Knockdown of miR-21-5P targets and regulates PDCD4-induced apoptosis in ovarian granulosa cells and ameliorates in sulin resistance in polycystic ovary syndrom

Hengzhen He; Peng Ning; Xiaohong Chen; Jing Lin

doi:10.1371/journal.pone.0343735

Abstract

This study examined the role of miRNA-21-5p in a rat model of polycystic ovary syndrome with insulin resistance (PCOS-IR) and its potential involvement in ovarian granulosa cell apoptosis. Female Sprague-Dawley rats were divided into four groups, with the PCOS-IR model established using dehydroepiandrosterone combined with a high-sugar, high-fat diet. Lentiviral transduction was utilized to silence miRNA-21-5p. Serum hormone levels were assessed via ELISA, while the protein expression of PDCD4, Bcl-2, and Caspase-3 in ovarian tissues was analyzed through Western blotting. Granulosa cell apoptosis was evaluated using CCK-8 assay, flow cytometry, and TUNEL staining. The targeting relationship between miRNA-21-5p and PDCD4 was confirmed via dual-luciferase reporter assay and further supported by AlphaFold3 and RNA immunoprecipitation (RIP) prediction. Compared to the PCOS-IR and si-NC groups, the si-miRNA-21-5p group displayed improved ovarian morphology, partially restored hormone levels, moderately enhanced insulin sensitivity, and reduced granulosa cell apoptosis, alongside altered PDCD4 expression. These findings suggest that miRNA-21-5p may play a role in the pathogenesis of PCOS-IR by regulating PDCD4 and influencing granulosa cell apoptosis. Inhibition of miRNA-21-5p shows potential in alleviating certain pathological features within this experimental model; however, further validation in human studies is needed to assess its clinical relevance and therapeutic applicability.

Citation: He H, Ning P, Chen X, Lin J (2026) Knockdown of miR-21-5P targets and regulates PDCD4-induced apoptosis in ovarian granulosa cells and ameliorates in sulin resistance in polycystic ovary syndrom. PLoS One 21(3): e0343735. https://doi.org/10.1371/journal.pone.0343735

Editor: Jean-Marc A. Lobaccaro, Université Clermont Auvergne - Faculté de Biologie, FRANCE

Received: October 7, 2025; Accepted: February 10, 2026; Published: March 6, 2026

Copyright: © 2026 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The datasets analyzed in this study are publicly available in the NCBI Gene Expression Omnibus (GEO) under accession numbers GSE72274 and GSE80432. The original plotting data, as well as the original uncropped and unadjusted images underlying all blot and gel results, have been made publicly available on Figshare at the following DOI: https://doi.org/10.6084/m9.figshare.30610571.

Funding: This study was supported by the Guangxi Natural Science Foundation (Grant Nos. 2023GXNSFBA026257, 2024GXNSFAA010146, and 2024GXNSFAA010468), the National Natural Science Foundation of China (Grant No. 82560972), and the Guangxi Key Research and Development Program (Grant No. Guike AB19110022). No additional external funding was received for this research.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder affecting 5–10% of women of reproductive age [1,2]. It is characterized by hyperandrogenemia, menorrhagia or amenorrhea, anovulatory cycles, and the presence of multiple ovarian cysts detected through ultrasonography [2,3]. PCOS is the most common cause of anovulatory infertility, with its pathogenesis driven by disruptions in sex hormone secretion, insulin resistance, and abnormal follicular development [4]. Insulin resistance represents a central pathological and clinical feature of PCOS [5–7]. Consequently, establishing a reliable animal model is critical for progressing research in this field [8]. This disease is primarily treated with Western medicine drugs to promote ovulation or through surgery [9]. However, these treatments often come with adverse reactions, and the disease can easily recur after stopping the medication or surgery, leading to poor long-term efficacy [9–11]. miRNAs play a crucial role in regulating various cellular functions, including DNA repair, differentiation, proliferation, development, cell death, and immune regulation, and they are involved in the development of many diseases [12,13].

Programmed cell death 4 (PDCD4) expression is significantly upregulated in patients with polycystic ovary syndrome (PCOS) and is closely associated with various metabolic abnormalities [14,15]. PDCD4 promotes apoptosis in granulosa cells, which impairs follicular development and maturation [16]. This leads to accelerated follicular atresia, hinders the formation of dominant follicles, and contributes to ovulatory dysfunction. Moreover, PDCD4-induced apoptosis of granulosa cells may interfere with embryo development and endometrial receptivity, ultimately reducing fertility outcomes [17,18]. Beyond its role in reproductive dysfunction, PDCD4 is linked to the metabolic disturbances commonly observed in PCOS, such as exacerbated insulin resistance and lipid metabolism disorders [19,20]. These changes further increase the risk of developing type 2 diabetes mellitus and cardiovascular disease [21]. Notably, elevated PDCD4 expression may also diminish ovarian responsiveness to ovulation induction therapies, complicating treatment strategies [22]. Given these multifaceted roles, PDCD4 represents a potential therapeutic target for PCOS. Importantly, recent studies have indicated a regulatory link between miRNAs and PDCD4. Specifically, it has been observed that knockdown of miRNA-21-5p leads to downregulation of PDCD4, suggesting a potential therapeutic axis.

miRNAs are considered potential targets for alleviating insulin resistance in patients with polycystic ovary syndrome (POCS), and to regulate follicular development and ovarian function effectively [23], but the specific mechanism of their action is still unclear. The present study focused on the ovarian polycystic insulin resistance rat model to investigate the specific mechanism by which miRNA-21-5p targets and regulates POCS. This research aimed to examine the effect of miRNA-21-5p on reproductive endocrine levels in rats with PCOS, as well as the impact of miRNA-21-5p-targeted regulation of PDCD4 gene expression on the apoptosis of ovarian granulosa cells. The goal was to understand how these processes may ameliorate insulin resistance and to explore the therapeutic potential of knocking down miRNA-21-5p in treating insulin resistance associated with PCOS.

Materials and methods

Experimental animals

Experimental animals were selected from 6-week-old fertile female Sprague-Dawley (SD) rats, obtained from Hunan Slaughter Laboratory Animal Co. Ltd (Hunan, China) [License No: SYXK (Gui) 2024−0004]. The rats were housed under a 12-hour light/dark cycle, with a room temperature of 20 ± 0.5°C and humidity levels between 30.0% and 50.0%. They had free access to food and water. Rat nests and cages were changed daily before any experimental manipulation, and the rats were acclimatized indoors for at least one week after their arrival in the laboratory. The experimental design was a single study and was approved by the Ethics Committee of Guangxi University of Traditional Chinese Medicine, People’s Republic of China (Animal Ethics Approval No. DW20240603−145).

Establishment of the PCOS insulin resistance rat model

The PCOS insulin resistance (PCOS-IR) rat model was established based on the protocol described by Lee et al [24]. Dehydroepiandrosterone (DHEA; China National Pharmaceutical and Chemical Import and Export Corporation) was administered via intraperitoneal injection at a dosage of 60 mg/100 g body weight once daily for 21 consecutive days. DHEA was dissolved in 0.3 mL of castor oil (same supplier) and given alongside a high-fat, high-sugar diet. The control group of rats received daily intraperitoneal injections of 0.3 mL of castor oil for the same duration. The model was induced on day 20 of the oestrus cycle. Three rats were euthanized under mild anesthesia with 3% pentobarbital sodium (100 mg/kg), and PCOS diagnosis was confirmed through vaginal cytology, fasting insulin and blood glucose measurements, serum hormone analysis, and ovarian histology. Sixty rats met the diagnostic criteria for insulin-resistant polycystic ovary syndrome [25].Euthanasia was ultimately performed via cervical dislocation on anesthetized subjects following administration of 3% pentobarbital sodium (100 mg/kg).

Grouping and lentivirus injection

Fifty SD rats were randomly assigned to five groups (n = 10 per group): CONTROL group, PCOS-IR group, small hairpin RNA (shRNA) negative control group (si-NC), miRNA-21-5p-shRNA lentiviral group (si-miRNA-21-5p), and PCOS-IR+PDCD4-IN-1 group.Rats in the si-NC and si-miRNA-21-5p groups were fasted for 12 hours. Lentiviral injection was performed according to the PCOS rat model [26]. Anaesthesia was induced using 2% isoflurane at a rate of 0.41 ml/min with a fresh gas flow of 4 L/min. The rats were positioned laterally for stereotaxic electrostimulation. Bilateral abdominal incisions of 1–2 cm were made through sharp dissection. The skin, fascia, and muscle layers were carefully divided using scissors or blunt instruments, revealing the moist white adipose tissue underneath. Upon retracting the folds of adipose tissue, the ovaries were visible as pink, glistening structures with a distinctive lobulated morphology. A 10 μl syringe was employed to inject si-miRNA-NC or si-miRNA-21-5p lentivirus from (Shanghai Jikang GenePharma Co., Ltd., Shanghai, China, with a viral titer 5 × 10⁸ transducing units/mL) directly into the ovaries of the rats. The needle was inserted slowly and held in place for 5 minutes. Each ovary received two injections at different sites, with 10 μl administered each time, resulting in a total of 50 μl of lentivirus per rat. To prevent infection, an appropriate dosage of penicillin (Sichuan, China, 80,000 units/mL) was injected intramuscularly before suturing the incisions.

Real-time PCR

RNA was extracted from rat ovarian granulosa cells using the Eastep® Super Total RNA Extraction Kit (Promega, Shanghai, China). 2 ul of RNA was reverse transcribed to cDNA using the Superscript Reverse Transcriptase Kit (Takara Bio, Beijing, China) as a template. cDNA samples were processed on a Light Cycler/Light Cycler480 System (Roche, Basel, Switzerland) using the TB Green® Premix Ex Taq™ II PCR Kit (Takara Bio). The cDNA samples were subjected to real-time PCR on a Light Cycler/Light Cycler480 system (Roche, Basel, Switzerland) using the TB Green® Premix Ex Taq™ II PCR kit (Takara Bio, Beijing, China). The PCR reaction conditions were: pre-denaturation for 30 seconds at 95°C (30 seconds at 95°C, 10 seconds at 60°C, and reading of 1 plate at the end of the extension) × 40 cycles, 50°C autoclave, 50°C autoclave. The relative expression of the PCR products was calculated as (2 ^{- ΔΔct}). The primer sequences are shown in Table 1. The primer sequences are shown in Table 1.

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Table 1. PCR primer sequences.

https://doi.org/10.1371/journal.pone.0343735.t001

AlphaFold3 predicts direct interaction of miRNA-21–5- with PDCD4

The nucleotide sequences of miRNA-21-5p and PDCD4 were obtained from public databases [27], and their three-dimensional structures were generated using the AlphaFold Server, with the corresponding models retrieved from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk). The predicted structures were provided in CIF format and subsequently visualized and analyzed using PyMOL, thereby supporting downstream structural characterization and functional investigation.

RNA immunoprecipitation (RIP) assay

RNA immunoprecipitation (RIP) was performed to examine the interaction between miR-21-5p and PDCD4 in ovarian granulosa cells from PCOS-IR rats. Cell lysates were prepared in RIP buffer and clarified by centrifugation. For immunoprecipitation, 5 µg of anti-PDCD4 antibody (9535; Cell Signaling Technology, Danvers, MA, USA) or IgG control (2729; Cell Signaling Technology) was added to the lysate and incubated overnight at 4℃. Following extensive washing, the immunoprecipitated complexes were treated with proteinase K, and the co-precipitated RNAs were isolated using TRIzol. The extracted RNA was reverse-transcribed and subsequently analyzed by qRT-PCR with SYBR Green Master Mix. The enrichment of miR-21-5p in PDCD4 complexes was quantified via the 2^−ΔΔCt method. All experiments were conducted in triplicate.The primer sequences are shown in Table 1.

Primary granulosa cell collection

Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (3% Pelltobarbitalum Natricum, Merck, Germany, 0.03 ml/100 g), and then ovaries were collected to extract granulosa cells, following the protocol of a previous study [25]. A sterile area was established to access the abdomen of rats, which have bilateral ovaries located below their bilateral kidneys. Ovarian tissues were isolated by repeatedly washing and removing ovarian blood vessels, surface membranes, and other tissues using pre-warmed phosphate-buffered saline (PBS) containing double antibodies. Ten rat unilateral ovaries were randomly assigned to each group for primary granulosa cell extraction. The stripped rat ovaries were punctured with a 1 mL syringe needle to transfer the granulosa cells into DMEM/F12 culture medium through repeated aspiration. The ovaries were then cut open with ophthalmic scissors and incubated with 0.25% collagenase for 1 hour at 37℃ in a 5% CO₂ incubator. Granulosa cells were added to DMEM/F12 (15% fetal bovine serum). The digestion was terminated by adding either DMEM/F12 (15% fetal bovine serum) or F12 (15% fetal bovine serum) medium. The cells were filtered through a 220-mesh cell sieve and centrifuged at 1000 rm/5 min in a 50 ml centrifuge tube. The supernatant was discarded to collect the cells. The loose cell mass at the bottom of the centrifuge tube was then resuspended in DMEM/F12 (15% fetal bovine serum, GIBCO, New York, USA; double antibody, Solarbio, Beijing, China), and incubated in a 5% CO₂ incubator at 37°C for 24 h. The medium was changed once to remove any unadhered cells, and subsequently every other day.

Enzyme-linked immunosorbent assay (ELISA) for FINS、FBG and sex hormones (T, E2, LH, FSH)

Fasting insulin (FINS), fasting blood glucose (FBG) and sex hormones (T, E2, LH, FSH) in rat serum were determined by enzyme-linked immunosorbent assay (ELISA) kit (Elabscience, Wuhan, China). The procedure was followed according to the manufacturer’s instructions. After freezing the rat serum from minus 80°C to room temperature for 2 h, the thawed rat serum was subjected to the relevant indexes in a 96-well enzyme-labeled plate. The thawed rat serum was assayed on 96-well enzyme labeled plates [25] First, 100 uL of various concentrations of standards were added to the standard wells, and 100 uL of the samples to be tested were added to the sample wells. Additionally, 100 uL of enzyme labelling reagent was added to each well, except for the blank wells. The plates were then sealed and incubated at 37°C for 90 min. After blocking the plates, the washing solutions was diluted and added to each well. After allowing it to stand for 1 min, the solution was discarded, and the wells were patted dry. This process was repeated three times. Next, 100 µL of substrates A and B were added to each well, and the plates were incubated at 37°C for 15 min, avoiding exposure to light. Finally, the reaction was terminated, and the absorbance of each well was measured at 450 nm to calculate the concentration of the samples.

Protein extraction and Western-blot analysis were performed to measure the expression of caspase-3, PDCD4and Bcl-2 in ovarian granulosa cells

The cultured primary ovarian granulosa cells were prepared on ice and homogenized at a ratio of 100:1. RIPA lysate (Beyotime, Shanghai, China), PMSF (Sangon Biotech, Shanghai, China) and protease inhibitor (MCE, China) were added to extract ovarian granulosa cell protein concentrations, and BCA assay kits (Beyotime, Shanghai, China. China) to measure ovarian granulosa cell protein concentrations, and the boiled protein samples were separated using SDS-PAG gel electrophoresis, transferred to a membrane, then blocked with 10% skim milk. The membrane was also incubated with the goat anti-rabbit caspase-3 polyclonal antibody (1: lysed caspase-3 polyclonal antibody (1:700, Cell Signaling Technology, Danvers Massachusetts, USA), Bax polyclonal antibody (1:2000, Proteintech, Wuhan, China),anti-PDCD4 antibody (1;2000, Cell Signaling Technology, Danvers, MA, USA) and β-actin (1:5000, Proteintech, Wuhan, China). Proteintech, Wuhan, China) were refrigerated overnight at 4℃. The membranes were washed three times with TBST and then incubated with a horseradish peroxidase-labeled secondary antibody (goat anti-rabbit IgG, 1:5000, Proteintech, Wuhan, China). After additional washes with TBST, the target protein bands were detected using an enhanced chemiluminescence detection system (Millipore, Burlington, MA, USA). Grayscale values were analyzed using Image-J software, and the results were recorded.

Detection of apoptosis by flow cytometry

Apoptosis detection by flow cytometry typically employs the Annexin V and PI double staining method. The procedure involves the following steps: 1) Collect cells and resuspend them in PBS, then centrifuge and discard the supernatant; 2) Add Binding Buffer to resuspend the cells and adjust the concentration; 3) Add Annexin V and PI for staining, incubate in a light protected area, and then add the Binding Buffer; 4) Detect the samples using a flow cytometer. By analyzing the fluorescence signals of Annexin V and PI, you can differentiate between live cells, early apoptotic cells, late apoptotic cells, and necrotic cells. It is important to handle the cells gently, protect them from light, and conduct the detection as soon as possible to prevent fluorescence decay.

Dual-luciferase reporter gene analysis

The miRNA-21-5p fragment containing the specific PDCD4 binding site was cloned into the pmirGLO dual luciferase expression vector (Promega Corporation, Madison, WI, USA), through which pmirGLO-miRNA-21-5p-Wt was formed. in addition, the same binding site of PDCD4 in miRNA-21-5p was mutated at the same binding site in PDCD4 to construct pmirGLO-miRNA-21-5p-Wt. pmirGLO-miRNA-21-5p-Wt was transfected with PDCD4 mimics and miR-NC transfections, respectively. pmirGLO-miRNA-21-5p-Mut or pmirGLO vector cells, respectively. About 48 h after transfection, luciferase activity was measured using a dual-luciferase reporter assay system (Promega Corporation).

PDCD4 inhibitor treatment in primary rat ovarian granulosa cells (OGC)

To investigate the effects of PDCD4 inhibition on the biological functions of Primary Rat Ovarian Granulosa Cells (UCSI246Ra01), this study utilized an ovarian granulosa cell line for ex vivo experiments. The cells were seeded onto culture plates, allowing them to adhere and stabilize. Following this, they were treated with a 10 mM PDCD4 inhibitor, PDCD4-IN-1 (CAS No.: 494763-64-3; MedChemExpress, Monmouth Junction, NJ, USA), was dissolved in DMSO and used to treat cells at the indicated concentrations. After 24–72 hours of treatment, cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. Simultaneously, cellular proteins and total RNA were extracted for Western blot analysis and quantitative polymerase chain reaction (qPCR), respectively, to evaluate changes in the expression of PDCD4 and its associated pathway proteins and genes.

Statistical analysis

Statistical analyses were performed using the SPSS statistical package (version 24.0, IBM Armonk, NY, USA). All data are reported in the form of mean ± standard deviation (±s). One-way analysis of variance (ANOVA) was employed to compare measurement data across multiple groups. For pairwise comparisons between groups, a t-test was used when variances were equal, while a rank-sum test was applied for those with unequal variances and differences were considered statistically significant at P < 0.05.

Results

Overexpression of miRNA-21-5p in Ovarian granulosa cells of a rat polycystic ovary syndrome model with insulin resistance

To confirm the upregulation of miRNA-21-5p expression in granulosa cells associated with polycystic ovary syndrome (PCOS), an in vivo PCOS model was created by administering DHEA to female Sprague-Dawley (SD) rats. By day 21, the body weight of the PCOS rats (Fig 1A) was significantly greater than that of the control group. Furthermore, histological analysis of the ovarian tissues, using hematoxylin and eosin (H&E) staining, showed that the ovaries of the PCOS rats displayed pronounced polycystic-like changes, along with thickening of the follicular theca cells when compared to the control group (Fig 1B). Starting on day 14 of the modeling period, vaginal smears were taken from both groups, revealing irregular estrous cycles in the PCOS rats (Fig 1C-D).

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Fig 1. Overexpression of miRNA-21-5p in Ovarian Granulosa Cells of a Rat Polycystic Ovary Syndrome Model with Insulin Resistance.

(A) Body weight of the PCOS-IR model group and control group. Significant differences in body weight began to appear 14 days after DHEA administration. By day 21, the body weight of the PCOS-IR group was significantly higher than that of the control group (n = 10 per group). (B) Histological analysis of ovarian tissue. H&E staining revealed marked polycystic changes in the ovaries of PCOS-IR rats compared to the control group (magnification 100 × , n = 4 per group), with thickened follicular membrane cells. (C-D) Estrous cycle staging in the PCOS-IR group and control group. Irregular estrous cycles were observed in PCOS-IR rats starting from day 14, while the control group exhibited regular estrous cycles (n = 10 per group). (E-a) LH levels in the PCOS-IR group and control group. Compared with the control group, LH levels were significantly elevated in the PCOS-IR group (n = 10 per group). (E-b) FSH levels in the PCOS-IR group and control group. Compared with the control group, FSH levels were significantly reduced in the PCOS-IR group (n = 10 per group). (E-c) Testosterone levels in the PCOS-IR group and control group. Testosterone levels were significantly higher in the PCOS-IR group (n = 10 per group). (E-d) E2 levels in the PCOS-IR group and control group. Compared with the control group, the E2 levels in the PCOS-IR group were significantly lower (n = 10 per group). (E-e) FINS levels in the PCOS-IR group and the control group. Compared with the control group, the FINS levels in the PCOS-IR group were significantly higher (n = 10 per group). (E-f) FBG levels in the PCOS-IR group and the control group. Compared with the control group, the FBG levels in the PCOS-IR group were significantly elevated (n = 10 per group) (E-g) HOMA-IR ratios in the PCOS-IR group and control group. Compared with the control group, the HOMA-IR ratios in the PCOS-IR group were significantly elevated (n = 10 per group). (F) Transcriptome analysis of human granulosa cells based on the Gene Expression Omnibus (GEO) database (GSE80432) showed that the mRNA levels of miRNA-21-5p in granulosa cells of PCOS-IR patients were significantly elevated compared with healthy controls. (G) Expression of miRNA-21-5p in ovarian granulosa cells of the rat PCOS-IR model was detected by RT-qPCR.Data are presented as mean ± standard deviation (SD). *P < 0.05,**P < 0.01,***P < 0.001, ****P < 0.0001 compared with the control group. E2 stands for estradiol, T stands for testosterone, FSH stands for follicle stimulating hormone, and LH stands for luteinizing hormone. LH stands for luteinizing hormone,FBG stands for fasting blood glucose and FINS stands for fasting insulin (10.6084/m9.figshare.30654101).

https://doi.org/10.1371/journal.pone.0343735.g001

Serum analysis revealed that levels of follicle-stimulating hormone (FSH) and estradiol (E2) were significantly lower in the PCOS group compared to the controls. In contrast, levels of luteinizing hormone (LH) and testosterone were markedly elevated (Fig 1E a-d). Furthermore, fasting insulin (FINS), fasting blood glucose (FBG), and the homeostasis model assessment of insulin resistance (HOMA-IR) index were significantly higher in the PCOS group (Fig 1E e-g), confirming the successful establishment of a PCOS with insulin resistance (PCOS-IR) mode (all P < 0.05). To better understand the regulatory role of miRNA-21-5p in PCOS-IR, we analyzed granulosa cell transcriptome data (GSE72274) from the Gene Expression Omnibus (GEO) database. mRNA profiling revealed that miRNA-21-5p (Fig 1F) was significantly upregulated in the ovarian granulosa cells of PCOS-IR patients compared to controls (all P < 0.001). Additionally, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) confirmed a significant increase in miRNA-21-5p expression (Fig 1G) in the rat PCOS model compared to controls (P < 0.001).

Inhibition of miRNA-21-5p ameliorates ovarian pathological changes, hormonal imbalance, and insulin resistance in polycystic ovary syndrome (PCOS)-IR rats

To further investigate the histopathological changes in PCOS rats, hematoxylin and eosin (HE) staining indicated that the ovaries of the si-NC group displayed diffuse cystic dilation along with typical polycystic alterations. Following the injection of the silencing si-miRNA-21-5p virus (S1 Fig in S1 File), the si-miRNA-21-5p group’s ovaries exhibited diffuse cystic dilation, accompanied by only a few luteinized and atretic follicles (Fig 2A). The primary pathological manifestations of PCOS include abnormal follicular development, metabolic dysfunction, and endocrine disturbances, which are closely linked to the coordinated regulation of hypothalamic gonadotropin-releasing hormone (GnRH) and ovarian estrogen [28]. In this study, enzyme-linked immunosorbent assay (ELISA) was employed to measure the secretion levels of estradiol (E2), testosterone (T), follicle-stimulating hormone (FSH), luteinizing hormone (LH), as well as insulin-related indices, including fasting blood glucose (FBG), fasting insulin (FINS), and homeostasis model assessment of insulin resistance (HOMA-IR). The results demonstrated that the si-NC group exhibited decreased secretion levels of FSH and E2, whereas the levels of T and LH were elevated (all P < 0.05 vs. the normal control group). Compared to the sh-NC group, the sh-miRNA-21-5p group showed higher secretion levels of FSH and E2, while levels of T and LH were reduced (all P < 0.05) (Fig 2B-2E). Concurrently, the serum levels of FBG, FINS, and HOMA-IR were significantly elevated in the si-NC group (P < 0.05). In contrast, the si-miRNA-21-5p group exhibited reduced serum levels of FBG, FINS, and HOMA-IR compared with the si-NC group (P < 0.05) (Fig 2F–2H).

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Fig 2. Suppression of miRNA-21-5p ameliorates ovarian pathological alterations, hormonal imbalance, and insulin resistance in PCOS-IR rats.

(A a-d) Hematoxylin and eosin (H&E) staining showed that miRNA-21-5p downregulation improved ovarian morphology in PCOS-IR rats, reducing cystic follicles and restoring granulosa cell layers (scale bar = 250μm). (B) Silencing miRNA-21-5p significantly lowered serum luteinizing hormone (LH) levels in PCOS rats. (C) Conversely, serum follicle-stimulating hormone (FSH) levels increased markedly after miRNA-21-5p suppression. (D) Serum testosterone (T) concentrations declined following miRNA-21-5p silencing. (E) Serum estradiol (E2) levels rose significantly in PCOS rats after miRNA-21-5p silencing. (F) Serum fasting insulin (FINS) levels decreased notably upon miRNA-21-5p knockdown. (G) Serum fasting blood glucose (FBG) concentrations were also reduced after miRNA-21-5p inhibition. (H) The homeostatic model assessment for insulin resistance (HOMA-IR) index decreased, indicating improved insulin sensitivity following miRNA-21-5p silencing in PCOS-IR rats.The number of groups is n = 6 for all groups. Compared with the si-NC group, *P < 0.05, **P < 0.01, ****P < 0.0001 and Tukey’s post-hoc test. E2 stands for estradiol, T stands for testosterone, FSH stands for follicle stimulating hormone, and LH stands for luteinizing hormone. LH stands for luteinizing hormone,FBG stands for fasting blood glucose and FINS stands for fasting insulin (10.6084/m9.figshare.30654101).

https://doi.org/10.1371/journal.pone.0343735.g002

Silencing miRNA-21-5p regulates proliferation and apoptosis of ovarian granulosa cell

Granulosa cells (GCs) are epithelial cells that surround the oocyte and play a crucial role in regulating follicle growth, development, and maturation through autocrine and paracrine mechanisms, which are essential for maintaining corpus luteum function. Dysfunction of granulosa cells is commonly observed in polycystic ovary syndrome (PCOS) and may contribute to the pathophysiology of the condition. In this study, granulosa cells extracted from follicles of PCOS-IR model rats were cultured in a 37°C incubator for 72 hours. We assessed the effects of miRNA-21-5p silencing on the proliferation and apoptosis of ovarian granulosa cells in the PCOS-IR model. After knocking down miRNA-21-5p in these cells, we observed a significant reduction in miRNA-21-5p mRNA levels, as illustrated in Fig 3A. To evaluate late-stage apoptosis in ovarian granulosa cells, we employed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The results indicated that the apoptotic fluorescence intensity in the si-miRNA-21-5p group was significantly lower than that in the lentiviral negative control group (si-NC), with only a few apoptotic granulosa cells exhibiting green fluorescence (P < 0.01) (Fig 3B). Concurrently, the CCK-8 assay revealed that compared with the si-NC group, the proliferation rate of granulosa cells in the si-miRNA-21-5p group was increased, indicating that miRNA-21-5p inhibition substantially enhanced the proliferative capacity of ovarian granulosa cells (P < 0.001) (Fig 3C). Flow cytometry similarly revealed a significant reduction in the apoptosis rate of ovarian granulosa cells following the silencing of miRNA-21-5p (Fig 3D). Furthermore, WB analysis was conducted to assess the expression of apoptosis-related proteins. The si-miRNA-21-5p group showed decreased levels of caspase-3 and PDCD4 compared to the si-NC group, while Bcl-2 expression was increased (all P < 0.05) (Fig 3E). Collectively, these findings suggest that miRNA-21-5p promotes the proliferation of ovarian granulosa cells in PCOS-IR models while suppressing apoptosis.

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Fig 3. Silencing miRNA-21-5p Regulates Proliferation and Apoptosis of Ovarian Granulosa Cell.

(A) miRNA-21-5p mRNA expression in primary ovarian granulosa cells of PCOS-IR model rats transfected with siNC or simiRNA-21-5p lentiviral vectors. (B) Apoptosis of ovarian granulosa cells in PCOS-IR rats following miRNA-21-5p downregulation, as detected by TUNEL assay (scale bar = 100 μm). Apoptotic cells were labeled with FITC (green fluorescence), while nuclei were counterstained with DAPI (blue fluorescence). The image represents a merged fluorescence signal. (C) Proliferative capacity of primary ovarian granulosa cells post-lentiviral transfection, assessed by CCK-8 assay. (D) Flow cytometric determination of apoptosis rates in primary ovarian granulosa cells after miRNA-21-5p downregulation. (E) Western blot analysis of Caspase-3, PDCD4, and Bcl-2 protein expression in granulosa cells of PCOS-IR rats following miRNA-21-5p silencing (n = 6). All quantitative data are presented as mean ± SD and compared with the normal control group. *P < 0.05,**P < 0.01,***P < 0.001 versus si-NC group; statistical analysis was performed using Tukey’s post hoc test (10.6084/m9.figshare.30654116).

https://doi.org/10.1371/journal.pone.0343735.g003

miRNA-21-5p targets and regulates PDCD4

Although miRNA-21-5p has been linked to the pathogenesis of polycystic ovary syndrome with insulin resistance (PCOS-IR) in rat models, its specific mechanism in regulating the proliferation and apoptosis of ovarian granulosa cells remains unclear. By analyzing granulosa cell transcriptomic data from the GEO database (GSE80432), we identified abnormal expression of PDCD4 in PCOS patients. mRNA analysis indicated that PDCD4 expression levels were significantly higher in the ovarian granulosa cells of PCOS patients compared to the control group (all P < 0.001; Fig 4A). RT-qPCR further confirmed that both PDCD4 (Fig 4B) and RNA immunoprecipitation (RIP) assays demonstrated that miRNA-21-5p and PDCD4 were co-enriched within the same RISC complex in ovarian granulosa cells of PCOS-IR rats, indicating a direct intracellular interaction or shared binding relationship between the two molecules (Fig 4C). Pearson correlation analysis showed a strong positive correlation between miRNA-21-5p and PDCD4 expression in the granulosa cells of PCOS-IR model rats (R² = 0.9667, P < 0.0001; Fig 4D). To further explore the relationship between miRNA-21-5p and PDCD4 in PCOS-IR, we examined the molecular structure of their binding pocket. The results suggested that PDCD4 may be a target of miRNA-21-5p (Fig 4E). Additionally, we investigated the interaction between miRNA-21-5p and PDCD4 using pmirGLO-miRNA-21-5p-WT and pmirGLO-miRNA-21-5p-Mut plasmids (Fig 4F). Dual-luciferase reporter assays demonstrated that rno-miR-21-3p significantly downregulated luciferase expression in the rno-miRNA-21-5p-WT group compared to the NC group (P < 0.001), indicating a binding interaction. However, after mutation, PDCD4 did not downregulate luciferase expression in the rno-miRNA-21-5p-MUT group (P > 0.05), confirming the success of the mutation. Collectively, these findings demonstrate that PDCD4 is a direct target of miRNA-21-5p (Fig 4G).

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Fig 4. miRNA-21-5p Targets and Regulates PDCD4.

(A) Transcriptome analysis of human granulosa cells based on the Gene Expression Omnibus (GEO) database (GSE80432) revealed that the mRNA levels of PDCD4 in granulosa cells from PCOS patients were significantly elevated compared to healthy controls. (B) RT-qPCR was used to detect PDCD4 expression in granulosa cells of PCOS-IR rat ovaries; (C) RNA immunoprecipitation (RIP) assays demonstrated co-localization of miRNA-21-5p and PDCD4 in ovarian granulosa cells of polycystic ovary syndrome with insulin resistance (PCOS-IR) rats. (D) Pearson correlation analysis revealed an association between miRNA-21-5p and PDCD4 in granulosa cells from PCOS-IR rat ovaries. (E) Protein-protein interaction binding sites between miRNA-21-5p and PDCD4; AlphaFold3 validated the protein-protein interaction correlation coefficient between miRNA-21-5p and PDCD4; (F-G) Potential complementary binding sites between miRNA-21-5p and PDCD4; luciferase activity assays validated the target relationship between PDCD4 and miRNA-21-5p; B-C, N = 6. Data are measured values, expressed as mean ± standard deviation. Statistical analysis between groups was performed using a t-test. Compared with the CONTROL group or the NC mimic + miRNA-21-5p-wt group, ***P < 0.001; ****P < 0.0001 (10.6084/m9.figshare.30654137).

https://doi.org/10.1371/journal.pone.0343735.g004

miRNA-21-5p regulates ovarian granulosa cell proliferation and apoptosis by targeting PDCD4

As demonstrated in previous studies, activation of the PDCD4 gene promotes insulin resistance in PCOS patients by inducing apoptosis in ovarian granulosa cells [14,29]. We have confirmed a positive correlation between miRNA-21-5p expression and PDCD4 activation. This leads us to hypothesize that miRNA-21-5p may regulate the proliferation and apoptosis of ovarian granulosa cells through the PDCD4 gene. To investigate this, we measured PDCD4 mRNA and protein levels in primary ovarian granulosa cells isolated from PCOS-IR model rats after silencing miRNA-21-5p using si-miRNA-21-5p lentivirus. As shown in Figs 5A and 5B, PDCD4 expression was significantly reduced in the si-miRNA-21-5p group, indicating that miRNA-21-5p positively regulates PDCD4 in the ovarian granulosa cells of PCOS-IR rats (all P < 0.05). To further investigate the role of PDCD4 in the miRNA-21-5p-mediated regulation of ovarian granulosa cell proliferation and apoptosis, PCOS-IR ovarian granulosa cells were treated with the PDCD4 inhibitor PDCD4-IN-1 (10 mM). Primary ovarian granulosa cells obtained from PCOS-IR model rats were divided into two groups: the PCOS-IR group and the PCOS-IR + PDCD4-IN-1 group. After the intervention with PDCD4-IN-1, miRNA-21-5p mRNA levels were significantly reduced(Fig 5A,B).Concurrently, PDCD4-IN-1 treatment elevated the levels of the apoptotic factors caspase-3 and PDCD4 and reduced Bcl-2 expression (Fig 5C). Furthermore, CCK-8 assay results indicated that PDCD4-IN-1 treatment increased the viability of primary ovarian granulosa cells from PCOS-IR model rats (Fig 3D) (P < 0.01). Additionally, PDCD4-IN-1 significantly decreased the apoptosis rate in these cells (Fig 3E) (P < 0.01).

Download:

Fig 5. miRNA-21-5p Regulates Ovarian Granulosa Cell Proliferation and Apoptosis by Targeting PDCD4.

(A and B) PDCD4 andmiRNA-21-5p mRNA expression levels in ovarian granulosa cells from si-NC, si-miRNA-21-5p, PCOS-IR, and PCOS-IR+PDCD4-IN-1 groups, where primary granulosa cells from PCOS-IR model rats underwent miRNA-21-5p silencing and PDCD4 inhibition. (C) Caspase-3, PDCD4, and Bcl-2 protein expression in ovarian granulosa cells from the same experimental groups following identical interventions. (D) Cell proliferation was measured using CCK-8 assay. (E) Apoptosis rates were determined by TUNEL assay (n = 6). Data represent mean ± SD. Significant differences versus si-NC group: **P < 0.01, ***P < 0.001; versus PCOS-IR group: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 (10.6084/m9.figshare.30654146).

https://doi.org/10.1371/journal.pone.0343735.g005

Discussion

PCOS is a leading cause of infertility in women of childbearing age. More than 50% of women with PCOS experience severe insulin resistance. Additionally, metabolic disorders such as insulin sensitivity and dyslipidemia can further impact infertility in these patients [30], therefore, the prevention and treatment of insulin resistance and the improvement of dyslipidemia and endocrine metabolism are the key to the treatment of PCOS. Since the pathogenesis of insulin resistance in PCOS has not yet been elucidated, many researchers believe that it involves various factors such as metabolism, immunity, endocrinology, and familial inheritance, and affects many organs such as the hypothalamus, pituitary, ovary, heart, and adrenal glands etc [31–33]. This has therefore become a difficult and prominent research problem in the field of gynaecology.

miRNAs play a crucial role in regulating various cellular processes, including DNA repair, differentiation, proliferation, development, cell death, and immunomodulation. Additionally, they are implicated in the progression of numerous diseases [34,35] Studies have demonstrated that specific miRNA differential expression profiles are present in the serum, follicular fluid, granulosa cells, follicular membrane cells, and adipose tissue of PCOS patients. These miRNAs play a role in PCOS by regulating insulin sensitivity, androgen synthesis, and follicular development. Future research may leverage miRNAs as potential biomarkers for the clinical diagnosis and treatment of PCOS [36]. Recent studies have confirmed that miRNA-21 is significantly overexpressed in the serum of obese PCOS patients [37,38], by regulating the expression of PTEN protein in adipocytes, it enhances cellular glucose uptake and utilization, thereby ameliorating insulin resistance induced by hyperglycemia and hyperinsulinemia [36,39], therefore, miR-21 may represent a potential mechanism and novel therapeutic target for improving insulin resistance. In our study, a PCOS–insulin resistance (IR) model was established using dehydroepiandrosterone (DHEA) in combination with a high-fat high-sucrose diet. This model demonstrated high expression of miRNA-21-5p and exhibited typical features of PCOS-IR, including insulin resistance, hormonal imbalance, and elevated levels of miRNA-21-5p. In comparison to the PCOS-IR group and the si-NC group, the si-miRNA-21-5p group displayed restored ovarian morphology, a more balanced hormone profile, and improved insulin resistance.

Programmed cell death 4 (PDCD4) is a recently identified apoptosis-associated gene. Research has demonstrated that PDCD4 can inhibit the translation process by directly binding to the mRNA coding region of target genes. Additionally, it can compete with the Eukaryotic Translation Initiation Factor (elF) 4G for binding to eIF4A, thereby blocking the initiation of translation for target proteins. This inhibition impacts various biological, including cell proliferation, apoptosis, transformation, invasion, insulin resistance, lipid metabolism disorders, and autophagy [40–42]. More importantly, the deletion of the PDCD4 gene was found to disrupt lipid metabolism by up-regulating the expression of the lipid metabolism factor liver X receptor (LXR-α). This action provides a protective effect against obesity induced by a high-fat diet in mice [43]. Given the presence of obesity, insulin resistance, dyslipidemia and other metabolic syndromes in women with PCOS, we hypothesize that PDCD4 plays a role in the development of insulin resistance and lipid metabolism disorders in this population [44]. In inflammatory diseases and tumors, PDCD4 promoter activity increases following miRNA-21 overexpression, indicating that miRNA-21 directly regulates PDCD4 expression in these pathological contexts [45,46]. Another study showed that miRNA-21 was able to reverse hyperglycemia- and hyperinsulin-induced insulin resistance in 3T3-L1 adipocytes via the PTEN-AKT pathway [47,48]. It was hypothesized that regulating the activity of miRNA-21 and PDCD4 genes in ovarian granulosa cells could inhibit apoptosis in these cells, thereby improving insulin resistance in PCOS. Our study revealed elevated PDCD4 expression in a polycystic ovary syndrome model with insulin resistance (PCOS-IR). The direct targeting relationship between miRNA-21-5p and PDCD4 was confirmed through dual-luciferase and RNA immunoprecipitation assay, further supported by AlphaFold3 predictions. Compared to the PCOS-IR and si-NC groups, the si-miRNA-21-5p group showed reduced apoptosis in ovarian granulosa cells.

In the PCOS-IR rat model, ovarian tissues exhibited distinct pathological features, such as dysregulation of sex hormones, stromal hyperplasia, and polycystic changes. Correspondingly, serum levels of fasting blood glucose (FBG), fasting insulin (FINS), and HOMA-IR were elevated, confirming the successful establishment of the model. At the molecular level, both miRNA-21-5p and PDCD4 were upregulated in ovarian granulosa cells. The dual-luciferase assay confirmed that miRNA-21-5p directly targets PDCD4. Functional experiments showed that knocking out miRNA-21-5p improved ovarian cyst architecture, altered insulin sensitivity parameters, and reduced granulosa cell apoptosis. These findings indicate that the miRNA-21-5p/PDCD4 axis may play a role in the pathophysiology of PCOS-IR and deserves further investigation. However, this study has several limitations. First, the findings were derived from a rat model, and their relevance to human polycystic ovary syndrome–related insulin resistance (PCOS-IR) remains unclear. Given potential species-specific differences in miRNA regulation and metabolic pathways, further validation in clinical populations is necessary. Second, while the dual-luciferase and RNA immunoprecipitation assay confirmed a direct targeting relationship between miRNA-21-5p and PDCD4, comprehensive mechanistic validation through rescue experiments was not conducted. Future studies should prioritize validation in human clinical samples and perform rigorous mechanistic studies, along with expanded metabolic profiling, to establish the therapeutic significance of this pathway in PCOS-IR.

Conclusion

In summary, this study identifies the miRNA-21-5p/PDCD4 axis as a significant factor in the pathogenesis of PCOS-IR. This provides a preliminary foundation for exploring its molecular mechanisms and potential therapeutic targets. However, these findings require further validation through more rigorous experimental and clinical studies.

Supporting information

S1 File. The original plotting data, the original uncropped and unadjusted images underlying all blot or gel results for https://doi.org/10.6084/m9.figshare.30610571.

https://doi.org/10.1371/journal.pone.0343735.s001

(DOCX)

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Figures

Abstract

Introduction

Materials and methods

Experimental animals

Establishment of the PCOS insulin resistance rat model

Grouping and lentivirus injection

Real-time PCR

AlphaFold3 predicts direct interaction of miRNA-21–5- with PDCD4

RNA immunoprecipitation (RIP) assay

Primary granulosa cell collection

Enzyme-linked immunosorbent assay (ELISA) for FINS、FBG and sex hormones (T, E2, LH, FSH)

Protein extraction and Western-blot analysis were performed to measure the expression of caspase-3, PDCD4and Bcl-2 in ovarian granulosa cells

Detection of apoptosis by flow cytometry

Dual-luciferase reporter gene analysis

PDCD4 inhibitor treatment in primary rat ovarian granulosa cells (OGC)

Statistical analysis

Results

Overexpression of miRNA-21-5p in Ovarian granulosa cells of a rat polycystic ovary syndrome model with insulin resistance

Inhibition of miRNA-21-5p ameliorates ovarian pathological changes, hormonal imbalance, and insulin resistance in polycystic ovary syndrome (PCOS)-IR rats

Silencing miRNA-21-5p regulates proliferation and apoptosis of ovarian granulosa cell

miRNA-21-5p targets and regulates PDCD4

miRNA-21-5p regulates ovarian granulosa cell proliferation and apoptosis by targeting PDCD4

Discussion

Conclusion

Supporting information

S1 File. The original plotting data, the original uncropped and unadjusted images underlying all blot or gel results for https://doi.org/10.6084/m9.figshare.30610571.

References