NSUN2通过m^(5)C修饰介导系统性红斑狼疮患者中CD4^(+)T细胞的活化  

NSUN2 mediates the activation of CD4^(+)T cells in systemic lupus erythematosus through m^(5)C modification

作　　者：陈稳稳 张敏 方苏 郭刚强 薛向阳 毛孙忠 CHEN Wenwen;ZHANG Min;FANG Su;GUO Gangqiang;XUE Xiangyang;MAO Sunzhong(School of Basic Medicine,Wenzhou Medical University,Wenzhou 325035,China)

机构地区：[1]温州医科大学基础医学院,浙江温州325035

出　　处：《温州医科大学学报》2024年第8期603-613,共11页Journal of Wenzhou Medical University

基　　金：国家自然科学基金项目(32200476);浙江省自然科学基金项目(LQ23C060002);温州市基础性科研项目(Y20220044)。

摘　　要：目的:探究mRNA的m^(5)C甲基转移酶NSUN2对系统性红斑狼疮(SLE)患者CD4^(+)T细胞活化的影响。方法:通过慢病毒感染和有限稀释法构建NSUN2稳定敲除的Jurkat细胞单克隆株,Western blot和免疫荧光检测Jurkat细胞中NSUN2的敲除效果,Dot blot检测NSUN2敲除后m^(5)C的修饰水平变化。使用抗人CD3/CD28抗体刺激Jurkat细胞活化,分别在24 h和48 h使用流式细胞术检测Jurkat细胞活化标志物CD69和CD25的表达水平,RT-qPCR检测Jurkat细胞活化后IL-2和TNF-α的转录水平;建立NSUN2^(+/-)小鼠模型,使用磁珠分离CD4^(+)T细胞,通过体外诱导细胞活化,进一步评价NSUN2的敲低对CD4^(+)T细胞活化的影响。使用Arraystar mRNA表观转录组芯片检测NSUN2敲除后Jurkat细胞中基因m^(5)C修饰水平和表达水平,获得受NSUN2调控的m^(5)C修饰的靶基因,利用GO和KEGG富集分析获得NSUN2调控的重要信号通路,结合SLE患者CD4^(+)T细胞中差异的m^(5)C修饰基因集,筛选获得受NSUN2调控的且与SLE疾病相关的靶基因,并进一步利用GO和KEGG等分析NSUN2调控系统性红斑狼疮CD4^(+)T细胞功能的潜在通路。结果:采用CRISPR-Cas9技术,成功获得NSUN2敲除的Jurkat细胞单克隆株,与NSUN2-NC组相比,NSUN2-KO组m^(5)C修饰水平下调,Jurkat细胞的活化标志物CD69和CD25表达水平上调,活化相关细胞因子IL-2和TNF-α表达水平上调(P<0.05);与野生型小鼠相比,NSUN2^(+/-)小鼠Naive CD4^(+)T细胞的活化标志物CD69的表达水平上调。机制上发现,Jurkat细胞中存在受NSUN2调控的基因集,GO和KEGG富集显示这些基因与T细胞活化过程相关。结合SLE患者CD4^(+)T细胞中差异的m^(5)C修饰基因集,筛选获得受NSUN2调控且与SLE疾病相关的靶基因集,GO和KEGG富集分析证实这些基因集与CD4^(+)T细胞功能相关。结论:NSUN2的敲除/敲低促进了Jurkat细胞和小鼠Naive CD4^(+)T细胞的活化,初步筛选出NSUN2调控SLE患者CD4^(+)T细胞活化的靶基因集及其潜在信号�Objective:To investigate the effect of mRNA m^(5)C methyltransferase NSUN2 on CD4^(+)T cell activation in patients with systemic lupus erythematosus(SLE).Methods:The NSUN2-stable knockout Jurkat cell monoclonal strain was constructed by lentivirus infection and limited dilution method,and the knockout effectiveness of NSUN2 in Jurkat cells was detected by Western blot and immunofluorescence.Dot blot analysis was made of m^(5)C modification level after NSUN2 knockout.Jurkat cells were activated with anti-human CD3/CD28 antibodies,and the expression levels of Jurkat cell activation markers CD69 and CD25 were detected by flow cytometry at 24 h and 48 h,respectively.The transcription levels of IL-2 and TNF-αafter Jurkat cell activation were detected by RT-qPCR.A mouse model of NSUN2^(+/-)was established,CD4^(+)T cells were isolated by magnetic beads,and the effect of NSUN2 knockdown on CD4^(+)T cell activation was further evaluated by inducing cell activation in vitro.Arraystar mRNA epigenome chip was used to detect the m^(5)C modification and gene expression level in Jurkat cells after NSUN2 knockout,and m^(5)C modified target genes regulated by NSUN2 were obtained.Combined with the m^(5)C modified gene set of differences in CD4^(+)T cells of SLE patients set that differs in CD4^(+)T cells of SLE patients,target genes regulated by NSUN2 and associated with SLE disease were screened,and then GO and KEGG enrichment was further used to analyze the potential pathway of NSUN2 regulating CD4^(+)T cell function in SLE.Results:The NSUN2-knockout Jurkat cell monoclonal strain was successfully obtained using CRISPR-Cas9 technology.Compared with the NSUN2-NC group,the m^(5)C modification level was down-regulated in the NSUN2-KO group,while the expression levels of Jurkat cell activation markers CD69 and CD25 were up-regulated.The expression levels of activation-related cytokines IL-2 and TNF-αwere up-regulated(P<0.05).Compared with wild-type mice,the expression level of CD69,an activation marker of Naive CD4^(+)T cells in NS

关 键 词：系统性红斑狼疮 m^(5)C NSUN2 CD4^(+)T细胞 

分 类 号：R392.1[医药卫生—免疫学]