Thrombin-induced, TNFR-dependent miR-181c downregulation promotes MLL1 and NF-κB target gene expression in human microglia

J Neuroinflammation. 2017 Jun 29;14(1):132. doi: 10.1186/s12974-017-0887-5.

Abstract

Background: Controlling thrombin-driven microglial activation may serve as a therapeutic target for intracerebral hemorrhage (ICH). Here, we investigated microRNA (miRNA)-based regulation of thrombin-driven microglial activation using an in vitro thrombin toxicity model applied to primary human microglia.

Methods: A miRNA array identified 22 differential miRNA candidates. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) identified miR-181c as the most significantly downregulated miRNA. TargetScan analysis identified mixed lineage leukemia-1 (MLL1) as a putative gene target for miR-181c. qRT-PCR was applied to assess tumor necrosis factor-alpha (TNF-α), miR-181c, and MLL1 levels following thrombin or proteinase-activated receptor-4-specific activating peptide (PAR4AP) exposure. Anti-TNF-α antibodies and tumor necrosis factor receptor (TNFR) silencing were employed to test TNF-α/TNFR dependence. A dual-luciferase reporter system and miR-181c mimic transfection assessed whether mir-181c directly binds to and negatively regulates MLL1. Nuclear factor kappa-B (NF-κB)-dependent luciferase reporter assays and NF-κB target gene expression were assessed in wild-type (MLL1+) and MLL1-silenced cells.

Results: Thrombin or PAR4AP-induced miR-181c downregulation (p < 0.05) and MLL1 upregulation (p < 0.05) that were dependent upon TNF-α/TNFR. miR-181c decreased wild-type MLL1 3'-UTR luciferase reporter activity (p < 0.05), and a miR-181c mimic suppressed MLL1 expression (p < 0.05). Thrombin treatment increased, while miR-181c reduced, NF-κB activity and NF-κB target gene expression in both wild-type (MLL1+) and MLL1-silenced cells (p < 0.05).

Conclusions: Thrombin-induced, TNF-α/TNFR-dependent miR-181c downregulation promotes MLL1 expression, increases NF-κB activity, and upregulates NF-κB target gene expression. As miR-181c opposes thrombin's stimulation of pro-inflammatory NF-κB activity, miR-181c mimic therapy may show promise in controlling thrombin-driven microglial activation following ICH.

Keywords: ICH; Intracerebral hemorrhage; MicroRNA; Microglia; Thrombin.

MeSH terms

  • Adult
  • Cells, Cultured
  • Down-Regulation / drug effects
  • Down-Regulation / physiology
  • Female
  • Gene Expression
  • HEK293 Cells
  • Histone-Lysine N-Methyltransferase / biosynthesis*
  • Histone-Lysine N-Methyltransferase / genetics
  • Humans
  • Male
  • MicroRNAs / metabolism*
  • Microglia / drug effects
  • Microglia / metabolism*
  • Middle Aged
  • Myeloid-Lymphoid Leukemia Protein / biosynthesis*
  • Myeloid-Lymphoid Leukemia Protein / genetics
  • NF-kappa B / biosynthesis*
  • NF-kappa B / genetics
  • Receptors, Tumor Necrosis Factor / metabolism*
  • Thrombin / pharmacology*

Substances

  • KMT2A protein, human
  • MIrn181 microRNA, human
  • MicroRNAs
  • NF-kappa B
  • Receptors, Tumor Necrosis Factor
  • Myeloid-Lymphoid Leukemia Protein
  • Histone-Lysine N-Methyltransferase
  • Thrombin