title: MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability.
abstract: OBJECTIVE
  MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages.
  
  
  METHODS
  Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3'-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip).
  
  
  RESULTS
  LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability.
  
  
  CONCLUSIONS
  miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases.
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Qin
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Wan
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Ming-He
authors.initials: MH
authors.lastname: Cheng
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Peng-Yuan
authors.initials: PY
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Ding-Feng
authors.initials: DF
authors.lastname: Su
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Jian-Guang
authors.initials: JG
authors.lastname: Yu
authors.affiliation: Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D055785:Gene Knockdown Techniques; D006207:Half-Life; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000172:deficiency* / Q000235:genetics; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; C509207:MIRN124 microRNA, human; D035683:MicroRNAs; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C469268:Usp14 protein, mouse; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; C579224:Usp2 protein, mouse