摘要
目的探究矽肺早期炎症中环状RNA(circRNA)的氮6-腺苷酸甲基化修饰(m6A)差异,分析circRNA参与矽肺进程的分子机制。方法体外培养人单核细胞系(THP-1)诱导的巨噬细胞,蛋白免疫印迹法(Western blot)检测巨噬细胞活化标志物,细胞计数试剂盒8(CCK8)法检测细胞活力,确定二氧化硅(SiO_(2))刺激浓度。比色法测定矽肺模型中总RNA的m6A甲基化修饰变化,检测m6A甲基化酶、去甲基酶和阅读蛋白表达情况。Arraystar m6A-circRNA表观转录组芯片检测二氧化硅模型组和对照组小鼠肺组织中m6A修饰差异表达的circRNA,并用荧光实时定量PCR(RT-qPCR)验证遴选出高表达circRNA。结果SiO_(2)浓度为50μg/cm^(2)时对THP-1诱导的巨噬细胞活化标志物和细胞活性影响最为明显(P<0.05)。与对照组比较,SiO_(2)引起小鼠原代巨噬细胞和THP-1诱导的巨噬细胞总RNA的m6A水平升高,甲基化酶METTL3和阅读蛋白YTDHF3表达量明显升高,差异均有统计学意义(P<0.05)。与对照组比较,矽肺模型小鼠肺中132个circRNA的m6A甲基化修饰水平升高,296个circ RNA的m6A甲基化修饰水平下降,基于基础表达量筛选得到靶标-circSLC2A13;SiO_(2)引起巨噬细胞中circSLC2A13的表达和m6A化修饰明显升高(P<0.05)。结论矽肺早期炎症阶段存在RNA的m6A甲基化修饰异常现象,其中circSLC2A13的m6A甲基化修饰参与矽肺早期炎症中巨噬细胞的激活。
Objective 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.Methods 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.Results The concentration of SiO_(2) at 50μg/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.Conclusion The methylation of circRNA m6A is involved in the activation of macrophages in early inflammation of silicosis.
作者
罗薇
王莎
李永琪
王静
杨少奇
巢杰
Luo Wei;Wang Sha;Li Yongqi;Wang Jing;Yang Shaoqi;Chao Jie(School of Medicine,Southeast University,Nanjing 210009,China;Department of Physiology,School of Medicine,Southeast University,Nanjing 210009,China;School of Public Health,Southeast University,Nanjing 210009,China)
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
北大核心
2021年第12期899-902,共4页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
国家自然科学基金项目(81803182、81972987、81773796)。
