目的研究DNA甲基化在结核分枝杆菌（MTB）裂解物诱导CD4 +T细胞白细胞介素6受体（IL-6R）表达下调中的作用及机制。
方法本研究属于前瞻性研究。收集并分选2019—2020年深圳市第三人民医院10名健康人（对照组）和10例结核病患者（TB组）外周血单个核细胞（PBMC）中CD4 +T细胞，亚硫酸氢盐测序法分析 IL-6R启动子区和3′非翻译区（UTR）区CpG岛甲基化变化；RT-qPCR和Western blotting分别检测IL-6R和DNA甲基转移酶（DNMT）表达；进一步通过MTB裂解物刺激anti-CD3/CD28抗体活化的对照组PBMC和Jurkat E6-1细胞，检测 IL-6R不同区域CpG岛甲基化和IL-6R、DNMT表达的变化；双荧光素酶报告基因系统检测 IL-6R 3′UTR区甲基化状态对其转录活性的影响；两组间采用非配对 t检验，三组及以上多组运用one-way ANOVA分析。
结果在对照组和TB 组外周血CD4 +T细胞中，TB组中IL-6R表达低于对照组，而DNMT1和DNMT3B则高于对照组；且与对照组比， IL-6R启动子CpG岛甲基化水平差异无统计学意义；3′ UTR区CpG岛甲基化率分别为54.3%±4.7%和69.5%±3.4%，差异有统计学意义（ P<0.001）；在体外，MTB裂解物刺激活化对照组PBMC后，IL-6R表达低于未刺激的，而DNMT1和DNMT 3B表达高于未刺激的；同时CD4 +T细胞中 IL-6R 3′ UTR区CpG岛甲基化率由58.9%±11.6%增加至79.4%±10.9%，差异有统计学意义（ P<0.001）；同样地，MTB刺激活化Jurkat E6-1细胞后的结果与对照组PBMC相一致。进一步发现DNA甲基转移酶抑制剂地西他滨（5-aza）与MTB裂解物共处理后IL-6R 的表达均高于MTB 裂解物单独刺激的；DNA甲基转移酶抑制剂地西他滨（5-aza）与MTB裂解物共处理后 IL-6R 3′ UTR区CpG岛甲基化水平低于MTB 裂解物单独刺激的，并且完全非甲基化修饰的 IL-6R 3′UTR报告基因的转录活性高于完全甲基化修饰的 IL-6R 3′ UTR区CpG岛。
结论MTB裂解物刺激通过诱导CD4 +T细胞 IL-6R 3′UTR区CpG岛高甲基化抑制 IL-6R转录活性进而下调其表达；MTB诱导CD4 +T细胞 IL-6R 3′UTR区CpG岛高甲基化可能与DNMT1、DNMT3B表达增加有关。
ObjectiveTo investigate the role and mechanism of DNA methylation in Mycobacterium tuberculosis (MTB lysate) -induced downregulation of interleukin-6 receptor(IL-6R) expression in CD4 +T cells.
MethodsA prospective study was conducted. Bisulfite sequencing (BSP) was applied to determine the methylation levels of CpG island in IL-6R promoter region and 3′untranslated region (3′UTR) region in CD4 +T cells from peripheral blood mononuclear cells (PBMC) of control group (healthy person, n=10) and TB group (tuberculosis patients, n=10) in Shenzhen Third People′s Hospital between 2019 and 2020. Quantitative reverse transcription-PCR (RT-qPCR) and Western blotting were used to detect the expression of IL-6R, DNMT1, DNMT3A and DNMT3B in MTB lysate-stimulated CD4 +T cells and Jurkat E6-1 cells. Furthermore, PBMC in control group and Jurkat E6-1 cells activated by anti-CD3/CD28 antibody were stimulated by MTB lysates to detect the methylation levels of CpG island and IL-6R and DNMT expression. Transcriptional activity of differently methylation regions of IL-6R 3′UTR was detected by using luciferase reporter gene system.
ResultsIL-6R expression in TB group was lower than that in control group, but DNMT1 and DNMT3B expressions were higher than those in control group in CD4 +T cells isolated from PBMC. There was no significant difference in the methylation rate of IL-6R promoter CpG island of CD4 +T cells between control and TB group. However, the methylation rates of CpG island in 3′UTR region were significantly higher ( P<0.001) in TB (69.5%±3.4%), compared with control (54.3%±4.7%). Besides, IL-6R expression was lower than unstimulated, while DNMT1 and DNMT3B expression was higher than unstimulated after MTB lysate-stimulation of activated control PBMC in vitro. The methylation rate of CpG island in IL-6R 3′UTR region of CD4 +T cells increased from 58.8%±11.6% to 79.4%±10.9% ( P<0.001) after MTB lysate-stimulated PBMC of the control. The same results were observed in the MTB lysate-stimulated CD4 +T cells isolated from PBMC in control and Jurkat E6-1 cell line. Furthermore, IL-6R expression after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysate was higher than that stimulated by MTB lysate alone. In addition, the methylation levels of CpG islands in the 3′ UTR region of IL-6R were lower than those stimulated by MTB lysates alone after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysates. The transcriptional activity of the fully unmethylated IL-6R 3′UTR CpG island reporter gene was higher than that of the fully methylated IL-6R 3′UTR CpG island.
ConclusionsMTB lysates stimulation inhibited IL-6R expression transcriptionalely as well as on the protein level by inducing hypermethylation of CpG island in IL-6R 3′UTR region of CD4 +T cells. The hypermethylation of CpG island in IL-6R 3′UTR region of CD4 +T cells induced by MTB may be related to the increased expression of DNMT1 and DNMT3B.
Mo Siwei and Zhu Chuanzhi contributed equally to the article
莫斯维,朱传智,刘晓倩,等. 结核分枝杆菌对CD4 +T细胞白细胞介素6受体3′非翻译区甲基化的作用及机制研究 [J]. 中华结核和呼吸杂志,2022,45(04)：379-386.
DOI:10.3760/cma.j.cn112147-20211206-00859版权归中华医学会所有。
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莫斯维：实验数据收集及整理、统计学分析、论文撰写及修改；朱传智：实验数据整理、统计学分析、参与论文撰写及修改；刘晓倩、万浩强、李富祥、邓国防：临床样本收集；张宗德、陈心春：研究指导、论文修改、经费支持
莫斯维和朱传智对本文有同等贡献
莫斯维, 朱传智, 刘晓倩, 等. 结核分枝杆菌对CD4 +T细胞白细胞介素6受体3′非翻译区甲基化的作用及机制研究[J]. 中华结核和呼吸杂志, 2022, 45(4): 379-386. DOI: 10.3760/cma.j.cn112147-20211206-00859.
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