多种自身免疫性疾病,包括红斑性狼疮、类风湿关节炎和多发性硬化症中已经发现多种miRNA的表达改变。然而,miRNA在SLE的发病机制中的作用还需要进一步研究。
miR-142被认为是T细胞特异性miRNA,在调节T细胞的发育中起着重要的作用。miR-142前体可以产生两个成熟miRNA,从前体5’端的臂加工而来的命名为miR-142-5p,从前体3’端的臂加工而来的命名为miR-142-3p。本课题之前的miRNA芯片研究发现,与健康对照组相比,miR-142-3p和miR-142-5p在SLE患者CD4+T细胞中表达降低。我们通过扩大样本量进行real-time PCR验证了miR-142-3p和miR-142-5p在SLE CD4+T细胞表达下调。另外,我们证实了CD84是miR-142-3p的靶基因,而SAP为miR-142-5p的靶基因。用miR-142-3p/-5p抑制剂处理健康对照组CD4+T细胞,导致CD84和SAP表达上调。此外,抑制CD4+T细胞miR-142-3p/5p表达使T细胞过度活化,并促使B细胞产生抗体增多。相反,过度表达miR-142-3p/-5p,导致SLE患者CD4+T细胞中CD84和SAP的表达水平下调,T细胞活性降低,抗体产生减少。此外,我们还发现miR-142前体序列上游500bp内的组蛋白修饰和DNA甲基化可能调节miR-142-3p/5p的表达。通过这些研究揭示了miR-142-3p/5p低表达可能是导致SLE CD4+T细胞自身反应的重要分子机制,为寻找治疗SLE有效的新靶点提供了理论依据。
第一部分SLE患者CD4+T细胞miR-142-3p/5p及其靶基因表达
第一节SLE患者CD4+T细胞miR-142-3p/5p表达
目的：检测SLE患者CD4+T细胞miR-142-3p/5p表达改变
方法：
1.密度梯度离心分离正常人、SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞；
2.试剂盒提取miRNA;
3.逆转录合成cDNA;
4.实时定量聚合酶链反应(real-time PCR)检测miRNA的表达水平。
结果：与正常对照组相比,SLE患者CD4+T细胞miR-142-3p、 miR-142-5p均明显下调(p<0.001；p<0.001)。
结论：SLE患者CD4+T细胞中miR-142-3p/5p表达下调。
第二节miR-142-3p/5p靶基因与验证
目的：验证CD84、SAP分别为miR-142-3p、miR-142-5p的靶基因。
方法：
1.运用Mirbase/Targetscans/PicTar等靶基因预测软件查找miR-142-3p/5p可能的靶基因,筛选出可能与SLE发病过程密切相关的基因作为候选靶基因,我们选定CD84为niR-142-3p靶基因,SAP为miR-142-5p靶基因。
2.构建萤火虫荧光素酶表达载体：将包含CD84的3’-UTR区克隆至pMIR-REPORTER Luciferase vector (Ambion)载体编码荧光素酶基因序列下游,构建CD84野生型(CD84WT-luciferase)荧光素酶报告基因质粒,同时采用点突变的方法使CD84-3'-UTR中miR-142-3p的结合位点发生碱基突变,构建突变型(CD84Mut-luciferase)荧光素酶报告基因质粒。同理,构建野生型(SAPWT-luciferase)荧光素酶报告基因和与miR-142-5p的结合位点发生碱基突变的突变型(SAPMut-luciferase)荧光素酶报告基因。
3.转染：CD84WT-luciferase和CD84Mut-luciferase与miR-142-3p模拟物(miR-142-3p mimic)和阴性对照(Negative control)用电穿孔法瞬时共转染至Jurkat细胞；SAPWT-luciferase和SAPMut-luciferase与miR-142-5p模拟物(miR-142-5p mimic)和阴性对照(Negative control)用电穿孔法瞬时共转染至Jurkat细胞.
4.转染48小时后收集细胞,运用双荧光素酶报告基因检测系统,以海肾荧光素酶作为内参照,检测萤火虫荧光素酶的活性。
结果：
1.通过生物信息学预测软件我们发现多种miR-142-3p/5p的可能靶基因,我们选定可能与SLE发病过程密切相关的CD84、SAP基因作为靶基因；
2.在Jurkat细胞中共转染miR-142-3p模拟物,可以抑制CD84野生型(CD84WT-luciferase)荧光素酶活性,而当CD84-3'-UTR突变修饰后(CD84Mut-luciferase), miR-142-3p不能抑制其活性。
3.在Jurkat细胞中共转染miR-142-5p模拟物,可以抑制SAP野生型(SAPWT-luciferase)荧光素酶活性,而当SAP-3'-UTR突变修饰后(SAPMut-luciferase), miR-142-5p不能抑制其活性。
结论：CD84、SAP分别为miR-142-3p、miR-142-5p的靶基因,miR-142-3p/5p通过作用于3’端UTR区抑制靶基因CD84/SAP表达。
第三节过表达与抑制miR-142-3p/5p表达后靶基因CD84/SAP的表达改变
目的：研究CD4+T细胞miR-142-3p/5p的改变对其靶基因的影响
方法：
1.密度梯度离心分离各三例正常人、SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞；
2.转染：miR-142-3p抑制物(miR-142-3p inhibitor)、miR-142-5p抑制物(miR-142-5p inhibitor)或阴性对照(Negative control)用电穿孔法瞬时转染至正常人CD4+T细胞；miR-142-3p模拟物(miR-142-3p mimic)、miR-142-5p模拟物(miR-142-5p mimic)或阴性对照(Negative control)用电穿孔法瞬时转染至SLE患者CD4+T细胞；
3.试剂盒提取miRNA和总蛋白；
4.实时定量聚合酶链反应(real-time PCR)检测miRNA的表达水平；
5.蛋白质印迹(Western blot)检测蛋白表达水平。
结果：
1.转染miR-142-3p抑制物的正常人CD4+T细胞miR-142-3p水平降低,CD84蛋白水平增高；
2.转染miR-142-5p抑制物的正常人CD4+T细胞miR-142-5p水平降低,SAP蛋白水平增高；
3.转染miR-142-3p模拟物的SLE患者CD4+T细胞miR-142-3p水平增高,CD84蛋白水平降低；
4.转染miR-142-5p模拟物的SLE患者CD4+T细胞miR-142-5p水平增高,SAP蛋白水平降低。
结论：抑制miR-142-3p/5p表达可增高正常人CD4+T细胞CD84/SAP蛋白表达水平；miR-142-3p/5p过表达可降低SLE患者CD4+T细胞CD84/SAP蛋白表达水平。
第四节SLE患者CD4+T细胞miR-142-3p/5p靶基因CD84/SAP表达水平
目的：检测miR-142-3p/5p靶基因CD84/SAP在SLE患者CD4+T细胞的表达水平
方法：
2.密度梯度离心分离正常人、SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞；
3.试剂盒提取总蛋白；
4.蛋白质印迹(Western blot)检测CD84和SAP蛋白表达水平。
结果：
2.与健康对照组相比,SLE患者CD4+T细胞CD84蛋白表达水平升高(P<0.01),且与miR-142-3p表达水平呈显著负相关(R=-0.621,P=0.003)。
3.与健康对照组相比,SLE患者CD4+T细胞SAP蛋白表达水平升高(P<0.01),且与miR-142-5p表达水平呈显著负相关(R=-0.708,P=0.001)。
结论：miR-142-3p/5p靶基因CD84/SAP在SLE患者CD4+T细胞表达增高,且与]miR-142-3p/5p表达水平呈显著负相关。
第二部分miR-142-3p/5p表达异常与自身免疫反应
第一节抑制miR-142-3p/5p表达诱导自身免疫反应
目的：研究抑制miR-142-3p/5p表达对正常人CD4+T细胞活化以及自身免疫反应的影响。
方法：
1.密度梯度离心分离正常人的外周血单个核细胞,磁珠分离CD4+T细胞、B细胞；
2.转染：正常人CD4+T细胞分成四组,分别用电穿孔法瞬时转染阴性对照(Negative control)、miR-142-3p抑制物(miR-142-3p inhibitor)、miR-142-5p抑制物(miR-142-5p inhibitor)或miR-142-3p抑制物加miR-142-5p抑制物；
3.流式细胞仪检测CD40L和ICOS蛋白表达；
4.ELISA方法检测IL4.IL10和IL21蛋白表达；
5.细胞增殖实验检测CD4+T细胞增殖水平；
6.流式细胞仪检测CD4+T细胞与B细胞粘附频率；
7.ELISA方法检测自身B细胞IgG抗体产生水平。
结果：转染抑制物的三组CD4+T细胞均表现为CD40L和ICOS蛋白表达升高,IL4、IL10和IL21蛋白表达增高,细胞增殖活性增加,CD4+T细胞与B细胞结合频率升高,自身B细胞的IgG抗体产生水平增加,差异均有显著统计学意义(P<0.05或P<0.01)。
结论：抑制miR-142-3p/5p表达可诱导正常CD4+T细胞异常激活,具有自身免疫反应性。
第二节过表达miR-142-3p/5p抑制自身免疫反应
目的：探讨过表达miR-142-3p/5p能否抑制或逆转SLE CD4+T细胞活化以及自身免疫反应。
方法：
1.密度梯度离心分离SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞、B细胞；
2.转染：SLE患者CD4+T细胞分成四组,分别用电穿孔法瞬时转染阴性对照(Negative control)、miR-142-3p模拟物(miR-142-3p mimic)、miR-142-5p模拟物(miR-142-5p mimic)或miR-142-3p模拟物加miR-142-5p模拟物；
3.细胞增殖实验检测CD4+T细胞增殖水平；
4.流式细胞仪检测CD40L和ICOS蛋白表达；
5.ELISA方法检测IL4、IL10和IL21蛋白表达；
6.流式细胞仪检测CD4+T细胞与B细胞粘附率；
7.ELISA方法检测自身B细胞IgG抗体产生水平。
结果：转染模拟物的三组SLE患者CD4+T细胞均表现为CD40L和ICOS蛋白表达降低,IL4、IL10和IL21蛋白表达减少,细胞增殖活性降低,CD4+T细胞与B细胞结合频率降低,自身B细胞的IgG抗体产生水平减少,差异均有显著统计学意义(P<0.05或P<0.01)。
结论：过表达miR-142-3p/5p可抑制SLE患者CD4+T细胞活化及自身免疫反应性。
第三部分MIR-142基因转录调控区域的组蛋白甲基化修饰与DNA甲基化状态
第一节MIR-142基因调控区域的组蛋白甲基化修饰
目的：研究SLE患者CD4+T细胞MIR-142基因转录调控区域的组蛋白甲基化修饰状态。
方法：采用密度梯度离心法分离外周血单个核细胞,免疫磁珠分离外周血CD4+T细胞,染色质免疫沉淀(ChIP)方法结合实时定量PCR (real-time PCR)法检测MIR-142基因转录调控区域H3K4二甲基化、H3K27三甲基化水平。
结果： MIR-142基因转录调控区域H3K4三甲基化水平在SLE患者CD4+T细胞和正常对照组间无显著差异(P=0.716)。而SLE患者CD4+T细胞中MIR-142基因转录调控区域H3K27三甲基化水平显著升高(P<0.001)。
结论：SLE患者CD4+T细胞中miR-142-3p/5p表达下降可能与MIR-142基因转录调控区域H3K27三甲基化水平升高有关。
第二节MIR-142基因转录调控区域的DNA甲基化状态
目的：探讨SLE患者CD4+T细胞MIR-142基因转录调控区域的DNA甲基化状态。
方法：采用密度梯度离心法分离外周血单个核细胞,免疫磁珠分离外周血CD4+T细胞,试剂盒提取DNA,亚硫酸氢钠基因组测序法检测MIR-142基因潜在转录调控区域的DNA甲基化状态。
结果：MIR-142基因转录调控区域(-409bp～-9bp)11个CpG位点平均甲基化水平在SLE患者CD4+T细胞和正常对照组间无显著差异(P=0.416)。而SLE患者CD4+T细胞靠近MIR-142基因转录起始位点(-15bp--9bp)3个CpG位点平均甲基化水平显著升高(P<0.05)。
结论：SLE患者CD4+T细胞中miR-142-3p/5p表达下降可能与MIR-142基因转录调控区域DNA高甲基化有关。
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 genes
Section I Abnormal miR-142-3p/5p expression in SLE CD4+T cells
Objective: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 Kit
4. 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 genes
Objective: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 cells
Objective: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 cells
Objective:To investigate expression of miR-142-3p/5p target genes 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 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 autoimmunity
Section I Inducing T cell autoreactivity by inhibiting miR-142-3p/5p expression
Objective: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/5p
Objective: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 cells
Section I Abnormal histone modification patterns in the MIR-142regulatory region of SLE CD4+T cells
Objective: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 cells
Objective: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 region
Results: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.
MicroRNAs (miRNAs)为表观遗传调控机制之一。miRNAs是一类长度约21-25个核苷酸的内源性非编码RNA分子,被认为是细胞生命活动中相关基因的潜在负调节子。miRNA可通过与靶mRNA的3’端非翻译区、编码区或5’端非翻译区结合进而抑制翻译或引发mRNA降解,阻遏基因转录后的翻译过程。miRNA是生物体自身的一套正常的调控机制,直接调节哺乳动物的基因组中30%以上蛋白编码基因的转录。最近的研究表明,miRNA参与调节人体各种免疫过程,可以作为多种疾病诊断的生物学标志物或治疗靶点。在人类多种自身免疫性疾病,包括红斑性狼疮、类风湿关节炎和多发性硬化症中已经发现多种miRNA的表达改变。然而,miRNA在SLE的发病机制中的作用还需要进一步研究。
miR-142被认为是T细胞特异性miRNA,在调节T细胞的发育中起着重要的作用。miR-142前体可以产生两个成熟miRNA,从前体5’端的臂加工而来的命名为miR-142-5p,从前体3’端的臂加工而来的命名为miR-142-3p。本课题之前的miRNA芯片研究发现,与健康对照组相比,miR-142-3p和miR-142-5p在SLE患者CD4+T细胞中表达降低。我们通过扩大样本量进行real-time PCR验证了miR-142-3p和miR-142-5p在SLE CD4+T细胞表达下调。另外,我们证实了CD84是miR-142-3p的靶基因,而SAP为miR-142-5p的靶基因。用miR-142-3p/-5p抑制剂处理健康对照组CD4+T细胞,导致CD84和SAP表达上调。此外,抑制CD4+T细胞miR-142-3p/5p表达使T细胞过度活化,并促使B细胞产生抗体增多。相反,过度表达miR-142-3p/-5p,导致SLE患者CD4+T细胞中CD84和SAP的表达水平下调,T细胞活性降低,抗体产生减少。此外,我们还发现miR-142前体序列上游500bp内的组蛋白修饰和DNA甲基化可能调节miR-142-3p/5p的表达。通过这些研究揭示了miR-142-3p/5p低表达可能是导致SLE CD4+T细胞自身反应的重要分子机制,为寻找治疗SLE有效的新靶点提供了理论依据。
第一部分SLE患者CD4+T细胞miR-142-3p/5p及其靶基因表达
第一节SLE患者CD4+T细胞miR-142-3p/5p表达
目的：检测SLE患者CD4+T细胞miR-142-3p/5p表达改变
方法：
1.密度梯度离心分离正常人、SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞；
2.试剂盒提取miRNA;
3.逆转录合成cDNA;
4.实时定量聚合酶链反应(real-time PCR)检测miRNA的表达水平。
结果：与正常对照组相比,SLE患者CD4+T细胞miR-142-3p、 miR-142-5p均明显下调(p<0.001；p<0.001)。
结论：SLE患者CD4+T细胞中miR-142-3p/5p表达下调。
第二节miR-142-3p/5p靶基因与验证
目的：验证CD84、SAP分别为miR-142-3p、miR-142-5p的靶基因。
方法：
1.运用Mirbase/Targetscans/PicTar等靶基因预测软件查找miR-142-3p/5p可能的靶基因,筛选出可能与SLE发病过程密切相关的基因作为候选靶基因,我们选定CD84为niR-142-3p靶基因,SAP为miR-142-5p靶基因。
2.构建萤火虫荧光素酶表达载体：将包含CD84的3’-UTR区克隆至pMIR-REPORTER Luciferase vector (Ambion)载体编码荧光素酶基因序列下游,构建CD84野生型(CD84WT-luciferase)荧光素酶报告基因质粒,同时采用点突变的方法使CD84-3'-UTR中miR-142-3p的结合位点发生碱基突变,构建突变型(CD84Mut-luciferase)荧光素酶报告基因质粒。同理,构建野生型(SAPWT-luciferase)荧光素酶报告基因和与miR-142-5p的结合位点发生碱基突变的突变型(SAPMut-luciferase)荧光素酶报告基因。
3.转染：CD84WT-luciferase和CD84Mut-luciferase与miR-142-3p模拟物(miR-142-3p mimic)和阴性对照(Negative control)用电穿孔法瞬时共转染至Jurkat细胞；SAPWT-luciferase和SAPMut-luciferase与miR-142-5p模拟物(miR-142-5p mimic)和阴性对照(Negative control)用电穿孔法瞬时共转染至Jurkat细胞.
4.转染48小时后收集细胞,运用双荧光素酶报告基因检测系统,以海肾荧光素酶作为内参照,检测萤火虫荧光素酶的活性。
结果：
1.通过生物信息学预测软件我们发现多种miR-142-3p/5p的可能靶基因,我们选定可能与SLE发病过程密切相关的CD84、SAP基因作为靶基因；
2.在Jurkat细胞中共转染miR-142-3p模拟物,可以抑制CD84野生型(CD84WT-luciferase)荧光素酶活性,而当CD84-3'-UTR突变修饰后(CD84Mut-luciferase), miR-142-3p不能抑制其活性。
3.在Jurkat细胞中共转染miR-142-5p模拟物,可以抑制SAP野生型(SAPWT-luciferase)荧光素酶活性,而当SAP-3'-UTR突变修饰后(SAPMut-luciferase), miR-142-5p不能抑制其活性。
结论：CD84、SAP分别为miR-142-3p、miR-142-5p的靶基因,miR-142-3p/5p通过作用于3’端UTR区抑制靶基因CD84/SAP表达。
第三节过表达与抑制miR-142-3p/5p表达后靶基因CD84/SAP的表达改变
目的：研究CD4+T细胞miR-142-3p/5p的改变对其靶基因的影响
方法：
1.密度梯度离心分离各三例正常人、SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞；
2.转染：miR-142-3p抑制物(miR-142-3p inhibitor)、miR-142-5p抑制物(miR-142-5p inhibitor)或阴性对照(Negative control)用电穿孔法瞬时转染至正常人CD4+T细胞；miR-142-3p模拟物(miR-142-3p mimic)、miR-142-5p模拟物(miR-142-5p mimic)或阴性对照(Negative control)用电穿孔法瞬时转染至SLE患者CD4+T细胞；
3.试剂盒提取miRNA和总蛋白；
4.实时定量聚合酶链反应(real-time PCR)检测miRNA的表达水平；
5.蛋白质印迹(Western blot)检测蛋白表达水平。
结果：
1.转染miR-142-3p抑制物的正常人CD4+T细胞miR-142-3p水平降低,CD84蛋白水平增高；
2.转染miR-142-5p抑制物的正常人CD4+T细胞miR-142-5p水平降低,SAP蛋白水平增高；
3.转染miR-142-3p模拟物的SLE患者CD4+T细胞miR-142-3p水平增高,CD84蛋白水平降低；
4.转染miR-142-5p模拟物的SLE患者CD4+T细胞miR-142-5p水平增高,SAP蛋白水平降低。
结论：抑制miR-142-3p/5p表达可增高正常人CD4+T细胞CD84/SAP蛋白表达水平；miR-142-3p/5p过表达可降低SLE患者CD4+T细胞CD84/SAP蛋白表达水平。
第四节SLE患者CD4+T细胞miR-142-3p/5p靶基因CD84/SAP表达水平
目的：检测miR-142-3p/5p靶基因CD84/SAP在SLE患者CD4+T细胞的表达水平
方法：
2.密度梯度离心分离正常人、SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞；
3.试剂盒提取总蛋白；
4.蛋白质印迹(Western blot)检测CD84和SAP蛋白表达水平。
结果：
2.与健康对照组相比,SLE患者CD4+T细胞CD84蛋白表达水平升高(P<0.01),且与miR-142-3p表达水平呈显著负相关(R=-0.621,P=0.003)。
3.与健康对照组相比,SLE患者CD4+T细胞SAP蛋白表达水平升高(P<0.01),且与miR-142-5p表达水平呈显著负相关(R=-0.708,P=0.001)。
结论：miR-142-3p/5p靶基因CD84/SAP在SLE患者CD4+T细胞表达增高,且与]miR-142-3p/5p表达水平呈显著负相关。
第二部分miR-142-3p/5p表达异常与自身免疫反应
第一节抑制miR-142-3p/5p表达诱导自身免疫反应
目的：研究抑制miR-142-3p/5p表达对正常人CD4+T细胞活化以及自身免疫反应的影响。
方法：
1.密度梯度离心分离正常人的外周血单个核细胞,磁珠分离CD4+T细胞、B细胞；
2.转染：正常人CD4+T细胞分成四组,分别用电穿孔法瞬时转染阴性对照(Negative control)、miR-142-3p抑制物(miR-142-3p inhibitor)、miR-142-5p抑制物(miR-142-5p inhibitor)或miR-142-3p抑制物加miR-142-5p抑制物；
3.流式细胞仪检测CD40L和ICOS蛋白表达；
4.ELISA方法检测IL4.IL10和IL21蛋白表达；
5.细胞增殖实验检测CD4+T细胞增殖水平；
6.流式细胞仪检测CD4+T细胞与B细胞粘附频率；
7.ELISA方法检测自身B细胞IgG抗体产生水平。
结果：转染抑制物的三组CD4+T细胞均表现为CD40L和ICOS蛋白表达升高,IL4、IL10和IL21蛋白表达增高,细胞增殖活性增加,CD4+T细胞与B细胞结合频率升高,自身B细胞的IgG抗体产生水平增加,差异均有显著统计学意义(P<0.05或P<0.01)。
结论：抑制miR-142-3p/5p表达可诱导正常CD4+T细胞异常激活,具有自身免疫反应性。
第二节过表达miR-142-3p/5p抑制自身免疫反应
目的：探讨过表达miR-142-3p/5p能否抑制或逆转SLE CD4+T细胞活化以及自身免疫反应。
方法：
1.密度梯度离心分离SLE患者的外周血单个核细胞,磁珠分离CD4+T细胞、B细胞；
2.转染：SLE患者CD4+T细胞分成四组,分别用电穿孔法瞬时转染阴性对照(Negative control)、miR-142-3p模拟物(miR-142-3p mimic)、miR-142-5p模拟物(miR-142-5p mimic)或miR-142-3p模拟物加miR-142-5p模拟物；
3.细胞增殖实验检测CD4+T细胞增殖水平；
4.流式细胞仪检测CD40L和ICOS蛋白表达；
5.ELISA方法检测IL4、IL10和IL21蛋白表达；
6.流式细胞仪检测CD4+T细胞与B细胞粘附率；
7.ELISA方法检测自身B细胞IgG抗体产生水平。
结果：转染模拟物的三组SLE患者CD4+T细胞均表现为CD40L和ICOS蛋白表达降低,IL4、IL10和IL21蛋白表达减少,细胞增殖活性降低,CD4+T细胞与B细胞结合频率降低,自身B细胞的IgG抗体产生水平减少,差异均有显著统计学意义(P<0.05或P<0.01)。
结论：过表达miR-142-3p/5p可抑制SLE患者CD4+T细胞活化及自身免疫反应性。
第三部分MIR-142基因转录调控区域的组蛋白甲基化修饰与DNA甲基化状态
第一节MIR-142基因调控区域的组蛋白甲基化修饰
目的：研究SLE患者CD4+T细胞MIR-142基因转录调控区域的组蛋白甲基化修饰状态。
方法：采用密度梯度离心法分离外周血单个核细胞,免疫磁珠分离外周血CD4+T细胞,染色质免疫沉淀(ChIP)方法结合实时定量PCR (real-time PCR)法检测MIR-142基因转录调控区域H3K4二甲基化、H3K27三甲基化水平。
结果： MIR-142基因转录调控区域H3K4三甲基化水平在SLE患者CD4+T细胞和正常对照组间无显著差异(P=0.716)。而SLE患者CD4+T细胞中MIR-142基因转录调控区域H3K27三甲基化水平显著升高(P<0.001)。
结论：SLE患者CD4+T细胞中miR-142-3p/5p表达下降可能与MIR-142基因转录调控区域H3K27三甲基化水平升高有关。
第二节MIR-142基因转录调控区域的DNA甲基化状态
目的：探讨SLE患者CD4+T细胞MIR-142基因转录调控区域的DNA甲基化状态。
方法：采用密度梯度离心法分离外周血单个核细胞,免疫磁珠分离外周血CD4+T细胞,试剂盒提取DNA,亚硫酸氢钠基因组测序法检测MIR-142基因潜在转录调控区域的DNA甲基化状态。
结果：MIR-142基因转录调控区域(-409bp～-9bp)11个CpG位点平均甲基化水平在SLE患者CD4+T细胞和正常对照组间无显著差异(P=0.416)。而SLE患者CD4+T细胞靠近MIR-142基因转录起始位点(-15bp--9bp)3个CpG位点平均甲基化水平显著升高(P<0.05)。
结论：SLE患者CD4+T细胞中miR-142-3p/5p表达下降可能与MIR-142基因转录调控区域DNA高甲基化有关。
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 genes
Section I Abnormal miR-142-3p/5p expression in SLE CD4+T cells
Objective: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 Kit
4. 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 genes
Objective: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 cells
Objective: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 cells
Objective:To investigate expression of miR-142-3p/5p target genes 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 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 autoimmunity
Section I Inducing T cell autoreactivity by inhibiting miR-142-3p/5p expression
Objective: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/5p
Objective: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 cells
Section I Abnormal histone modification patterns in the MIR-142regulatory region of SLE CD4+T cells
Objective: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 cells
Objective: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 region
Results: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.
引文
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