Luo W, Wang S, Li Y Q, Wang J, Yang S S, Chao J
School of Medicine, Southeast University, Nanjing 210009, China.
Department of Physiology, School of Medicine, Southeast University, Nanjing 210009, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2021 Dec 20;39(12):899-902. doi: 10.3760/cma.j.cn121094-20210312-00141.
To explore the difference of methylation of circRNA related m6A in early inflammation of silicosis and to elucidate the underlying molecular mechanism of circRNA involved in the process of silicosis. The activation markers of macrophages were detected by Western blotting (WB) in THP-1-derived macrophages. The cell viability was detected with CCK8, by which the stimulation concentration and time of silica were determined. The methylation of total RNA was determined by colorimetry, and the expression of RNA m6A methylase, demethylase and reading protein were detected by Western blotting in mouse model of silicosis. The differential expression of m6A modified circRNA in lung tissues form silicosis and control mice was obtained through Arraystar m6A circRNA epigenetic transcriptome Chip and verified by RT-PCR. The concentration of SiO(2) at 50 μg/cm(2) had the most significant effect on the activation markers and activity of macrophages. Compared with the control group, SiO(2) increased the total RNA m6A level of macrophages, and there were significant differences in the expression of methylase METTL3 and reading protein YTDHF3. High throughput sequencing analysis showed that compared with the control group, the methylation levels of 132 circRNA m6A in the lung of silicosis model mice were increased, while the methylation levels of 296 circRNA m6A were decreased, and then the target circSLC2A13 was screened based on the basic expression. Further verification showed that SiO(2) significantly increased the expression of circSLC2A13 and m6A modification in macrophages. The methylation of circRNA m6A is involved in the activation of macrophages in early inflammation of silicosis.
探讨矽肺早期circRNA相关m6A甲基化的差异,阐明circRNA参与矽肺发生过程的潜在分子机制。采用蛋白质免疫印迹法(WB)检测THP-1来源巨噬细胞中巨噬细胞的活化标志物。用CCK8检测细胞活力,以此确定二氧化硅的刺激浓度和时间。采用比色法测定总RNA的甲基化水平,用蛋白质免疫印迹法检测矽肺小鼠模型中RNA m6A甲基转移酶、去甲基化酶和阅读蛋白的表达。通过Arraystar m6A circRNA表观转录组芯片获得矽肺和对照小鼠肺组织中m6A修饰的circRNA差异表达,并通过RT-PCR进行验证。50 μg/cm(2) 的SiO(2) 浓度对巨噬细胞的活化标志物和活性影响最为显著。与对照组相比,SiO(2) 增加了巨噬细胞的总RNA m6A水平,甲基转移酶METTL3和阅读蛋白YTDHF3的表达存在显著差异。高通量测序分析显示,与对照组相比,矽肺模型小鼠肺组织中132个circRNA m6A的甲基化水平升高,296个circRNA m6A的甲基化水平降低,然后根据基础表达筛选出靶circSLC2A13。进一步验证表明,SiO(2) 显著增加了巨噬细胞中circSLC2A13的表达和m6A修饰。circRNA m6A甲基化参与矽肺早期炎症中巨噬细胞的活化。