Figure 3

RhoA and ERK1 are the respective target genes of miR-125a-3p and miR-483-5p.

(A) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-RhoA-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-RhoA-3′-UTR) was cotransfected with the miR-125a-3p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (B) Dual-luciferase reporter plasmid containing human wild-type (WT-Pmir-ERK1-3′-UTR) or mutant RhoA-3′-UTR (MUT-Pmir-ERK1-3′-UTR) was cotransfected with the miR-483-5p mimic (100 nM) or nontarget control (NC), respectively. Firefly and renilla luciferase activity was determined and normalized to firefly luciferase activity. (C) hADSCs were transfected with the miR-125a-3p mimic, inhibitor and controls for 72 h. Total protein was extracted for immunoblotting of RhoA and ROCK1. (D) hADSCs were transfected with the miR-125a-3p agomir, antagomir and controls for 72 h and induced to mature adipocytes for 12 days. Total protein was extracted for immunoblotting of RhoA and ROCK1. (E) hADSCs were transfected with the miR-483-5p mimic, inhibitor and controls for 72 h. Whole cell protein was extracted for immunoblotting to detect total ERK1/2 (T-ERK1/2) and total phosphorylated ERK1/2 (T-p-ERK1/2). (F) hADSCs were transfected with the miR-483-5p mimic, inhibitor and controls for 72 h. Whole nuclear protein was extracted to detect nuclear total ERK1/2 (n-T-ERK1/2) and nuclear phosphorylated ERK1/2 (n-p-ERK1/2).*p < 0.05, compared to 293T cells transfected with miR control mimics. All measurements were preformed by three independent experiments.