Fig. 5
MiR-9 regulated microglial activation by targeting HECTD1. a Putative miR-9 binding sites in HECTD1 (HECTD1 gene). b Relative luciferase activity of wild-type and 3’-UTR mutant constructs of HECTD1 co-transfected with miR-9 mimics and miRNA negative control. The data were presented as mean ± SEM. *p < 0.05 versus miR-NC WT UTR using Student’s t test. c Transduction-ACT-upregulated HECTD1 significantly inhibited the increased iNOS expression induced by LPS in primary mouse microglia. All the data were presented as mean ± SEM of 3 independent experiments. (HECTD1-ACT: F(1,8) = 5.391, P < 0.05; LPS: F(1,8) = 29.975, P < 0.01; interaction: F(1,8) = 11.766, P < 0.01. **P < 0.01 vs. Control-ACT Control group; #p < 0.05 versus Control-ACT treated with LPS group). d Transfection of cells with HECTD1-ACT significantly inhibited the increased iNOS expression induced by miR-9 in primary mouse microglia. All data were presented as mean ± SEM of 3 independent experiments. (HECTD1-ACT: F(1,8) = 7.627, P < 0.05; miR-9: F(1,8) = 39.874, P < 0.001; interaction: F(1,8) = 5.861, P < 0.05. **p < 0.01 vs. miR-Control transfected with Control-ACT group; #p < 0.05 vs. miR-9 transfected with Control-ACT group). e Effect of circDYM overexpression on the decreased expression HECTD1 induced by LPS. Cells were transduced with the circControl/circDYM-GFP lentivirus for 24 h and then treated with LPS (100 ng/ml) for another 24 h in primary mouse microglia. All data were presented as mean ± SEM of 3 independent experiments. (circDYM: F(1,8) = 8.703, P < 0.05; CUS: F(1,8) = 21.364, P < 0.01; interaction: F(1,8) = 12.326, P < 0.01. *p < 0.05 vs. circControl Control group; #p < 0.05 versus circControl treated with LPS group). f circDYM overexpression significantly attenuated the decreased expression of HECTD1 induced by miR-9 in primary mouse microglia. All the data were presented as mean ± SEM of 3 independent experiments. (circDYM: F(1,8) = 13.306, P < 0.01; miR-9: F(1,8) = 36.924, P < 0.001; interaction: F(1,8) = 14.735, P < 0.01. **p < 0.01 versus miR-Control transduced with circControl group; #p < 0.05 versus miR-9 transduced with circControl group). g, h Effect of circDYM overexpression on the decreased expression of HECTD1 in the CUS (g) and LPS (h) model. Mice were microinjected with the circControl/circDYM-GFP lentivirus in the hippocampus. One week after microinjection, mice were exposed to a CUS protocol for 5 weeks or intraperitoneally injected with LPS (1 mg/kg) for 5 successive days. Three representative immunoblots were presented from 6 mice/group. All data were presented as mean ± SEM. (CUS model, circDYM: F(1,20) = 50.088, P < 0.001; CUS: F(1,20) = 46.113, P < 0.001; interaction: F(1,20) = 5.085, P < 0.05. LPS model, circDYM: F(1,20) = 36.839, P < 0.001; LPS: F(1,20) = 48.185, P < 0.001; interaction: F(1,20) = 5.034, P < 0.05). ***p < 0.001 versus circControl Control group; ##p < 0.01 vs. circControl treated with CUS/LPS group. Control-ACT Control CRISPR Activation Plasmid (ACT), HECTD1-ACT HECTD1 CRISPR Activation Plasmid (ACT)
