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Published on: January 7, 2019
Thrombin-induced, TNFR-dependent miR-181c downregulation promotes MLL1 and NF-κB target gene expression in human
Min Yin1, Zhiying Chen2, Yetong Ouyang1
1Department of Neurology, The Second Affiliated Hospital of Nanchang University, No. 1 Minde Road, Nanchang, 330006, Jiangxi Province, China.
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.
Insights
MicroRNA-181c (miR-181c) downregulation promotes inflammation in brain injury by increasing MLL1 expression and NF-κB activity. Restoring miR-181c levels may offer a novel therapy for intracerebral hemorrhage (ICH).
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Microglial activation by thrombin is a key factor in intracerebral hemorrhage (ICH) pathogenesis.
- Targeting thrombin-driven microglial activation presents a potential therapeutic strategy for ICH.
Purpose of the Study:
- To investigate the role of microRNA (miRNA)-based regulation in thrombin-induced microglial activation.
- To identify specific miRNAs involved in the inflammatory response following ICH.
Main Methods:
- Utilized an in vitro model of thrombin-induced microglial activation using primary human microglia.
- Employed miRNA array, qRT-PCR, luciferase reporter assays, and gene silencing techniques.
- Assessed the impact of miR-181c on MLL1 expression and NF-κB signaling pathways.
Main Results:
- Thrombin exposure led to miR-181c downregulation and MLL1 upregulation, dependent on TNF-α/TNFR signaling.
- miR-181c was confirmed to directly target and downregulate MLL1 expression.
- Thrombin increased NF-κB activity, while miR-181c mimic transfection reduced it.
Conclusions:
- Thrombin-induced miR-181c downregulation promotes MLL1 expression and enhances pro-inflammatory NF-κB activity.
- miR-181c acts as a negative regulator of thrombin-driven microglial activation.
- miR-181c mimic therapy holds promise for managing ICH by controlling microglial inflammation.
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