miR-484 mediates oxidative stress-induced ovarian dysfunction and promotes granulosa cell apoptosis via SESN2 downregulation

Abstract

Ovarian dysfunction is a common cause of female infertility, which is associated with genetic, autoimmune and environmental factors. Granulosa cells (GCs) constitute the largest cell population of ovarian follicles. Changes in GCs, including oxidative stress (OS) and excessive reactive oxygen species (ROS), are involved in regulating ovary function. miR-484 is highly expressed in 3-NP-induced oxidative stress models of ovaries and GCs. miR-484 overexpression aggravated GCs dysfunction and thereby intensified ovarian oxidative stress injury in mice. Moreover, bioinformatic analyses, luciferase assays and pull-down assays indicated that LINC00958 acted as a competing endogenous RNA (ceRNA) for miR-484 and formed a signaling axis with Sestrin2(SESN2) under oxidative stress conditions, which in turn regulated mitochondrial functions and mitochondrial-related apoptosis in GCs. Additionally, the inhibition of miR-484 alleviated GCs dysfunction under ovarian oxidative stress condition. Our present study revealed the role of miR-484 in oxidative stress of ovaries and GCs and the function of LINC00958/miR-484/SESN2 axis in mitochondrial function and mitochondria-related apoptosis.

Keywords: Apoptosis; Granulosa cells; Mitochondria; Ovarian dysfunction; Oxidative stress; miR-484.

Graphical abstract

Fig. 1

miR-484 is highly expressed in 3-NP-induced oxidative stress (OS) models of ovaries and GCs. (A) Ovarian ROS levels of C57BL/6J mice were measured using DHE probe in the Control and OS group after 2 weeks of PBS or 3-NP treatment (n = 6). Scale bar, 50 μm. (B) H&E staining to show ovarian structures in the Control and OS group and (E) the number of follicles was quantified for each group (n = 5). Scale bar, 500 μm. (C–D) The expression levels of miR-484 in ovarian tissues in the Control and OS group were determined using FISH and RT-qPCR (n ≥ 10). Scale bar, 50 μm. (F) Intercellular ROS contents of GCs (SVOG cells) were measured using DCFH-DA probe and quantified in the Control and OS group for PBS or 10 mM 3-NP treatment for 24h (n = 5). Scale bar, 50 μm. (G–H) The expression levels of miR-484 in ovarian tissues in the Control and OS group were determined using FISH and RT-qPCR. (n ≥ 10). Scale bar, 20 μm. Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 2

Overexpression of miR-484 compromised GCs function by damaging mitochondrial functions and increasing apoptosis under oxidative stress conditions. (A) CCK-8 assays were performed to measure the cell viability in silenced- or overexpressed-miR-484 in GCs under oxidative stress (n = 5). (B) EdU assays of GCs and the percentage of EdU-positive GCs (Green) were measured (n = 5). Scale bar, 100 μm. (C) Cellular ROS (Green) and mtROS (Red) in GCs were assessed using DCFH-DA probe and MitoSOX Red mitochondrial superoxide indicator (n = 5). Scale bar, 50 μm. (D) The MDA level and total SOD and GSH-Px activities of GCs (n = 5). (E) MitoTracker (red) was used to stain mitochondria, and mitochondrial morphology is counted (n = 5). Scale bar, 10 μm. (F) ATP levels were recorded following miR-484 knockdown or overexpression in GCs under oxidative stress (n = 5). (G) Western blot was used to measure the expression of apoptosis-associated proteins in GCs. (H) Mitochondrial membrane potential (MMP, ΔΨm) was measured by the JC-1 staining (n = 5). Scale bar, 50 μm. (I) Apoptotic cells were measured by flow cytometry in GCs (n = 3). Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Prediction and confirmation of direct interaction between miR-484 and LINC00958 in GCs. (A) FISH was performed to detect the expression of LINC00958 in the Control and OS group (n = 6). Scale bar, 50 μm. (B–C) The predicted binding sites of LINC00958 and miR-484 and the luciferase reporter assay showed direct binding between the LINC00958-WT and the miR-484. (D–E) GCs were transfected with biotinylated wild-type miR-484 or biotinylated LINC00958. RNA pull-down assay showed that LINC00958 was associated with miR-484. (F–G) The RIP assay for LINC00958 was performed with an anti-AGO2 antibody in GCs transfected with miR-484 mimics or mimic-NC, and the expression of LINC00958 and miR-484 was measured. (H) EdU assays of GCs and the percentage of EdU-positive GCs (Green) were measured (n = 5). Scale bar, 100 μm. (I) Cellular ROS (Green) and mtROS (Red) in GCs were assessed using DCFH-DA probe and MitoSOX Red mitochondrial superoxide indicator (n = 5). Scale bar, 50 μm. (J) CCK-8 assays were performed to measure the cell viability in silencing LINC00958 of GCs under oxidative stress (n = 5). (K) MitoTracker (red) was used to stain mitochondria, and mitochondrial morphology is counted (n = 5). Scale bar, 10 μm. (L) ATP levels were measured following LINC00958 knockdown in GCs under oxidative stress (n = 5). (M) Western blot was used to measure the expression of apoptosis-associated proteins in GCs. (N) Mitochondrial membrane potential (MMP, ΔΨm) was measured by the JC-1 assays (n = 5). Scale bar, 50 μm. (O) Apoptotic cells were measured by flow cytometry in GCs (n = 3). Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Inhibiting miR-484 alleviated LINC00958 depletion-aggravated GC injuries under oxidative stress conditions. (A) Relative cell viability was measured by the CCK-8 assay (n = 5). (B) EdU assays of GCs and the percentage of EdU-positive GCs (Green) were measured (n = 5). Scale bar, 100 μm. (C) Cellular ROS (Green) and mtROS (Red) in GCs were assessed using DCFH-DA probe and MitoSOX Red mitochondrial superoxide indicator (n = 5). Scale bar, 50 μm. (D) The MDA level and total SOD and GSH-Px activities of GCs (n = 5). (E) MitoTracker (red) was used to stain mitochondria, and mitochondrial morphology is counted (n = 5). Scale bar, 10 μm. (F) ATP levels were measured (n = 5). (G) Western blot was used to measure the expression of apoptosis-associated proteins in GCs. (H) Mitochondrial membrane potential (MMP, ΔΨm) was measured by the JC-1 assays (n = 5). Scale bar, 50 μm. (I) Apoptotic cells were measured by flow cytometry in GCs (n = 3). Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Sestrin2(SESN2) is a direct downstream mRNA target of miR-484 in GCs. (A)Venn diagram showing predicted target gene of miR-484 by four algorithms (miRanda, TargetScan, RNAhybrid, and PITA). (B–C) Dual luciferase reporter analysis showed that miR-484 could bind to the 3′-UTR of SESN2. (D) Enrichment of SESN2 pulled down by biotin-miR-484 or negative control. (E–G) The mRNA and protein expression of SESN2 was decreased or increased in a dose-dependent manner when miR-484 overexpressed or knockdown by RT-qPCR and Western blot assays (n = 3). Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 6

SESN2 positively regulated GCs functions after oxidative stress injury. (A) CCK-8 assays were performed to measure the cell viability in silenced- or overexpressed-SESN2 in GCs under oxidative stress (n = 5). (B) EdU assays of GCs and the percentage of EdU-positive GCs (Green) were measured (n = 5). Scale bar, 100 μm. (C) Cellular ROS (Green) and mtROS (Red) in GCs were assessed using DCFH-DA probe and MitoSOX Red mitochondrial superoxide indicator (n = 5). Scale bar, 50 μm. (D) ATP levels were recorded following miR-484 knockdown or overexpression in GCs under oxidative stress (n = 5). (E) MitoTracker (red) was used to stain mitochondria, and mitochondrial morphology is counted (n = 5). Scale bar, 10 μm. (F) Mitochondrial membrane potential (MMP, ΔΨm) was measured by the JC-1 staining (n = 5). Scale bar, 50 μm. (G) Apoptotic cells were measured by flow cytometry in GCs (n = 3). (H) Western blot was used to measure the expression of apoptosis-associated proteins in GCs. (I) The protein expression levels of SESN2 downstream molecules and antioxidant related molecules. Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

SESN2 reversed miR-484 mediated mitochondrial damages and apoptosis in GCs under oxidative stress conditions. (A) Relative cell viability was measured by the CCK-8 assay (n = 5). (B) EdU assays of GCs and the percentage of EdU-positive GCs (Green) were measured (n = 5). Scale bar, 100 μm. (C) Cellular ROS (Green) and mtROS (Red) in GCs were assessed using DCFH-DA probe and MitoSOX Red mitochondrial superoxide indicator (n = 5). Scale bar, 50 μm. (D) MitoTracker (red) was used to stain mitochondria, and mitochondrial morphology is counted (n = 5). Scale bar, 10 μm. (E) Mitochondrial membrane potential (MMP, ΔΨm) was measured by the JC-1 assays (n = 5). Scale bar, 50 μm. (F) ATP levels were measured (n = 5). (G) Apoptotic cells were measured by flow cytometry in GCs (n = 3). (H) Western blot was used to measure the expression of apoptosis-associated proteins in GCs. (I) The protein expression levels of SESN2 downstream molecules and antioxidant related molecules. Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Inhibition of miR-484 ameliorated ovarian oxidative stress injuries in mice. (A) H&E staining to show ovarian structures in the Control, OS, OS + AAV-vector, OS + AAV-inhibitor, and OS + Melatonin groups and the number of follicles was quantified for each group (n = 5). Scale bar,500 μm. (B, D) The level of estradiol, FSH and AMH of serum in each group (n ≥ 5). (C) Ovarian ROS levels of mice were measured using DHE probe in each group (n = 5). Scale bar, 50 μm(E) TUNEL staining of ovaries and the percentage of TUNEL-positive section (Green) were quantified(n = 5). Scale bar, 50 μm. (F) Ki-67 staining of ovaries and the proportion of Ki-67 positive cells were measured(n = 5). Scale bar, 50 μm. Data represent the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

Schematic diagram showing the role of miR-484 mediates oxidative stress-induced ovarian dysfunction by suppressing granulosa cell function via mitochondrial-related apoptosis.