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

• Published in: Journal of neuroinflammation Vol. 14
• Main Authors: Yin, Min, Chen, Zhiying, Ouyang, Yetong, Zhang, Huiyan, Wan, Zhigang, Wang, Han, Wu, Wei, Yin, Xiaoping
• Format: Journal Article
• Language: English
• Published: London BioMed Central 01.01.2017
• Subjects: 3' Untranslated regions; Antibodies; Cell cycle; Cytokines; Epigenetics; Gene expression; Granulocytes; Hemorrhage; Inflammation; Kinases; Magnetic fields; Microglia; MicroRNAs; miRNA; NF-κB protein; Polymerase chain reaction; Proteinase; Reverse transcription; RNA polymerase; Thrombin; Toxicity; Transfection; Tumor necrosis factor; Tumor necrosis factor receptors; Tumor necrosis factor-TNF
• ISSN: 1742-2094
• DOI: 10.1186/s12974-017-0887-5

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.