Figure legends
Fig 1. Alterations in the maternal adrenal and placental functions, and the level of CORT in fetal male rats with PDE.(A) Maternal serum CORT concentration; (B) Maternal serum ACTH concentration; (C) The mRNA expression of maternal adrenal steroidogenic genes; (D) Fetal serum CORT concentration; (E) Placental Mdr1a, Mdr1b, 11β-HSD1, 11β-HSD2 mRNA expression; (G, H) Placental Mdr1a, Mdr1b, 11β-HSD1, 11β-HSD2 protein expression; (F, I) The ratio of placental 11β-HSD1/11β-HSD2 mRNA and protein expression; (J) Correlations between maternal serum CORT concentration and fetal serum CORT concentration. n=8 placentas or adrenals for qRT-PCR and n=3 for Western blot in each group. Comparisons between groups were performed using Student’s t -test or Pearson correlation analysis. Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective controls. CORT, corticosterone; PDE, prenatal dexamethasone exposure; ACTH, adrenocorticotropic hormone; Mdr1a, multidrug-resistance protein 1a; 11β-HSD1, hydroxysteroid 11-beta dehydrogenase 1; StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 cholesterol side-chain cleavage; 3β-HSD, 3β-hydroxysteroid dehydrogenase; P450c21, steroid 21-hydroxylase; P450c11, steroid 11β-hydroxylase.
Fig 2. Alterations in serum CORT level, histone acetylation and expression levels of IGF1, adrenal steroidogenesis, and related indicators in fetal male rats with PDE.(A, D, E, H) The mRNA expression of fetal adrenal steroidogenic genes, IGF1 signal pathway, GRα and Sirt3; (B, C) StAR protein expression (yellow represents StAR positive cells, 400×); (F) Clustering of miRNAs differentially expressed in fetal adrenal; (G) Adrenal miR-370-3p expression; (I) Enrichment of H3K9ac, H3K14ac and H3K27ac levels of IGF1 promoter region; preimmune IgG served as the negative control. n=8 adrenals for qRT-PCR and n=5 for IHC/ChIP assay in each group. Comparisons between groups were performed using Student’s t -test Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective controls. CORT, corticosterone; IGF1, insulin-like growth factor 1; PDE, prenatal dexamethasone exposure; GRα, glucocorticoid receptor alpha; H3K9ac, histone 3 lysine 9 acetylation; StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 cholesterol side-chain cleavage; 3β-HSD, 3β-hydroxysteroid dehydrogenase; P450c21, steroid 21-hydroxylase; P450c11, steroid 11β-hydroxylase; IGF1R, IGF1 receptor; Akt1, protein kinase B.
Fig 3. Alterations in serum CORT level, histone acetylation and expression levels of IGF1, adrenal steroidogenesis, and related indicators s in male offspring rats with PDE.(A) Adult serum CORT concentration; (B, D, E, G) Adrenal GRα, Sirt3, IGF1 signal pathway, StAR and P450scc mRNA expression; (F) miR-370-3p expression; (C) Adrenal StAR protein expression (yellow represents StAR positive cells, 400×); (H) Enrichment of H3K9ac, H3K14ac and H3K27ac levels of IGF1 promoter region; preimmune IgG served as the negative control. n=8 adrenals for qRT-PCR and n=5 for IHC/ChIP assay in each group. Comparisons between groups were performed using Student’st -test. Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective controls. CORT, corticosterone; IGF1, insulin-like growth factor 1; PDE, prenatal dexamethasone exposure; GRα, glucocorticoid receptor alpha; StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 cholesterol side-chain cleavage; H3K9ac, histone 3 lysine 9 acetylation; IGF1R, IGF1 receptor; Akt1, protein kinase B.
Fig 4. Effects of chronic stress on CORT level, the histone acetylation and expression levels of IGF1, steroidogenesis, and related indicators in male rats with PDE.(A) Serum CORT concentration; (B-D,F) Adrenal GRα, Sirt3, IGF1, StAR and P450scc mRNA expression; (E) miR-370-3p expression; (G) Enrichment of H3K27ac level of IGF1 promoter region; preimmune IgG served as the negative control. n=8 adrenals for qRT-PCR and n=5 for ChIP assay in each group. Mean ± S.E.M., *P <0.05,**P <0.01 vs . respective controls. CORT, corticosterone; IGF1, insulin-like growth factor 1; PDE, prenatal dexamethasone exposure; GRα, glucocorticoid receptor alpha; StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 cholesterol side-chain cleavage; H3K27ac, histone 3 lysine 27 acetylation.
Fig 5. Effects of dexamethasone on the histone acetylation and expression levels of IGF1 and steroidogenesis in NCI-H295R cells.(A) IGF1 mRNA expression; (B, E) StAR mRNA expression; (C) IGF1 and StAR protein expression; (D) Intracellular cortisol concentration; (F) Enrichment of H3K9ac, H3K14ac, H3K27ac levels of IGF1 promoter region; preimmune IgG served as the negative control. n=6 for qRT-PCR from triplicate independent experiments and n=5 for Western blot in each group. Mean ± S.E.M.,*P <0.05,**P <0.01 vs control;#P <0.05,##P <0.01 vs 500 nM dexamethasone by one-way ANOVA or Student’s t -test. IGF1, insulin-like growth factor 1; StAR, steroidogenic acute regulatory protein; H3K9ac, histone 3 lysine 9 acetylation.
Fig 6. Effects of low physiological concentrations of cortisol on the histone acetylation and expression levels of IGF1, steroidogenesis and related indicators in NCI-H295R cells. (A, C-E, G) StAR, GRα, miR-370-3p, Sirt3 and IGF1 expression; (B) Intracellular cortisol concentration; (G) Enrichment of H3K9ac, H3K14ac, H3K27ac levels of IGF1 promoter region; preimmune IgG served as the negative control; (H) StAR, Sirt3 and IGF1 protein expression. n=6 for qRT-PCR from triplicate independent experiments and n=5 for Western blot/ChIP assay in each group. Comparisons between groups were performed using Student’s t -test and ANOVA. Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective controls. IGF1, insulin-like growth factor 1; StAR, steroidogenic acute regulatory protein; GRα, glucocorticoid receptor alpha; H3K9ac, histone 3 lysine 9 acetylation.
Fig 7. Effects of low physiological concentrations of cortisol on IGF1 and StAR expression through the GRα/miR-370-3p/Sirt3 pathway in NCI-H295R cells. To confirm the roles of GRα, miR-370-3p, and Sirt3 in the gene regulation of cortisol, we incubated the cells with cortisol (300 nM) in the absence or presence of GRα plasmid, miR-370-3p mimics, Sirt3 siRNA, IGF1 plasmid, respectively, for 24 h and then harvested. (A) StAR mRNA expression; (B, E, J) Enrichment of H3K27ac level of IGF1 promoter region; preimmune IgG served as the negative control; (C, F, K) IGF1 expression; (D) Sirt3 expression; (G) Sequence of the WT or mutated miR-370-3p binding site within the 3’-UTR of Sirt3 mRNA; (H) The association between miR-370-3p and Sirt3 by luciferase assay; (I) miR-370-3p expression. n=6 for qRT-PCR from triplicate independent experiments and n=5 for Western blot/ChIP assay in each group. Mean ± S.E.M.,*P <0.05,**P <0.01 vs control;#P <0.05,##P <0.01 vs 75 nM cortisol by one-way ANOVA or Student’s t -test. IGF1, insulin-like growth factor 1; StAR, steroidogenic acute regulatory protein; GRα, glucocorticoid receptor alpha; H3K27ac, histone 3 lysine 27 acetylation.
Fig 8. The GRα/miR-370-3p/Sirt3 pathway mediated the inhibitory effect of low physiological concentrations of cortisol on the H3K27ac and expression levels of IGF1. GRα, glucocorticoid receptor alpha; GRE, glucocorticoid receptor element; IGF1, insulin-like growth factor 1; H3K27ac, histone 3 lysine 27 acetylation.