FOXP3型
流式细胞术
基因敲除
信使核糖核酸
免疫学
外周血单个核细胞
分子生物学
免疫印迹
生物
甲基化
细胞培养
体外
基因
免疫系统
遗传学
生物化学
作者
Linxia Fan,Jiqiang Wu,Hongjuan Wang,Qizhang Chen,Xiaodong He,Qiong Wang,Zhen Yang
出处
期刊:PubMed
日期:2022-11-01
卷期号:52 (6): 884-894
摘要
To investigate the regulatory effect and mechanism of methyltransferase-like protein 3 (METTL3)-mediated N6-methyladenosine methylation (m6A) on forkhead box protein 3 (Foxp3) levels and the proportion of regulatory T (Treg) cells in the peripheral blood of patients with asthma.Flow cytometry and ELISA were used to detect the differences in the proportions of Treg cells and serum interleukins (ILs) 4 and 7, respectively, in the peripheral blood between healthy individuals and patients with different asthma conditions. Reverse transcription-quantitative PCR (RT-qPCR) and Western blotting were used to detect the mRNA and protein expression levels, respectively, of METTL3 and Foxp3 in CD4+ T cells in the peripheral blood samples of different groups. M6A blot and m6A coimmunoprecipitation-PCR were used to detect the global and Foxp3 mRNA m6A levels, respectively, in the peripheral blood CD4+ T cells. CD4+ T cells collected from the peripheral blood of patients with asthma were subjected to in vitro transfection to knockdown the METTL3 levels and observe changes in the Foxp3 mRNA, protein, m6A levels, and RNA stability. Flow cytometry and ELISA were used to detect the changes in the Treg cell proportion and IL-4 and IL-17 levels in the cell culture supernatant.Compared with the healthy individuals, the ratio of Treg cells to peripheral blood CD4+ T cells was significantly decreased and the Foxp3 mRNA and protein expression was downregulated in patients with asthma with disease progression. The Foxp3 mRNA and protein expression levels were positively correlated with the Treg cell proportion and negatively correlated with IL-17 expression. The global and Foxp3 mRNA m6A levels were increased in the peripheral blood CD4+ T cells of patients with asthma. METTL3 expression was significantly higher in the peripheral blood CD4+ T cells of patients with asthma compared with healthy individuals. After METTL3 knockdown, the Foxp3 mRNA m6A level was reduced, and the stability of Foxp3 mRNA and protein expression was increased. YTHDF2 could bind to the m6A site in 3'UTR of Foxp3 mRNA. Knockdown of YTHDF2 regulated the level and stability of Foxp3 mRNA. METTL3 knockdown reduced the ratio of Treg cells to CD4+ T cells and the IL-4 and IL-17 secretion levels from CD4+ T cells in the peripheral blood of patients with asthma.High METTL3 expression in the peripheral blood CD4+ T cells of patients with asthma increased the m6A level and reduced the stability of Foxp3 mRNA in a YTHDF2-dependent way, thereby reducing the expression of Foxp3 and the proportion of Treg cells.
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