3.7 GRα/miR-370-3p/Sirt3/IGF1 pathway mediated the inhibition of StAR expression caused by low physiological concentrations of cortisol
To further confirm that the GRα/miR-370-3p/Sirt3/IGF1 pathway mediated the inhibitory effect of low physiological concentrations of cortisol on StAR expression, we conducted a series of interference experiments. Firstly, we treated the NCI-H295R cells with exogenous IGF1, and found that IGF1 could significantly reverse the inhibitory effect of 75 nM cortisol on StAR expression (P< 0.05,P< 0.01, Fig 7A). Secondly, we observed that 75 nM cortisol decreased the H3K27ac and expression levels of the IGF1, while treatment with the Sirt3 siRNA or miR-370-3p mimics reversed the above effects of cortisol (P< 0.05,P< 0.01, Fig 7B, C, E, F). And miR-370-3p over-expression also significantly reversed the effect of 75 nM cortisol on Sirt3 expression (P< 0.05, Fig 7D). Furthermore, we found that the 3’UTR of Sirt3 mRNA contains a potential miR-370-3p binding site via bioinformatics prediction, and successfully constructed the double luciferase expression vectors containing the Sirt3 wild type or mutant type (416–422 location) sequence (Fig 7G). The results showed that compared to the respective control, co-transfection of miR-370-3p mimics with Sirt3 WT 3’UTR luciferase reporter plasmids significantly reduced luciferase activity, while co-transfection of miR-370-3p mimics with Sirt3 MUT 3’UTR luciferase reporter plasmids showed no significant change in the luciferase activity (P< 0.05, P< 0.01, Fig 7H). In addition, 75 nM cortisol reduced the GRα expression, and transfection with GRα plasmid could significantly reverse the inhibitory effect of 75 nM cortisol on miR-370-3p expression and the H3K27ac and expression levels of IGF1 (P< 0.01, Fig 7I-K). These data indicated that low physiological concentrations of cortisol reduced GRα expression, downregulating miR-370-3p to target upregulation of Sirt3, which resulted in the decreased H3K27ac and expression levels of IGF1. Ultimately, the StAR expression was reduced.