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

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. © 2017 the author(s).