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.