Research Paper Volume 13, Issue 5 pp 6752—6764

Extracellular vesicle-encapsulated microRNA-23a from dorsal root ganglia neurons binds to A20 and promotes inflammatory macrophage polarization following peripheral nerve injury

miR-23a targets and negatively regulates A20. (A) predicted binding sites between miR-23a and A20 mRNA 3’-UTR; (B) detection of luciferase activity using dual-luciferase reporter assay; (C) A20 mRNA expression in macrophages after elevated expression of miR-185 by its specific agomir determined using RT-qPCR, normalized to β-actin; (D) A20 protein level in macrophages after elevated expression of miR-185 by its specific agomir determined using Western blot analysis, normalized to β-actin; (E) A20 mRNA expression in macrophages co-cultured with EVs determined using RT-qPCR, normalized to β-actin; (F) A20 protein level in macrophages co-cultured with EVs determined using Western blot analysis, normalized to β-actin. Values obtained from three independent experiments are expressed as mean ± SD and analyzed by unpaired t-test between two groups and by one-way ANOVA, followed by Bonferroni’s multiple comparison test among multiple groups; n = 3 cultures. * p vs. macrophages treated with antagomir-NC plasmids; # p vs. macrophages treated with antagomir-NC + miR-23a expression EVs. Cell experiment was independently repeated for three times.

Figure 4. miR-23a targets and negatively regulates A20. (A) predicted binding sites between miR-23a and A20 mRNA 3’-UTR; (B) detection of luciferase activity using dual-luciferase reporter assay; (C) A20 mRNA expression in macrophages after elevated expression of miR-185 by its specific agomir determined using RT-qPCR, normalized to β-actin; (D) A20 protein level in macrophages after elevated expression of miR-185 by its specific agomir determined using Western blot analysis, normalized to β-actin; (E) A20 mRNA expression in macrophages co-cultured with EVs determined using RT-qPCR, normalized to β-actin; (F) A20 protein level in macrophages co-cultured with EVs determined using Western blot analysis, normalized to β-actin. Values obtained from three independent experiments are expressed as mean ± SD and analyzed by unpaired t-test between two groups and by one-way ANOVA, followed by Bonferroni’s multiple comparison test among multiple groups; n = 3 cultures. * p < 0.05 vs. macrophages treated with antagomir-NC plasmids; # p < 0.05 vs. macrophages treated with antagomir-NC + miR-23a expression EVs. Cell experiment was independently repeated for three times.