miR-22 inhibits mouse ovarian granulosa cell apoptosis by targeting SIRT1

Abstract

Granulosa cell (GC) apoptosis has been shown to be involved in follicular atresia, which is a degenerative process in ovarian follicles of mammals. However, the mechanism underlying the regulation of follicular atresia, particularly by microRNAs, is not well known. Real-time PCR (RT-PCR) was used to detect the expression level of miR-22 in healthy follicles (HF), early atretic follicles (EAF), and progressively atretic follicles (PAF). Flow cytometry was performed to assess the apoptosis of mouse granulosa cells (mGCs) treated with miR-22 mimics or negative control (NC) mimics. Regulation of the expression of SIRT1 by miR-22 was evaluated using a luciferase reporter assay system. To investigate the roles of SIRT1 in mGC apoptosis, the endogenous SIRT1 gene in mGCs was knocked down using an siRNA specific for SIRT1. miR-22 was increased during follicular atresia and suppressed granulosa cell apoptosis. The results of the luciferase reporter assay indicated that SIRT1 was a target gene of miR-22. In addition, knockdown of SIRT1 attenuated apoptosis in mGCs. miR-22 inhibits mGC apoptosis by downregulating SIRT1 directly in vitro. This study provides important insights into understanding the regulation mechanism of ovarian follicle atresia.

Keywords: Apoptosis; Follicle atresia; Granulosa cell; SIRT1; miR-22.

Fig. 1.

The expression of miR-22 in mouse ovarian granulosa cells. The relative expression levels of miR-22 were measured in healthy follicles (HF), early atretic follicles (EAF), and progressively atretic follicles (PAF) using qRT-PCR. U6 was used as a loading control to normalize expression levels. Data were expressed as the mean±s.d. (n=3). *P<0.05, **P<0.01.

Fig. 2.

Apoptosis rate of mGCs when transfecting with miR-22 mimics or mimics NC. (A) Western blot was performed to detect the expression levels of Bax and Bcl-2. (B) mGC apoptosis level after miR-22 mimics or mimics NC treatment was determined by FACS. Data were expressed as the mean±s.d. (n=3). *P<0.05, **P<0.01.

Fig. 3.

miR-22 directly inhibits expression of SIRT1 via its 3′-UTR. (A) Putative binding sites for mouse miR-22 on SIRT1 3′-UTR are shown. The mutated region in SIRT1 3′-UTR is indicated with a horizontal line. (B) HEK293T cells were co-transfected with wild type (WT) or mutant (MUT) SIRT1 3′-UTR luciferase reporter plasmid, along with miR-22 mimics or mimics NC. The relative luciferase activities were detected 24 h after transfection. (C) The protein expression level of SIRT1 was determined by western blot. Data were expressed as the mean±s.d. (n=3). *P<0.05.

Fig. 4.

The effect of SIRT1 knockdown on mGC apoptosis. (A,B) Western blot was performed to detect the expression levels of SIRT1 (A), Bax and Bcl-2 (B) in mGCs transfected with SIRT1 siRNA or control siRNA. (C) mGC apoptosis level was measured by FACS 48 h after transfection with SIRT1 siRNA or control siRNA. Data were represented as the mean±s.d. (n=3). *P<0.05, **P<0.01.