SLE诱导多能干细胞及向B淋巴细胞分化过程中的甲基腺嘌呤修饰图谱及功能研究

Systemic lupus erythematosus (SLE) is a typical systemic autoimmune disease, involving in abnormal lymphocytes, especially the abnormality of B lymphocytes. The differentiation and development of B lymphocytes are regulated by epigenetic modification, such as methylation. And methyladenine (mA) modification may be one of the key control factors. By focusing on SLE, we found that 5-hydroxymethylcytosine were differentially expressed in SLE，and abnormal activation of B lymphocytes was associated with specific methylation in SLE, then we built SLE-iPSCs model. On this basis, we assume that mA is associated with abnormal activation B lymphocytes in SLE. Consequently, we propose to use the techniques of methylation immunoprecipitation assays and sequencing to analyze methyladenine on the level of DNA and mRNA in SLE-iPSCs, lymphoid stem/progenitor cells and pre-B lymphocytes, and correlate the mA map on the level of DNA and mRNA. The mechanism of gene expression, transcription and translation will be dynamically analyzed by differentially expressed mA, and then the function of differentially expressed mA will be validated by CRISPER and RNAi technology. Combining with our previous study, the candidate of mA will be found in regulation of differentiation and development of B lymphocytes in SLE. Differentially expressed mA may be subsequently elucidated for the understanding of the molecular mechanism and clinical drug development of SLE.

SLE是一种累及全身的免疫性疾病,大多数SLE患者出现淋巴细胞异常，尤其是与B淋巴细胞异常密切相关。B淋巴细胞分化发育受甲基化等表观遗传调控，其中甲基腺嘌呤(mA)修饰可能是关键调控因素之一。本课题组前期研究发现SLE中甲基胞嘧啶差异表达，特异甲基化与异常B淋巴细胞活化相关，并构建了SLE-iPSCs模型。基于此，本课题假设mA与SLE中异常B淋巴细胞活化相关。因而，本项目拟采用甲基化免疫共沉淀测序技术分别在DNA和mRNA水平上对SLE-iPSCs、淋巴干/祖细胞及pre-B淋巴细胞的mA进行分析，绘制mA图谱并关联分析，获得差异表达mA，动态分析差异mA对基因表达、转录及翻译的调控机制。进而结合前期研究寻找调控SLE B淋巴细胞分化发育相关候选mA，利用CRISPER、RNAi等实验技术对候选mA的功能进行验证，以期获得SLE致病相关关键靶点，为该病的发病机制及临床药物研发奠定基础。

SLE是一种累及全身的免疫性疾病，大多数SLE患者出现淋巴细胞异常，尤其是与B淋巴细胞异常密切相关。B淋巴细胞分化发育受甲基化等表观遗传调控，其中甲基腺嘌呤(mA)修饰可能是关键调控因素之一。本项目前期已成功构建源于SLE患者尿液中肾管状细胞的诱导多能干细胞，在此基础上利用SLE患者血液成功构建SLE-iPSCs模型。发现SLE-iPSCs和NC-iPSCs之间有4254个基因存在两倍以上差异性表达，进一步对4254个差异表达基因进行GO及KEGG分析，发现SLE-iPSCs中与嘌呤代谢有关的基因表达差异最为显着。采用测序结合质谱技术对SLE-iPSCs的mRNA, miRNA和蛋白质组进行整合研究，发现了1099个差异表达的mRNAs、223个差异表达的microRNAs和94个差异表达的蛋白质。重点研究转录和蛋白质组图谱的表达变化趋势发现一些重要的途径在SLE-iPSCs中显著富集，包括RNA转运、赖氨酸降解等过程。同时，本项目还进行SLE患者外周血单个核细胞中6mA位点预测及GO/KEGG分析，结果揭示6mA在系统性红斑狼疮中有潜在作用，表明高甲基化基因部分通过JAK-STAT途径在系统性红斑狼疮中发挥作用。利用MEME和DREME对6mA的偏好序列进行预测，结果显示的序列可为发展6mA作为诊断标记物和治疗靶点提供一条新途径。此外，通过多组学的整合分析，我们揭示了SLE疾病中一些常见的调控信号传导途径，包括在外周血单核细胞内的TNF信号传导，JAK-STAT，氧化（GTPase活性，线粒体）等，提示这些通路在SLE疾病发病机理中扮演关键角色。从单细胞水平对SLE患者外周血单个核细胞的转录本进行分析，结果表明，SLE患者外周血中不同原始树突状细胞的丰度增加，而T细胞的丰度和亚群减少。利用scATAC-seq技术研究SLE患者的外周血单个核细胞可及性染色质，发现介导T细胞活化的基因BCL11B, CCR7, CD83, ELF4, ITPKB, NCK2, NKAP, RAB27A, RUNX3, ZMIZ1在多种免疫细胞亚群中显著开放，还发现了9个关键转录因子参与调控介导T细胞活化的基因，为我们深入认识SLE的发生发展机制及药物靶点治疗的确定打下基础。本课题已发表9篇SCI论文和2篇中文论文，申报发明专利5项；联合培养研究生1名，指导培养研究生1名。