Abstract
Oxidative stress impairs follicular development by inducing granulosa cell (GC) apoptosis, which involves enhancement of the transcriptional activity of the pro-apoptotic factor Forkhead box O1 (FoxO1). However, the mechanism by which oxidative stress promotes FoxO1 activity is still unclear. Here, we found that miR-181a was upregulated in hydrogen peroxide (H2O2)-treated GCs and a 3-nitropropionic acid (NP)-induced in vivo model of ovarian oxidative stress. miR-181a overexpression promoted GC apoptosis, whereas knockdown of endogenous miR-181a blocked H2O2-induced cell apoptosis. Moreover, we identified that Sirtuin 1 (SIRT1), a deacetylase that suppresses FoxO1 acetylation in GCs, was downregulated by miR-181a and reversed the promoting effects of H2O2 and miR-181a on FoxO1 acetylation and GC apoptosis. Importantly, decreased miR-181a expression in the in vivo ovarian oxidative stress model inhibited apoptosis by upregulating SIRT1 expression and FoxO1 deacetylation. Together, our results suggest that miR-181a mediates oxidative stress-induced FoxO1 acetylation and GC apoptosis by targeting SIRT1 both in vitro and in vivo.
Highlights
In mammals, the neonatal ovaries contain numerous resting primordial follicles, of which only a limited number successfully develop to ovulation, and more than 99% undergo degeneration at any stage following development
We found that the level of acetylated FoxO1 in primary mouse granulosa cells (mGCs) was decreased by SIRT1 activator 3 (SA3) treatment (Supplementary Figure S3C)
Despite the necessity of FoxO1 transcriptional activation for oxidative stress-induced granulosa cell (GC) apoptosis,[4] little is known about the detailed mechanism
Summary
MiR-181a promotes GC apoptosis via modulation of SIRT1-dependent FoxO1 deacetylation in GCs. To elucidate the functional target that mediates the effect of miR-181a on FoxO1 acetylation and activation, we first demonstrated that miR-181a directly bound to and downregulated SIRT1 expression in KGN cells and mGCs (Supplementary Figure S2). We found that the level of acetylated FoxO1 in primary mGCs was decreased by SIRT1 activator 3 (SA3) treatment (Supplementary Figure S3C) These data suggest that SIRT1 can mediate FoxO1 deacetylation in GCs. In addition, SIRT1 expression was dose-dependently reduced by H2O2 in KGN cells and mGCS, suggesting that SIRT1 may be a functional target of miR-181a in the regulation of H2O2-induced GC apoptosis (Figure 4e). (i) The protein levels of SIRT1, acetylated FoxO1, total FoxO1, and apoptosis-related genes were determined by western blot analysis apoptotic gene activation were blocked by SIRT1 overexpression in KGN cells (Figure 4h).
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