摘要
目的探索赖氨酸乙酰基转移酶6B(KAT6B)在巨噬细胞中对脂多糖(LPS)诱导的白细胞介素6(IL-6)分泌的调控作用及机制。方法体外培养小鼠原代腹腔巨噬细胞与RAW264.7巨噬细胞并以100 ng/mL LPS刺激0、2、4、6 h,采用实时定量PCR(qRT-PCR)检测KAT6B mRNA表达水平;利用RNA干扰技术敲低小鼠腹腔巨噬细胞KAT6B的表达,qRT-PCR检测LPS诱导的小鼠原代巨噬细胞IL-6 mRNA水平,ELISA检测IL-6蛋白水平;同时在RAW264.7细胞中过表达KAT6B,qRT-PCR检测其产生IL-6 mRNA的水平,ELISA检测分泌IL-6蛋白的水平。Western blot法检测对LPS诱发的核因子κB(NF-κB)与丝裂原活化蛋白激酶(MAPK)信号通路的影响,通过双荧光素酶报告基因试验检测KAT6B对IL-6基因的启动子区的影响;利用染色质免疫沉淀技术(Ch IP)检测KAT6B对IL-6基因启动子区第23位赖氨酸乙酰化的组蛋白3(H3K23ac)募集的影响。结果LPS上调小鼠腹腔巨噬细胞与RAW264.7细胞中KAT6B mRNA的表达;敲低KAT6B水平后,抑制腹腔巨噬细胞IL-6的分泌;过表达V5-KAT6B促进RAW264.7细胞IL-6的分泌;NF-κB与MAPK相关分子活化无差异;KAT6B在NF-κBp65下游发挥对IL-6的调控作用;KAT6B增强IL-6启动子区H3K23ac的募集促进IL-6的转录。结论KAT6B通过增强IL-6基因启动子区H3K23的乙酰化修饰促进LPS诱导的IL-6的产生。
Objective To explore the regulatory role of lysine acetyltransferase 6B(KAT6B)in lipopolysaccharide(LPS)-triggered interleukin 6(IL-6)production in macrophages and the mechanism.Methods Real-time quantitative PCR(qRT-PCR)was performed to detect and quantitate KAT6B mRNA level in mouse peritoneal macrophages and RAW264.7 cells under LPS stimulation for 0,2,4,6 hours.RNA interference technology was used to knock down the expression of KAT6B in peritoneal macrophages,the expression of IL-6 in LPS-stimulated murine macrophages was detected by qRT-PCR at the mRNA level and ELISA at the protein level;meanwhile,the levels of IL-6 mRNA and protein were tested by the same means in RAW264.7 cells with over-expressed KAT6B.The transfection efficiency and signal pathway activation were examined by Western blot analysis.Dual-luciferase reporter assay was used to investigate the role of KAT6B in IL-6 transcription.Chromatin immunoprecipitation(Ch IP)was done to evaluate the effect of KAT6B on the recruitment of acetylation of histone 3 lysine 23(H3K23 ac)within IL-6 promoter region.Results LPS stimulation up-regulated KAT6B expression in both peritoneal macrophages and RAW264.7 cells.Silence of KAT6B suppressed LPS-induced IL-6 production in murine peritoneal macrophages,overexpression of V5-KAT6B promoted the production of LPS-triggered IL-6 in RAW264.7 cells.The change of KAT6B level did not affect the activity of NF-κBp65 and MAPK induced by LPS.KAT6B increased the recruitment of H3K23 ac on IL-6 gene DNA promoter.Conclusion KAT6B can enhance LPS-triggered IL-6 production by promoting the recruitment of H3K23 ac to IL-6 gene promoter region.
作者
孙东豪
温巧莲
王春梅
SUN Donghao;WEN Qiaoian;WANG Chunmei(Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences,School of Basic Medicine,Peking Union Medical College,Beijing 100005,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第11期1441-1447,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31270931)
