Abstract
BackgroundMicroglia are important immune cells, which can be induced by lipopolysaccharide (LPS) into M1 phenotype that express pro-inflammatory cytokines. Some studies have shown that microRNAs play critical roles in microglial activation.ObjectiveThis study was designed to investigate the role of miR-200c-3p in regulating inflammatory responses of LPS-treated BV2 cells.MethodsThe expression of miR-200c-3p in BV2 cells was detected by real-time PCR. Receptor-interacting protein 2 (RIP2) was predicted as a target gene of miR-200c-3p. Their relationship was verified by dual-luciferase reporter assay. The function of miR-200c-3p and RIP2 in microglial polarization and NF-κB signaling was further evaluated.ResultsLPS treatment reduced miR-200c-3p expression in a dose-dependent and time-dependent manner in BV2 cells. LPS treatment increased the expression of M1 phenotype markers inducible nitric oxide synthase (iNOS) and major histocompatibility complex class (MHC)-II, promoted the release of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and enhanced the nuclear translocation and phosphorylation of nuclear factor-kappaB (NF-κB) p65. Reversely, miR-200c-3p mimics down-regulated the levels of these inflammatory factors. Furthermore, RIP2 was identified to be a direct target of miR-200c-3p. RIP2 knockdown had a similar effect to miR-200c-3p mimics. Overexpression of RIP2 eliminated the inhibitory effect of miR-200c-3p on LPS-induced M1 polarization and NF-κB activation in BV2 cells.ConclusionsMiR-200c-3p mimics suppressed LPS-induced microglial M1 polarization and NF-κB activation by targeting RIP2. MiR-200c-3p/RIP2 might be a potential therapeutic target for the treatment of neuroinflammation-associated diseases.
Highlights
Microglia are a unique group of cells in central nervous system that originate in myeloid system
Data were represented as mean ± SD (n = 3) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. #p < 0.05 vs. Con group; *p < 0.05 vs. Con mimics; *p < 0.05 vs. LPS or the indicated group levels of inflammatory cytokines IL-1β, IL-6 and tumor necrosis factor (TNF)-α in cell supernatant (p < 0.05), but these increases were reversed by miR-200c-3p mimics (p < 0.05) (Fig. 2D)
Data were represented as mean ± SD (n = 3) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. #p < 0.05 vs. Con group; *p < 0.05 vs. LPS or the indicated group was used to assess Receptor-interacting protein 2 (RIP2) mRNA expression in BV2 cells with miR200c-3p mimics transfection
Summary
Microglia are a unique group of cells in central nervous system that originate in myeloid system. Microglia are important immune cells, which can be induced by lipopolysaccharide (LPS) into M1 phenotype that express pro-inflammatory cytokines. Receptor-interacting protein 2 (RIP2) was predicted as a target gene of miR-200c-3p. Their relationship was verified by dual-luciferase reporter assay. Results LPS treatment reduced miR-200c-3p expression in a dose-dependent and time-dependent manner in BV2 cells. LPS treatment increased the expression of M1 phenotype markers inducible nitric oxide synthase (iNOS) and major histocompatibility complex class (MHC)-II, promoted the release of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and enhanced the nuclear translocation and phosphorylation of nuclear factor-kappaB (NF-κB) p65. Overexpression of RIP2 eliminated the inhibitory effect of miR-200c-3p on LPS-induced M1 polarization and NF-κB activation in BV2 cells. Conclusions MiR-200c-3p mimics suppressed LPS-induced microglial M1 polarization and NF-κB activation by targeting RIP2. MiR-200c-3p/RIP2 might be a potential therapeutic target for the treatment of neuroinflammation-associated diseases
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