Figure 6.

Overexpression of miR-23b-5p impaired brown adipocytes thermogenesis mainly through decreasing lipolysis-and β-oxidation-related genes expression. (A) TargetScan predicted miR-23b-5p binds the 3′ UTR of Slc27a4 (Fatp4), Adam12, and Ern1. (B) The relative expression of targets genes after miR-23b-5p overexpression in mouse brown adipocytes. (C) Ern1 expression level upon cold exposure in BAT and its expression level in the brown adipocytes treated with CL (10 µM) and Fsk (10 μM) for 4 h. (D) The psi-CHECK2 luciferase reporter plasmids containing Ern1 WT (WT1 and WT2) 3′UTR or mutant 3′UTR (MUT1 and MUT2) were transfected into mouse 3T3-L1 cells (six replicates per group) for 16 h followed by miR-23b-5p or NC lentivirus infection. After 48 h infection, cells were harvested and renilla luciferase activity was analyzed using the dual-luciferase reporter Assay System and normalized to firefly luciferase activity. (E, F, and G) The relative expression of lipolysis, the electron transport chain (ETC)-, fatty acid oxidation-related genes involved in brown adipocytes thermogenesis were detected in NC and miR-23b-5p overexpression groups by qPCR and normalized to Ppia expression. (H and I) The relative protein amount of AMPKa (predicted in 63 kDa), HSL (predicted in 81–83 kDa), ATGL (predicted in 54 kDa), GLUT4 (predicted in 55 kDa), ACCSL1 (predicted in 78 kDa), ACSL1 (predicted in 78 kDa), ACADM (predicted in 45 kDa), and ACADS (predicted in 43 kDa) among NC and miR-23b-5p overexpression groups. Values are the means ± s.d. of three separated experiments. *P < 0.05; **P < 0.01; ***P < 0.001.