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. Author manuscript; available in PMC: 2020 Dec 8.
Published in final edited form as: Gastroenterology. 2020 May 22;159(3):999–1014.e9. doi: 10.1053/j.gastro.2020.05.056

Figure 3.

Figure 3.

A newly identified RORA agonist RS-2982 affects liver and systemic MIR122 levels in mice. (A) Schematic presentation of MIR122 promoter reporter plasmid (pMIR122 luciferase) containing the RORA binding site. (B) Huh7 cells were transfected with the pMIR122, and 1 day later, cells were treated with 1 μmol/L of 1 of 10 different compounds. The compound solvent, 0.2% DMSO, was used as control, and 10 mol/L SR1078, a commercial RORA agonist, was used as a positive control. Luciferase activity was measured 48 hours after transfection. (C) The chemical structure of compound 1: RS-2982. (D) Huh7 cells were transfected with pMIR122 carrying a wild-type or mutated RORA binding site. One day later, cells were treated with different RS-2982 concentrations. The RS-2982 solvent, 0.2% DMSO, was used as control. Luciferase activity was measured 48 hours after transfection. Luciferase was normalized to Renilla luciferase activity expressed from a cotransfected pRL plasmid. (E) qRT-PCR analysis of microRNAs extracted from Huh7 cells or cell media after treatment with 1 μmol/L RS-2982 for 24 hours. miR-18 and miR-93 served as negative controls. (F–M) The RORA agonist RS-2982 affects MIR122 expression and secretion in mice. (F) C57BL/6 mice (n = 6) were administered 7.5 mg/kg RS-2982 or saline + 5% DMSO intraperitoneally, and 1, 2, and 7 days later, mice were killed, and RNA was extracted from various tissues for qRT-PCR analysis. MicroRNA levels were measured by qRT-PCR in (G) the liver and (H) the plasma. (I) qRT-PCR analysis of pri- and pre-MIR122, the MIR122 target gene AldoA, and the RORA target gene G6PC in the liver. (J–L) qRT-PCR analysis of microRNAs (J) in WAT, (K) in muscle, and (L) heart tissues. miR-126, miR-21, miR-34, and miR-18 levels were not increased after RS-2982 treatment. (M) qRT-PCR of MIR122 target genes in heart tissues. Plasma microRNA levels were normalized to spiked C elegans miR-39; microRNA levels in tissues were normalized to RNU6. mRNA levels were normalized to HPRT. Data are represented as mean ± SD. *P < .05, **P < .01. ***P < .001. cmpd, compound; M, mol/L; mut, mutated; wt, wild type.