摘要
目的探讨miR-155在THP-1巨噬细胞炎性反应中的作用及调控机制。方法使用脂多糖(LPS)处理THP-1来源的巨噬细胞构建体外巨噬细胞炎性模型,分为control组、LPS组、miR-155 mimics+LPS组和mimics NC+LPS组;采用实时荧光定量PCR(RT-qPCR)检测上述各组中miR-155、炎性细胞因子(TNF-α、IL-1β)mRNA、STAT3 mRNA表达水平,酶联免疫吸附法(ELISA)检测细胞上清中炎性细胞因子(TNF-α、IL-1β)含量,免疫印迹(Western blot)法检测细胞中STAT3蛋白及STAT3磷酸化(p-STAT3)蛋白水平。结果LPS诱导THP-1巨噬细胞中miR-155表达增加,同时炎性细胞因子(TNF-α、IL-1β)的表达增加;转染miR-155 mimics组进一步促进LPS诱导的巨噬细胞中上述炎性细胞因子的表达;Western blot法分析显示miR-155 mimics+LPS组中p-STAT3蛋白表达水平较LPS组进一步增加。结论miR-155可上调THP-1巨噬细胞炎症中TNF-α、IL-1β的表达,其调控机制是增加转录因子STAT3的磷酸化活化,进而靶向JAK/STAT3信号通路促进LPS诱导的THP-1巨噬细胞炎性反应。
Objective To investigate the role and regulatory mechanism of miR-155 in THP-1 macrophage inflammation.Methods THP-1-derived macrophages were treated with lipopolysaccharide(LPS)to construct an in vitro macrophage inflammation model,which was divided into control group,LPS group,miR-155 mimics+LPS group and mimics NC+LPS group.Real-time fluorescence quantitative PCR(RT-QPCR)was used to detect the mRNA expression levels of miR-155,inflammatory factors(TNF-α,IL-1β)and STAT3 mRNA in the above groups,and enzyme-linked immunosorbent assay(ELISA)was used to detect the content of inflammatory factors(TNF-α,IL-1β)in cell supernatant.The levels of STAT3 protein and STAT3 phosphorylation(P-STAT3)in cells were detected by Western blot.Results The expression of miR-155 and inflammatory factors(TNF-αand IL-1β)were increased in THP-1 macrophages induced by LPS.Transfection of miR-155 mimics group further promoted the expression of the above inflammatory factors in LPS-induced macrophages.Western blot analysis showed that p-STAT3 protein expression level in miR-155 mimics+LPS group was further increased compared with that in LPS group.Conclusion miR-155 up-regulates the expression of TNF-αand IL-1βin THP-1 macrophage inflammation by increasing the phosphorylation activation of transcription factor STAT3,and then targeting the JAK/STAT3 signaling pathway to promote LPS-induced THP-1 macrophage inflammation.
作者
谭仕廉
李艳丽
何永勋
郭乐
申元英
TAN Shilian;LI Yanli;HE Yongxun(Department of Microbiology and Immunology,College of Basic Medicine,Dali University,Yunnan 671000,China)
出处
《医学研究杂志》
2022年第4期41-45,98,共6页
Journal of Medical Research
基金
国家自然科学基金资助项目(81641095,81960371)
云南省科技厅青年项目(202001AU070014)。
