摘要
目的主要研究巨噬细胞中核受体结合SET结构域蛋白3(NSD3)对脂多糖(LPS)触发的肿瘤坏死因子α(TNF-α)的调控作用,并探讨其调控机制。方法以小鼠腹腔巨噬细胞和RAW264. 7细胞作为细胞模型。100 ng/m L LPS刺激小鼠腹腔巨噬细胞,采用实时定量PCR检测NSD3 mRNA水平,Western blot法检测NSD3蛋白水平;在RAW264. 7细胞中过表达NSD3或采用RNA干扰技术敲低腹腔巨噬细胞中NSD3的表达,ELISA检测NSD3对LPS触发的TNF-α分泌的影响; Western blot法检测敲低NSD3对LPS触发的核因子κB p65(NF-κBp65)信号通路的影响;荧光素酶法检测NSD3对NF-κBp65介导的TNF-α基因转录活性的影响;染色质免疫沉淀实验检测TNF-α基因启动子区组蛋白H3的36位赖氨酸(H3K36)甲基化的募集。结果 LPS抑制巨噬细胞中NSD3的表达;过表达NSD3抑制LPS触发的TNF-α的产生,敲低NSD3则促进LPS触发的TNF-α的产生,但对NF-κB活化无影响; NSD3抑制NF-κBp65介导的TNF-α基因的转录活化,促进TNF-α基因启动子区的H3K36二甲基化。结论 NSD3促进TNF-α基因启动子区H3K36的双甲基化,抑制TNF-α的表达。
Objective To investigate the role of nuclear receptor-binding SET domain protein 3（ NSD3） in lipopolysaccharide（ LPS）-triggered tumor necrosis factor α（ TNF-α） production in macrophages and the underlying epigenetic mechanism. Methods The experiment used murine peritoneal macrophages and RAW264. 7 cells as cell models. The mRNA and protein level of NSD3 were detected by real-time quantitative PCR and Western blot analysis in mouse peritoneal macrophages stimulated with LPS（ 100 n/ L）. ELISA was used to detect the production of TNF-α in NSD3-overexpressing RAW264. 7 cells or NSD3-silencing peritoneal macrophages. Western blot analysis was performed to test the activation of LPS-triggered NF-κBp65 in NSD3-silencing macrophages. Luciferase assay was used to assess NF-κBp65-mediated transcriptional activation of TNF-α gene. Ch IP assay was used to detect the recruitment of H3 K36 methylation to TNF-α gene promoter. Results LPS inhibited the expression of NSD3 in the macrophages. Over-expression of NSD3 suppressed LPS-triggered TNF-α production,and silencing NSD3 promoted LPS-triggered TNF-α production. However,NSD3 had no effect on the activation of LPS-triggered NF-κBp65. NSD3 enhanced NF-κBp65-mediated transcriptional activation of TNF-αgene. NSD3 enhanced the dimethylation of H3 K36 of TNF-α gene promoter. Conclusion NSD3 can promote the dimethylation of H3 K36 of TNF-α gene promoter and suppress TNF-α transcription and production.
作者
刘兆龙
程起江
杨乐
孙燕翔
LIU Zhaolong;CHENG Qijiang;YANG Le;SUN Yanxiang(Department of General Surgery,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203;Department of Immunology,Second Military Medical University,Shanghai 200433,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2018年第6期481-487,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81672798)
医学免疫学国家重点实验室开放课题(NKLMI2017K11)
