Fig. 3: Lin28/let-7 regulates the ketogenesis repressor NCoR1.
a Ketogenesis pathway. b Expression of ketogenic genes from C1632-treated 7-week-old, male, wild-type mice after 5 daily IP injections (50 mg/kg) (n = 4 mice/group; Pparα: *P = 0.0285; Cpt1a: *P = 0.0338; Hmgcs2: ***P = 0.0006; Hmgcl: *P = 0.0272). c NCoR1 represses PPARα-controlled ketogenesis. d Immunoblot analysis for NCoR1 and Lin28B in liver tissues from treated mice as described in (b). e Immunoblot analysis for NCoR1 in HepG2 cells after 1632-treatment. f Analysis for NCoR1 in HepG2 cells with/without Lin28A/B overexpression. g Immunoblot analysis for NCoR1 and Lin28B in HepG2 cells after pre-let-7 transfections (pre-miR-16-2 as control). h Predicted base-pairing between let-7 and NCoR1 3’UTR. Wild-type or mutated (mut) NCoR1 sequences inserted into dual-luciferase vectors for assays in HEK293T cells (n = 3 biologically independent samples/group; let-7a: *P = 0.0447; let-7c: **P = 0.0064). i HEK293T cells were treated with C1632 and wild-type plasmid and luciferase activity was assayed (n = 4 independent samples; 0 vs 25 μM: *P = 0,0400; 0 vs 50 μΜ: *P = 0,0340; 0 vs 100 μM: *P = 0,0299). j Relative PPARα and CPT1a mRNA levels in HepG2 cells transfected with pre-let-7 (n = 3 independent samples/group; (Ppara) mock vs let-7a1: *P = 0.0171; mock vs let-7c: *P = 0.0157; mock vs let-7e: ***P = 0.0002; mock vs. let-7g: ****P < 0.0001; (Cpt1a) mock vs let-7a1: **P = 0.0074; mock vs let-7c: **P = 0.0052; mock vs let-7d: *P = 0.0231; mock vs let-7e: *P = 0.0300; mock vs let-7g: **P = 0.0065). k, m HepG2 cells treated with C1632/rapamycin combinations. Rapamycin activity was validated by measuring effects on P-S6. NCoR1 levels were determined by immunoblot analysis. l, n β-OHB levels in the medium of cells treated as indicated (n = 3 independent samples/group; ****P < 0.0001; vehicle vs. Rapamycin (10 nM): **P = 0.0056; vehicle vs. Rapamycin (10 nM) +1632 (30 μM): ***P = 0.0006; vehicle vs. Rapamycin (10 nM)+1632 (60 μM): ***P = 0.0002; vehicle vs. Rapamycin (10 nM)+1632 (90 μM): ****P < 0.0001; vehicle vs. Rapamycin (10 nM)+1632 (120 μM): ****P < 0.0001). o Schematic showing effects of Rapamycin and C1632: Rapamycin inhibits mTORC1 and prevents nuclear translocation of NCoR152, whereas let-7 inhibits NCoR1 synthesis (this work). Densitometry of immunoblots are provided in Fig. S9a-f. Values are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001. Groups were compared using two-tailed unpaired Student’s t-test and 1-way ANOVA with Bonferroni’s post hoc test. Source data provided in Source Data file.
