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. 2026 Sep 11;105(12):107747. doi: 10.1016/j.psj.2026.107747

miR-182-5p regulates proliferation and senescence in chicken Leydig cells via the Wnt/β-catenin pathway by targeting FOXN3

Xin-yue Zhao a,b,1, Yu-qi Chen a,b,1, Yao Zhang a,b, Hua-dong Yin a,b,⁎
PMCID: PMC13625787  PMID: 42777356

Abstract

The reproductive performance of roosters is an important factor influencing production efficiency and genetic progress in the poultry industry. Leydig cells (LCs) support male reproductive function through testosterone synthesis and secretion; however, the post-transcriptional mechanisms regulating chicken LCs function remain incompletely understood. Previous studies have shown that testicular miR-182-5p expression is higher in 200-day-old than in 500-day-old roosters, suggesting a possible association with age-related changes in testicular function. This study aims to investigate the role of miR-182-5p in vitro. miR-182-5p overexpression promoted proliferation, inhibited senescence, and elevated testosterone levels in chicken LCs, whereas miR-182-5p inhibition produced the opposite effects. A dual-luciferase reporter assay performed in the chicken fibroblast cell line (DF-1 cells) demonstrated that miR-182-5p targets the 3′untranslated region (3′UTR) of FOXN3. FOXN3 knockdown produced effects consistent with those of miR-182-5p overexpression, supporting FOXN3 as a functional downstream mediator of miR-182-5p. Moreover, miR-182-5p enhanced Wnt/β-catenin signaling, and co-transfection with FOXN3 overexpression, which inhibited this pathway, attenuated the miR-182-5p-induced increases in proliferation and testosterone levels as well as the decrease in senescence. These findings support a miR-182-5p/FOXN3/Wnt/β-catenin regulatory axis that modulates proliferation, senescence, and testosterone levels in chicken LCs in vitro. Further age-comparative and in vivo studies are warranted to determine whether this mechanism contributes to natural reproductive aging or influences reproductive performance in roosters.

Keywords: miR-182-5p, Proliferation, Rooster Leydig cells, Senescence, Testosterone levels

Introduction

The reproductive performance of roosters is a key determinant of egg fertilization rates and offspring genetic quality, serving as a primary driver of production efficiency and economic profitability in the poultry industry (Ansari, 2024; Juiputta et al., 2026). As roosters age, particularly after approximately 45 weeks, testicular atrophy, reduced testicular weight, and declining testosterone levels are frequently observed (Sarabia Fragoso et al., 2013). These age-associated changes may arise from intrinsic alterations in Leydig cells (LCs), diminished stimulation by pituitary gonadotropins, or a combination of both (Chen et al., 2026; Avital-Cohen et al., 2013; Rosenstrauch et al., 1998). As the principal testosterone-producing cells in the testes, LCs play an essential role in supporting spermatogenesis and maintaining male fertility (Chen et al., 2026; Guo et al., 2026). Therefore, alterations in LCs function and its underlying regulatory networks, including those mediated by non-coding RNAs, may contribute to age-associated reproductive decline in roosters (Wang et al., 2026).

MicroRNAs (miRNAs) are a class of small non-coding RNAs, approximately 22 nucleotides in length, that function as crucial post-transcriptional regulators by binding to the 3′untranslated region (3′UTR) of target mRNAs, leading to translational repression or mRNA degradation (Bartel, 2004). In livestock and poultry, miRNAs have been reported to modulate LCs proliferation and testosterone synthesis. For instance, miR-7481-3p overexpression significantly suppresses CXCL14 expression and counteracts the melatonin-induced inhibition of testosterone production in rooster LCs (Xu et al., 2023). In Tibetan sheep, circCSNK1G3 promotes LCs proliferation and testosterone production through the miR-29b/IGF1 axis (Wu et al., 2026). Similarly, miR-27b-3p inhibits goat LCs proliferation and testosterone production by targeting PPARG and thereby modulating AMPK signaling (An et al., 2026). Collectively, these studies indicate that the proliferation and functional activity of LCs in livestock and poultry are closely associated with multiple miRNAs.

MiR-182-5p participates in the regulation of proliferation, senescence and differentiation processes in several mammalian cell types. Specifically, miR-182-5p promotes myogenic differentiation of C2C12 cells by ZBTB7A suppression (Zhang et al., 2025), directly targets FOXO3a to activate AKT/FOXO3a pathway and drive hepatocellular carcinoma proliferation (Cao et al., 2018), and attenuates lens epithelial cellular senescence by inhibiting NOX4 and p38 MAPK against oxidative injury (Li et al., 2020). In the context of reproduction, miR-182-5p has been shown to regulate ovarian steroidogenesis during sheep follicular development (Yuan et al., 2022); however, its role in testicular testosterone biosynthesis in mammals remains unexplored. In poultry, the functions of miR-182-5p are currently limited to reports on neural development, wooden breast myopathy, and follicular atresia (Heydari et al., 2024; Li et al., 2025; Yu et al., 2023), and whether it plays a role in chicken LCs is yet to be determined.

Our previous research conducted miRNA sequencing on the testes of 200-day-old and 500-day-old roosters (BioProject ID: PRJNA1484738), and found that miR-182-5p was significantly highly expressed in the 200-day-old group, suggesting that it may be involved in age-associated testicular function regulation. However, direct evidence for its effects on chicken LCs is still lacking. Based on the foregoing evidence, we hypothesized that miR-182-5p modulates its downstream target genes, which in turn mediate signaling pathways, ultimately regulating the proliferation, senescence, and testosterone levels of chicken LCs. To test this hypothesis, the present study was designed to elucidate the molecular pathways underlying miR-182-5p-mediated regulation of LCs function, with the goal of investigating a cell-intrinsic regulatory mechanism and providing a mechanistic basis for age-comparative and in vivo studies of rooster reproductive function.

Materials and methods

Animals and samples

All experimental animals were approved by the Animal Ethics Committee of Sichuan Agricultural University in Ya′an, and the permit number is 2024202019-1. The experiment used male chickens of the Tianfu P01 strain, which are 15-20 weeks and are currently being bred at Sichuan Agricultural University.

Isolation and culture of chicken LCs

Chicken LCs were isolated and cultured as described in our previous study (Chen et al., 2026). The testes were removed from roosters, and the epididymides and capsules were carefully stripped. Tissue blocks of approximately 1 cm³ were excised, washed in Dulbecco′s phosphate-buffered saline (D-PBS; Gibco, Grand Island, NY, USA) containing 1% penicillin-streptomycin solution (Servicebio, Hubei, China), and then minced into small pieces. The minced tissues were digested with 8 volumes of 1% collagenase type II (Biofroxx, Einhausen, Germany) in a 37 °C water bath for 30 min, with gentle agitation every 5-8 min. After digestion, an equal volume of complete medium (DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin) was added to terminate the reaction. The cell suspension was sequentially filtered through 70 μm and 40 μm nylon mesh filters and then centrifuged at 125 g for 5 min. The supernatant was discarded. After resuspending the pellet, Percoll (Solarbio, Beijing, China) density gradient centrifugation (30%, 60%, and 70%) was performed at 664 g for 30 min. LCs collected from the 30%-60% interface were washed with D-PBS and centrifuged again. Subsequently, the LCs were resuspended and seeded into six-well plates at a density of 1 × 106 cells/well, and then cultured in an incubator (Thermo, Waltham, MA, USA) at 37 °C with 5% CO₂ and saturated humidity.

RNA oligonucleotides, plasmids construction, and transfection

MiR-182-5p mimic, mimic negative control (NC), miR-182-5p inhibitor, inhibitor NC, three siRNAs for FOXN3 (si-FOXN3) and siRNA-NC were designed and synthesized by Sangon Biotech (Shanghai, China). The FOXN3 overexpression plasmid was constructed using pcDNA3.1 vector (Sangon Biotech). For the dual-luciferase reporter assay, wild-type (FOXN3-WT) and mutant (FOXN3-MT) sequences of FOXN3 were cloned into the pmirGLO vector (Sangon Biotech). Transfections were performed using LipofectamineTM 3000 transfection reagent (Thermo) according to the manufacturer′s instructions, when LCs reached 60%-70% confluence. All specific sequences are listed in Table S1.

RNA extraction, cDNA synthesis and qPCR

Total RNA was extracted from LCs at 24 h post-transfection using a commercial RNA extraction kit (Foregene, Chengdu, China). For miRNA analysis, reverse transcription was performed with the Mir-XTM miRNA First-Strand Synthesis Kit (Takara, Beijing, China), using U6 as the internal reference. The qPCR cycling conditions were as follows: pre-denaturation at 95 °C for 5 min; 39 cycles of denaturation at 95 °C for 10 s and annealing at X°C for 30 s (X is related to the target gene); followed by a melting curve analysis with a ramp rate of 0.5 °C/s from 65 °C to 95 °C. For mRNA analysis, reverse transcription was carried out using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara), with β-actin as the internal reference. The qPCR protocol consisted of pre-denaturation at 98 °C for 2 min; 40 cycles of denaturation at 98 °C for 2 s, annealing at X°C for 15 s (X is related to the target gene), and extension at 72 °C for 10 s; followed by melting curve generation from 65 °C to 95 °C at 0.5 °C/s. All qPCR primers were designed using Prime Premier 5 (PREMIER Biosoft, CA, USA) and synthesized by Tsingke (Tsingke, Beijing, China). The primer sequences are detailed in Table S2. Each sample was tested three times in duplicate. Relative expression levels were calculated using the 2-∆∆Ct method.

Western blot assay

Total protein was extracted from LCs at 48 h post-transfection using RIPA buffer (high) (Solarbio). Protein concentrations were determined with a Bicinchoninic Acid (BCA) Protein Assay Kit (BestBio, Shanghai, China). Different molecular weight proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with Western blocking solution (Beyotime, Shanghai, China) for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. After washing three times with Tris-buffered saline (Servicebio) containing Tween-20 (TBST; Adamas, Shanghai, China), the membranes were incubated with appropriate secondary antibodies at 4 °C for 1 h. The antibody information is listed in Table S3. Following three additional TBST washes, protein bands were visualized using an Enhanced Chemiluminescence (ECL) reagent (Beyotime). GAPDH was used as the internal reference, and band densities were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunofluorescence assay

To verify the expression of 3β‑hydroxysteroid dehydrogenase 1 (HSD3B1), a LCs‑specific marker, immunofluorescence staining was performed. Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, washed three times with D‑PBS, and permeabilized with 0.3% Triton X-100 for 10 min, followed by three additional D-PBS washes. Non-specific binding sites were blocked with Western blocking solution for 1 h at room temperature. Then, cells were incubated with anti-HSD3B1 primary antibody overnight at 4°C, washed three times with TBST, and incubated with Rhodamine (TRITC)-conjugated goat anti-Rabbit IgG secondary antibody at 37°C for 1 h in the dark. After three TBST washes, nuclei were counterstained with DAPI (1 μg/ml) for 5 min at room temperature. Finally, fluorescence images were captured using an inverted fluorescence microscope. The antibody information is listed in Table S4.

5-Ethynyl-2′-deoxyuridine (EdU) assay

Cell proliferation was assessed using the BeyoClickTM EdU Cell Proliferation Kit with AF584 (Beyotime). Briefly, LCs were incubated with EdU reagent A for 3 h, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 after three washes. The cells were then incubated with the Click reaction solution in the dark for 30 min, followed by nuclear counterstaining with Hoechst 33342. After additional washes, fluorescence images were captured using an inverted fluorescence microscope. The ratio of EdU-positive cells was quantified using ImageJ software (National Institutes of Health).

Flow cytometric cell cycle analysis

Cell cycle distribution was assessed using a Cell Cycle Assay Kit (Elabscience, Wuhan, China). Transfected LCs (48 h post-transfection) were collected, washed with D-PBS, fixed with 70% ethanol, thoroughly mixed, and stored at -20°C overnight. The next day, the cells were washed with D-PBS and the supernatant was discarded. RNase A reagent was then added, and the mixture was incubated in a 37 °C water bath for 30 min. Subsequently, PI reagent was added, and the cells were incubated at 4 °C in the dark for 30 min. Finally, the cells were analyzed using a flow cytometer (Challen Bio, Beijing, China), and cell cycle data were processed with Modfit LT 5.0 software (Verity Software House, Topsham, Maine, USA).

SA-β-Gal analysis

Cellular senescence was assessed using a Senescence β-Galactosidase Staining Kit (Beyotime). At 48 h post-transfection, LCs were washed once with D-PBS, fixed with the provided fixative solution at room temperature for 15 min, and then washed three times with D-PBS. The cells were subsequently incubated with the staining working solution (prepared at a ratio Component A: Component B: Component C: X-Gal solution = 1: 1: 93: 5) at 37 °C for 30 min. Stained cells were observed and photographed under a light microscope, and SA-β-Gal positive area was quantified using ImageJ software (National Institutes of Health).

ELISA

The ELISA kit (Meimian, Jiangsu, China) was used to detect testosterone concentrations in the cell culture supernatants. Following the manufacturer's instructions, we performed the following steps. 50 μL of standard solution was added to the standard wells, while sample wells received 10 μL of test sample followed by 40 μL of sample diluent. Subsequently, 100 μL of HRP‑Conjugate Reagent was dispensed into each well. The plate was sealed and incubated at 37 °C for 1 h. After incubation, the wells were washed five times with wash solution. Then, 50 μL of Chromogen Solution A and 50 μL of Chromogen Solution B were added to each well, mixed gently, and incubated at 37 °C in the dark for 15 min. Finally, 50 μL of Stop Solution was added, and the absorbance was measured at 450 nm within 15 min.

Dual-luciferase reporter assay

The dual-luciferase reporter assay was performed using chicken fibroblast cell line (DF-1 cells), owing to their high and reproducible transfection efficiency. DF-1 cells were seeded in 24-well plates and co-transfected with FOXN3-WT or FOXN3-MT, along with miR-182-5p mimic or mimic NC. At 48 h post-transfection, luciferase activity was measured using the Luc-PairTM Duo-Luciferase HS Assay Kit (Genecopoeia, Maryland, USA) according to the manufacturer′s instructions.

Statistical analysis

Statistical analyses were performed using SPSS version 26.0 (SPSS, Inc., Chicago, IL, USA). All experiments were repeated with three biological replicates. Differences between the two groups were conducted using the t-test, while comparisons among three or more groups were performed using one-way analysis of variance (ANOVA). The results were presented in the form of the mean ± the standard error of the mean (SEM). The visualization was achieved using GraphPad Prism (GraphPad, La Jolla, CA, USA). Statistical significance was defined as *P < 0.05, and **P < 0.01 was considered highly significant.

Results

miR-182-5p promotes proliferation and inhibits senescence in chicken LCs

Immunofluorescence staining revealed that the isolated LCs had sufficient purity for subsequent experiments. (Fig. 1A). To explore the role of miR-182-5p in chicken LCs, we constructed miR-182-5p mimic and miR-182-5p inhibitor, both of which exhibited high transfection efficiency (P < 0.01, Fig. 1B-C). The effects of miR-182-5p on LCs proliferation were then assessed. qPCR and western blot analyses showed that the miR-182-5p mimic significantly upregulated the expression of proliferation-related genes (P < 0.01, Fig. 1D-F), whereas the inhibitor downregulated their expression (P < 0.05, Fig. 1G-I). Consistently, the EdU assay revealed that miR-182-5p mimic markedly increased the ratio of EdU-positive cells (P < 0.05, Fig. 1J-K), while the inhibitor decreased it (P < 0.01, Fig. 1L-M). Furthermore, flow cytometry cell cycle analysis indicated that miR-182-5p mimic promoted the transition of LCs into S and G2 phases (P < 0.01, Fig. 1N-O), whereas the inhibitor induced cell cycle arrest at the G0/G1 phase (P < 0.01, Fig. 1P-Q). Collectively, these findings demonstrate that miR-182-5p promotes chicken LCs proliferation.

Fig. 1.

Fig. 1

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MiR-182-5p promotes chicken LCs proliferation. (A) Immunofluorescence staining for HSD3B1, a LCs‑specific protein. (B, C) Transfection efficiency of miR-182-5p mimic and inhibitor in LCs. n = 3. (D) Relative mRNA expression of proliferation-related genes following miR-182-5p overexpression. n = 3. (E, F) Relative protein levels of of CDK2 and PCNA after transfection with miR-182-5p mimic. n = 3. (G) Relative mRNA expression of proliferation-related genes following miR-182-5p interference. n = 3. (H, I) Relative protein levels of CDK2 and PCNA after transfection with miR-182-5p inhibitor. n = 3. (J-M) EdU positive cell ratios after transfection with miR-182-5p mimic and inhibitor. n = 3. (N-Q) Effects of miR-182-5p overexpression and interference on cell cycle progression. n = 3. Statistical results are represented as mean ± SEM. *P < 0.05, **P < 0.01.

Cell proliferation and senescence are two core processes that are mutually opposing yet closely related in the fate of cells (Zhou et al., 2025). The effect of miR-182-5p on chicken LCs senescence was then examined. qPCR and western blot analyses revealed that the miR-182-5p mimic significantly inhibited the expression of senescence-associated genes (P < 0.05, Fig. 2A-C), whereas the inhibitor significantly increased their expression (P < 0.01, Fig. 2D-F). Consistently, the mimic markedly reduced the SA-β-Gal positive area (P < 0.01, Fig. 2G-H), while the inhibitor significantly enlarged it (P < 0.05, Fig. 2I-J). In addition, miR-182-5p mimic significantly increased testosterone levels (P < 0.01, Fig. 2K), whereas the inhibitor had the opposite effect (P < 0.01, Fig. 2L). Overall, these results demonstrate that miR-182-5p promotes proliferation, inhibits senescence, and elevates testosterone levels in chicken LCs.

Fig. 2.

Fig. 2

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MiR-182-5p inhibits senescence and elevates testosterone levels in chicken LCs. (A) Relative mRNA expression of senescence-associated genes following miR-182-5p overexpression. n = 3. (B, C) Relative protein levels of P53 and P16 after transfection with miR-182-5p mimic. n = 3. (D) Relative mRNA expression of senescence-associated genes following miR-182-5p interference. n = 3. (E, F) Relative protein levels of P53 and P16 after transfection with miR-182-5p inhibitor. n = 3. (G-J) SA-β-Gal positive area changes after miR-182-5p overexpression and interference. n = 3. (K, L) ELISA analysis of testosterone levels following miR-182-5p overexpression and interference. n = 3. Statistical results are represented as mean ± SEM. *P < 0.05, **P < 0.01.

miR-182-5p directly target FOXN3 and inhibits expression

To elucidate the mechanism by which miR-182-5p regulates chicken LCs proliferation and senescence, potential target genes were predicted using the miRDB database (https://mirdb.org/cgi-bin/search.cgi). Among these candidate genes (SH3BGRL2, PCDH8, CBX7, ACAP3, FOXN3), FOXN3 was selected based on qPCR screening results (Fig. 3A-B). To verify the direct targeting relationship between miR-182-5p and FOXN3, a dual-luciferase reporter assay was performed. FOXN3-WT and FOXN3-MT sequences were cloned into the reporter vector (Fig. 3C) and co-transfected with mimic NC and miR-182-5p mimic into DF-1 cells. The results demonstrated that the miR-182-5p mimic significantly reduced firefly/renilla luciferase ratio in cells transfected with FOXN3-WT, whereas no significant effect was observed in cells transfected with FOXN3-MT (P < 0.01, Fig. 3D). Collectively, these findings confirm that miR-182-5p directly targets the 3′UTR of FOXN3 and negatively regulates its expression.

Fig. 3.

Fig. 3

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MiR-182-5p targets FOXN3 and inhibits its expression. (A, B) Effects of miR-182-5p mimic and inhibitor on target gene mRNA expression level. n = 3. (C) Diagram of the miR-182-5p binding sites on the wild type and mutant type of FOXN3. (D) Firefly/Renilla luciferase ratio in DF-1 cells co-transfected with FOXN3-WT or FOXN3-MT and mimic NC or miR-182-5p mimic. n = 3. Statistical results are represented as mean ± SEM. *P < 0.05, **P < 0.01.

FOXN3 inhibits proliferation and promotes senescence in chicken LCs

To clarify the role of FOXN3 in chicken LCs, three specific siRNAs targeting FOXN3 were designed. qPCR analysis showed that all three siRNAs significantly reduced FOXN3 mRNA level, with si-FOXN3-77 exhibiting the highest knockdown efficiency (P < 0.01, Fig. 4A); thus, this siRNA was selected for subsequent experiments. Meanwhile, an overexpression plasmid (pcDNA3.1-FOXN3) was constructed and its effectiveness was confirmed by qPCR (P < 0.01, Fig. 4B). To investigate the effect of FOXN3 on the LCs proliferation, we used the same experimental methods as described previously. qPCR and western blot results revealed that pcDNA3.1-FOXN3 significantly downregulated the expression of proliferation-related genes (P < 0.05, Fig. 4C-E), whereas FOXN3 knockdown exerted the opposite effect (P < 0.05, Fig. 4F-H). Consistently, EdU assay demonstrated that pcDNA3.1-FOXN3 markedly reduced the ratio of EdU-positive cells (P < 0.01, Fig. 4I-J), while knockdown increased it (P < 0.01, Fig. 4K-L). Moreover, cell cycle analysis indicated that pcDNA3.1-FOXN3 induced G0/G1 phase arrest (P < 0.01, Fig. 4M-N), whereas FOXN3 knockdown promoted cell cycle progression into S and G2 phases (P < 0.01, Fig. 4O-P). Collectively, these results demonstrate that FOXN3 functions as a negative regulator of LCs proliferation.

Fig. 4.

Fig. 4

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FOXN3 inhibits chicken LCs proliferation. (A, B) knockdown and overexpression efficiency of FOXN3 in LCs. n = 3. (C) Relative mRNA expression of proliferation-related genes following FOXN3 overexpression. n = 3. (D, E) Relative protein levels of CDK2 and PCNA after transfection with pcDNA3.1-FOXN3. n = 3. (F) Relative mRNA expression of proliferation-related genes following FOXN3 knockdown. n = 3. (G, H) Relative protein levels of CDK2 and PCNA after transfection with si-FOXN3. n = 3. (I-L) EdU positive cell ratios after transfection with pcDNA3.1-FOXN3 and si-FOXN3. n = 3. (M-P) Effects of FOXN3 overexpression and knockdown on the cell cycle progression. n = 3. Statistical results are represented as mean ± SEM. *P < 0.05, **P < 0.01.

The effect of FOXN3 on chicken LCs senescence was then examined. pcDNA3.1-FOXN3 significantly upregulated the mRNA and protein levels of senescence-associated genes (P < 0.01, Fig. 5A-C), whereas FOXN3 knockdown exerted the opposite effect (P < 0.05, Fig. 5D-F). Consistently, SA-β-Gal staining revealed that pcDNA3.1-FOXN3 markedly increased the SA-β-Gal positive area (P < 0.05, Fig. 5G-H), while FOXN3 knockdown significantly decreased it (P < 0.05, Fig. 5I-J). Furthermore, FOXN3 overexpression significantly reduced testosterone levels (P < 0.01, Fig. 5K), whereas FOXN3 knockdown had the opposite effect (P < 0.01, Fig. 5L). These results indicate that FOXN3 inhibits proliferation, promotes senescence, and decreases testosterone levels in chicken LCs, which is opposite to the functional role of miR-182-5p.

Fig. 5.

Fig. 5

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FOXN3 promotes senescence and reduces testosterone levels in chicken LCs. (A) Relative mRNA expression of senescence-associated genes following FOXN3 overexpression. n = 3. (B, C) Relative protein levels of P53 and P16 after transfection with pcDNA3.1-FOXN3. n = 3. (D) Relative mRNA expression of senescence-associated genes following FOXN3 knockdown. n = 3. (E, F) Relative protein levels of P53 and P16 after transfection with si-FOXN3. n = 3. (G-J) SA-β-Gal positive area changes after FOXN3 overexpression and knockdown. n = 3. (K, L) ELISA analysis of testosterone levels following FOXN3 overexpression and knockdown. n = 3. Statistical results are represented as mean ± SEM. *P < 0.05, **P < 0.01.

miR-182-5p mediates the Wnt/β-catenin signaling pathway by targeting FOXN3

To determine whether FOXN3 functionally mediates the effects of miR-182-5p on chicken LCs proliferation and senescence, we performed a co-transfection assay. Compared with miR-182-5p overexpression alone, co-transfection of the miR-182-5p mimic with pcDNA3.1-FOXN3 significantly reduced the expression of proliferation-related genes (P < 0.01, Fig. 6A-C), and decreased EdU-positive cells ratio (P < 0.05, Fig. 6D-E). Conversely, co-transfection elevated the expression of senescence-associated genes (P < 0.01, Fig. 6F-H), and increased SA-β-Gal positive area (P < 0.01, Fig. 6I-J). Furthermore, co-transfection reduced the testosterone levels compared with miR-182-5p mimic transfection alone (P < 0.01, Fig. 6K). These results indicate that FOXN3 overexpression attenuates the pro-proliferative, anti-senescence, and testosterone-increasing effects of miR-182-5p in chicken LCs. Thus, the co-transfection results support FOXN3 as a functional downstream mediator of miR-182-5p in chicken LCs.

Fig. 6.

Fig. 6

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FOXN3 acts as a functional downstream mediator of miR-182-5p in chicken LCs. (A) Relative mRNA expression of proliferation-related genes following co-transfection of mimic NC or miR-182-5p mimic with pcDNA3.1 or pcDNA3.1-FOXN3. n = 3. (B, C) Relative protein levels of CDK2 and PCNA after co-transfection. n = 3. (D-E) EdU positive cell ratio following co-transfection. n = 3. (F) Relative mRNA expression of senescence-associated genes after co-transfection. n = 3. (G-H) Relative protein levels of P53 and P16 following co-transfection. n = 3. (I-J) SA-β-Gal positive area changes after co-transfection. n = 3. (K) ELISA analysis of testosterone levels following co-transfection. n = 3. Statistical results are represented as mean ± SEM. *P < 0.05, **P < 0.01.

The Wnt/β-catenin signaling pathway is evolutionarily conserved and centrally regulates cell proliferation, senescence, and differentiation throughout the cellular life cycle (Garcin and Habib, 2017). To investigate the downstream pathways of FOXN3, we examined key components of the Wnt/β-catenin cascade by western blotting. FOXN3 Knockdown significantly increased active β-catenin levels, decreased phosphorylated-β-catenin (p-β-catenin) ratio (P < 0.05, Fig. 7A-C). Similarly, miR-182-5p overexpression significantly inhibited β-catenin phosphorylation and elevated its active protein expression (P < 0.05, Fig. 7D-F). However, this effect was reversed upon co-transfection of the miR-182-5p mimic with pcDNA3.1-FOXN3 (P < 0.01, Fig. 7D-F). Collectively, both miR-182-5p overexpression and FOXN3 knockdown activated the Wnt/β-catenin pathway, whereas co-transfection attenuated this activation. Taken together, these findings indicate that miR-182-5p targets FOXN3 and, through the Wnt/β-catenin pathway, promotes proliferation, inhibits senescence and elevates testosterone levels in chicken LCs.

Fig. 7.

Fig. 7

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MiR-182-5p activates the Wnt/β-catenin signaling pathway by targeting FOXN3. (A, B) Relative protein levels of active β-catenin, P-β-catenin and total β-catenin following FOXN3 knockdown. n = 3. (C) Relative protein levels of P-β-catenin / total β-catenin after FOXN3 knockdown. n = 3. (D, E) Relative protein levels of active β-catenin, P-β-catenin and total β-catenin after co-transfection of mimic NC or miR-182-5p mimic with pcDNA3.1 or pcDNA3.1-FOXN3. n = 3. (F) Relative protein levels of P-β-catenin / total β-catenin after co-transfection. n = 3. Statistical results are represented as mean ± SEM. *P < 0.05, **P < 0.01.

Discussion

The present study identifies miR-182-5p as a regulator of proliferation, senescence, and testosterone levels in chicken LCs in vitro. Bidirectional manipulation of miR-182-5p showed that miR-182-5p overexpression promoted proliferation, inhibited LCs senescence, and elevated testosterone levels in chicken LCs, whereas its inhibition yielded the opposite effects. FOXN3 exerts regulatory effects contrary to those of miR-182-5p, and the co-transfection experiments showed that FOXN3 overexpression attenuated the effects of miR-182-5p. However, since the cells used in this study were obtained from 15‑20 weeks roosters and cultured in an isolated system, the present experiments address cell‑autonomous regulation in vitro. The observed difference in testicular miR-182-5p expression between 200-day-old and 500-day-old roosters provided rationale for investigating this miRNA, but it does not establish that the current cell model reproduces age-related testicular decline.

Testosterone output is the most physiologically relevant functional endpoint of LCs. Testosterone production is the most physiologically relevant functional output of LCs (Zirkin and Papadopoulos, 2018). Increased miR-182-5p expression was accompanied by higher testosterone levels, whereas FOXN3 overexpression reduced testosterone levels; the rescue experiments further supported a functional relationship between miR-182-5p and FOXN3. Nevertheless, the concurrent changes in testosterone levels, proliferation, and senescence-associated measures do not establish that one of these cellular outcomes directly caused the others. The observed testosterone differences could reflect altered steroidogenic activity per cell, differences in the number or viability of LCs, or a combination of these factors. Moreover, testosterone production in vivo is not determined by LCs in isolation. It is regulated by pituitary luteinizing hormone (LH) stimulation and by interactions with Sertoli cells, germ cells, peritubular cells, immune cells, and other components of the testicular microenvironment (O'Donnell et al., 2022; Mishra et al., 2012; Piprek et al., 2024). Accordingly, the present results show that the miR-182-5p/FOXN3 regulatory relationship can influence testosterone levels in cultured LCs, but they do not determine its contribution to circulating testosterone levels or reproductive function in intact roosters.

More broadly, miRNAs contribute to male reproductive function by regulating the state and behavior of both germ cells and testicular somatic cells. In dairy goats, miR-204 regulates spermatogonial stem cell proliferation through Sirt1 (Niu et al., 2016). Sertoli cell-derived exosomal miR-486-5p promotes mouse spermatogonial stem cell differentiation by regulating PTEN, illustrating how miRNAs can mediate communication within the testicular microenvironment (Li et al., 2021). In rooster LCs, miR-7481-3p has been reported to regulate CXCL14 expression and modify the effect of melatonin on testosterone production (Xu et al., 2023). Together, these studies indicate that miRNAs can coordinate reproductive cell proliferation, differentiation, and endocrine activity, although their effects depend on species, cell type, and target availability. The pro-proliferative and anti-senescence effects of miR-182-5p observed in chicken LCs are directionally consistent with its reported promotion of hepatocellular carcinoma cell proliferation and attenuation of senescence-associated changes in lens epithelial cells (Cao et al., 2018; Li et al., 2020). This cross context agreement suggests that miR-182-5p may engage partially conserved cell state regulatory programs. It does not, however, demonstrate functional specificity or cell type independent activity, because the targets and regulatory networks available to a miRNA differ across cellular contexts.

Mechanistically, the dual-luciferase reporter assay conducted in DF-1 cells demonstrated that miR-182-5p regulates the FOXN3 3′UTR region through a recognition site dependent mechanism. In primary chicken LCs, miR-182-5p and FOXN3 exert opposing effects on cell proliferation, senescence, and testosterone levels. Together with the co-transfection rescue results, these findings support that FOXN3 serves as a functional downstream mediator of miR-182-5p. These complementary findings do not, however, establish a direct endogenous physical interaction between miR-182-5p and FOXN3 mRNA in LCs. Nevertheless, FOXN3 has been linked to male reproductive biology: it has been identified as a candidate gene for human azoospermia, and disruption of its homolog in Drosophila affects germ cell homeostasis (Yu et al., 2016). Previous studies in tumor-derived cells have also described antiproliferative and senescence-associated functions of FOXN3 (Sun et al., 2016; Yang et al., 2023; Wang et al., 2024; Kong et al., 2024). These reports provide mechanistic context, but transformed tumor cells differ substantially from normal rooster LCs and do not establish the physiological role of FOXN3 in the rooster testis. By contrast, the functional evidence obtained in primary chicken LCs suggests that FOXN3 restrains the miR‑182‑5p associated cellular effects under the present experimental conditions.

The present findings further support the Wnt/β-catenin pathway as a downstream component of this regulation. FOXN3 knockdown increased active β-catenin and reduced p-β-catenin ratio, changes consistent with enhanced pathway related signaling, whereas FOXN3 overexpression attenuated the effect of the miR-182-5p mimic. Together, these support the interpretation that Wnt/β-catenin signaling mediates, at least in part, the effects of the miR-182-5p/FOXN3 axis in cultured chicken LCs. This interpretation is compatible with the established roles of Wnt/β-catenin signaling in cell-cycle control and the proliferation of spermatogonial stem cells and stromal stem cells (Tetsu and McCormick, 1999; Chan et al., 1999; Takase and Nusse, 2016; Liu et al., 2024), as well as reports that FOXN3 can negatively regulate β-catenin in other cellular contexts (Zhao et al., 2020; Lin et al., 2020). However, Wnt/β-catenin is a broadly acting, context-dependent pathway, and the present findings do not establish it as the only downstream mediator. Other targets of miR-182-5p and other FOXN3 associated pathways, including AKT/MDM2/p53, NF-κB, and MAPK signaling, may also contribute to the observed phenotypes, either independently or through pathway crosstalk (Wang et al., 2021; Zhu et al., 2023; Zhang et al., 2026). Furthermore, because key downstream effectors of Wnt/β-catenin signaling, including cyclin D1 and c-Myc, were not examined, the contribution of this pathway cannot yet be fully defined. Future investigations should distinguish the relative contributions and interactions of these signaling pathways to the cellular effects of the miR-182-5p/FOXN3 relationship.

Several limitations define the scope of these conclusions. Roosters aged 15-20 weeks were selected to facilitate the isolation of viable primary LCs and improve experimental consistency while minimizing pre-existing age associated cellular damage. This design is appropriate for examining an intrinsic regulatory mechanism, but it does not reproduce the natural reproductive decline that generally becomes evident after approximately 45 weeks. In addition, isolated cell culture removes endocrine input from the hypothalamic-pituitary-gonadal axis and paracrine, immune, and extracellular-matrix signals from the testis. Experimental overexpression and inhibition may also exceed physiological variation, and the multi-target of miRNAs make the involvement of additional pathways likely. Although the combined SA-β-Gal and molecular marker evidence supports a senescence-associated phenotype, it cannot establish organism level aging or reproductive decline. Future studies should compare LCs from roosters across reproductive ages, measure circulating LH and testosterone concurrently, examine LCs responsiveness to LH, and test the pathway in testicular tissue and in vivo. Therefore, the miR-182-5p/FOXN3/Wnt/β-catenin axis should be regarded as a candidate cell-autonomous mechanism regulating proliferation, senescence, and testosterone levels in chicken LCs in vitro, rather than as a direct reflection of the natural aging process.

Conclusion

In conclusion, the present study demonstrates that miR-182-5p promotes proliferation, inhibits senescence, and elevates testosterone levels in chicken LCs in vitro. As summarized in the proposed model (Fig. 8), these findings delineate a regulatory axis wherein miR-182-5p suppresses FOXN3 to activate the Wnt/β-catenin signaling pathway. However, further age-comparative and in vivo studies are warranted to determine whether this mechanism contributes to natural reproductive aging or influences reproductive performance in roosters.

Fig. 8.

Fig. 8

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Schematic model of miR-182-5p targeting FOXN3 to mediate the Wnt/β-catenin signaling pathway.

Ethics declaration

This study was conducted in accordance with the following guidelines for animal welfare and/or reporting: Sichuan Agricultural University Guidelines for Laboratory Animal Operation and Welfare. This study was approved by the Sichuan Agricultural University. (Approval No. 2024202019-1).

Author Contributions

Xin-yue Zhao and Yu-qi Chen designed the experiments, acquired and analyzed the data, and wrote the manuscript. Yao Zhang interpreted the data and results, critically reviewed the manuscript, and provided important revision suggestions. Hua-dong Yin guided the experimental framework, refined the core academic content through critical reviews, and finalized the manuscript. All authors reviewed and approved the final version of the manuscript and agree to be accountable for all aspects of the work.

Disclosures

The authors declare that they have no conflict of interest.

Acknowledgments

The authors thank Wen-juan Wang and Chi-mei Liao for their technical assistance with statistical analysis and data visualization.

This research was funded by the National Key Research and Development Program of China, grant number 2021YFD1300600; Sichuan Science and Technology Program (2024YFNH0025, 2026NXYZ0010), and China Agriculture Research System of MOF and MARA (CARS-40).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2026.107747.

Appendix. Supplementary materials

mmc1.pdf (1.4MB, pdf)
mmc2.pdf (73.4KB, pdf)

References

  1. An P., Cao M., Tang W., Wang Q., Ji Q., Wang Y., Feng Z., Zhang D., Chen X. MiR-27b-3p suppresses proliferation and testosterone synthesis in goat Leydig cells by activating the AMPK pathway through PPARG targeting. Anim. Reprod. Sci. 2026;291 doi: 10.1016/j.anireprosci.2026.108230. [DOI] [PubMed] [Google Scholar]
  2. Ansari M. Recent strategies to mitigate reproductive aging in male broiler breeders: a review. Anim. Reprod. Sci. 2024;268 doi: 10.1016/j.anireprosci.2024.107570. [DOI] [PubMed] [Google Scholar]
  3. Avital-Cohen N., Heiblum R., Argov-Argaman N., Rosenstrauch A., Chaiseha Y., Mobarkey N., Rozenboim I. Age-related changes in gonadal and serotonergic axes of broiler breeder roosters. Domest. Anim. Endocrinol. 2013;44:145–150. doi: 10.1016/j.domaniend.2013.01.002. [DOI] [PubMed] [Google Scholar]
  4. Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. doi: 10.1016/s0092-8674(04)00045-5. [DOI] [PubMed] [Google Scholar]
  5. Cao M.Q., You A.B., Zhu X.D., Zhang W., Zhang Y.Y., Zhang S.Z., Zhang K.W., Cai H., Shi W.K., Li X.L., Li K.S., Gao D.M., Ma D.N., Ye B.G., Wang C.H., Qin C.D., Sun H.C., Zhang T., Tang Z.Y. miR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a. J. Hematol. Oncol. 2018;11:12. doi: 10.1186/s13045-018-0555-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chan E.F., Gat U., Mcniff J.M., Fuchs E. A common human skin tumour is caused by activating mutations in beta-catenin. Nat. Genet. 1999;21:410–413. doi: 10.1038/7747. [DOI] [PubMed] [Google Scholar]
  7. Chen Y., Zhao X., Zheng J., Zhang Y., Chen H., Cui C., Yu C., Yang C., Yin H., Han S. Sport improves function of roosters testicular Leydig cells by mediating FUNDC1. Poult. Sci. 2026;105 doi: 10.1016/j.psj.2026.106541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Garcin C.L., Habib S.J. A comparative perspective on Wnt/β-catenin signalling in cell fate determination. Results Probl. Cell Differ. 2017;61:323–350. doi: 10.1007/978-3-319-53150-2_15. [DOI] [PubMed] [Google Scholar]
  9. Guo H., Deng X., Guo B., Li Z., Jin W., Li W., Kang X., Sun G. Synergistic roles of follicle-stimulating hormone and testosterone in Sertoli cell proliferation and testicular function in roosters. Poult. Sci. 2026;105 doi: 10.1016/j.psj.2026.106485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heydari B., Mashayekhi F., Kashani M.H.G. Effect of in ovo feeding of folic acid on Disabled-1 and gga-miR-182-5p expression in the cerebral cortex of chick embryo. J. Anim. Physiol. Anim. Nutr. 2024;108:285–290. doi: 10.1111/jpn.13889. [DOI] [PubMed] [Google Scholar]
  11. Juiputta J., Tuntiyasawasdikul V., Chankitisakul V., Boonkum W. Genomic selection and association analyses for improving semen traits in Thai native roosters. Poult. Sci. 2026;105 doi: 10.1016/j.psj.2026.107065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kong D., Wu Y., Tong B., Liang Y., Xu F., Chi X., Ni L., Tian G., Zhang G., Xu Z. CHES1 modulated tumorigenesis and senescence of pancreas cancer cells through repressing AKR1B10. BBA-Mol. Basis Dis. 2024;1870 doi: 10.1016/j.bbadis.2024.167214. [DOI] [PubMed] [Google Scholar]
  13. Li D., Hou T., Du X., Zhao L., Zhang L., Gao F., Xing T. Integrated analysis of miRNA and mRNA expression profiles associated with wooden breast myopathy in broiler chickens. Int. J. Biol. Macromol. 2025;284 doi: 10.1016/j.ijbiomac.2024.137990. [DOI] [PubMed] [Google Scholar]
  14. Li Q., Li H., Liang J., Mei J., Cao Z., Zhang L., Luo J., Tang Y., Huang R., Xia H., Zhang Q., Xiang Q., Y. Yang, Y. Huang Sertoli cell-derived exosomal MicroRNA-486-5p regulates differentiation of spermatogonial stem cell through PTEN in mice. J. Cell Mol. Med. 2021;25:3950–3962. doi: 10.1111/jcmm.16347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Li Z.N., Ge M.X., Yuan Z.F. MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling. BMC Ophthalmol. 2020;20:233. doi: 10.1186/s12886-020-01489-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lin Z., Chen M., Wan Y., Lei L., Ruan H. miR-574-5p targets FOXN3 to regulate the invasion of nasopharyngeal carcinoma cells via Wnt/β-catenin pathway. Technol. Cancer Res. Treat. 2020;19 doi: 10.1177/1533033820971659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu W., Du L., Cui Y., He C., He Z. WNT5A regulates the proliferation, apoptosis and stemness of human stem Leydig cells via the β-catenin signaling pathway. Cell. Mol. Life. Sci. 2024;81:93. doi: 10.1007/s00018-023-05077-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mishra J., Gautam M., Dadhich R., Kowtharapu B.S., Majumdar S.S. Peritubular cells may modulate Leydig cell-mediated testosterone production through a nonclassic pathway. Fertil. Steril. 2012;98:1308–1317.e1301. doi: 10.1016/j.fertnstert.2012.07.1124. [DOI] [PubMed] [Google Scholar]
  19. Niu B., Wu J., Mu H., Li B., Wu C., He X., Bai C., Li G., Hua J. miR-204 regulates the proliferation of dairy goat spermatogonial stem cells via targeting to Sirt1. Rejuvenation Res. 2016;19:120–130. doi: 10.1089/rej.2015.1719. [DOI] [PubMed] [Google Scholar]
  20. O'donnell L., Smith L.B., Rebourcet D. Sertoli cells as key drivers of testis function. Semin. Cell Dev. Biol. 2022;121:2–9. doi: 10.1016/j.semcdb.2021.06.016. [DOI] [PubMed] [Google Scholar]
  21. Piprek R.P., Kloc M., Porebska K., Mizia P.C., Rams-Pociecha I., Kubiak J.Z. Origin and role of testicular macrophages in testis development, steroidogenesis, and spermatogenesis. Results Probl. Cell Differ. 2024;74:137–157. doi: 10.1007/978-3-031-65944-7_5. [DOI] [PubMed] [Google Scholar]
  22. Rosenstrauch A., Weil S., Degen A.A., Friedländer M. Leydig cell functional structure and plasma androgen level during the decline in fertility in aging roosters. Gen Comp Endocrinol. 1998;109:251–258. doi: 10.1006/gcen.1997.7029. [DOI] [PubMed] [Google Scholar]
  23. Sarabia Fragoso J., Pizarro Díaz M., Abad Moreno J.C., Casanovas Infesta P., Rodriguez-Bertos A., Barger K. Relationships between fertility and some parameters in male broiler breeders (body and testicular weight, histology and immunohistochemistry of testes, spermatogenesis and hormonal levels) Reprod. Domest. Anim. 2013;48:345–352. doi: 10.1111/j.1439-0531.2012.02161.x. [DOI] [PubMed] [Google Scholar]
  24. Sun J., Li H., Huo Q., Cui M., Ge C., Zhao F., Tian H., Chen T., Yao M., Li J. The transcription factor FOXN3 inhibits cell proliferation by downregulating E2F5 expression in hepatocellular carcinoma cells. Oncotarget. 2016;7:43534–43545. doi: 10.18632/oncotarget.9780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Takase H.M., Nusse R. Paracrine Wnt/β-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse testis. Proc. Natl. Acad. Sci. U. S. A. 2016;113:E1489–E1497. doi: 10.1073/pnas.1601461113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tetsu O., Mccormick F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 1999;398:422–426. doi: 10.1038/18884. [DOI] [PubMed] [Google Scholar]
  27. Wang C., Tu H., Yang L., Ma C., Hu J., Luo J., Wang H. FOXN3 inhibits cell proliferation and invasion via modulating the AKT/MDM2/p53 axis in human glioma. Aging. 2021;13:21587–21598. doi: 10.18632/aging.203499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wang X., Huang Q., Li Z., Wang C., He X., Li J., Wang X., Lin Z., Yang B., Hu Q., Xia Q., Sun X., Yu J., Qi Y. LncRNA:CR43306 modulates testicular aging via cell adhesion in Drosophila. Insect Mol. Biol. 2026;35:284–295. doi: 10.1111/imb.70028. [DOI] [PubMed] [Google Scholar]
  29. Wang Y., Su H., Wang X., Tu C., Xiao T., Ren B., Wang S. FOXN3 Regulates autophagic activity to suppress drug resistance in melanoma cells. Clin. Cosmet. Investig. Dermatol. 2024;17:2505–2518. doi: 10.2147/ccid.S462854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wu Y., Qi X., Li Q., Ma Y. Circular RNA circCSNK1G3 induces Leydig cells proliferation and promotes testosterone production through the miR-29b/IGF1 axis. J. Anim. Sci. 2026;104 doi: 10.1093/jas/skag010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xu H., Pu J., Teng Y., Zhu Q., Guo L., Zhao J., Ding H., Fang Y., Ma X., Liu H., Guo J., Lu W., Wang J. Melatonin inhibits testosterone synthesis in rooster Leydig cells by targeting CXCL14 through miR-7481-3p. Int. J. Mol. Sci. 2023;24 doi: 10.3390/ijms242316552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang H., Li M., Qi Y. FOXN3 inhibits the progression of ovarian cancer through negatively regulating the expression of RPS15A. Hum Cell. 2023;36:1120–1134. doi: 10.1007/s13577-023-00876-9. [DOI] [PubMed] [Google Scholar]
  33. Yu C., Lin Z., Song X., Hu C., Qiu M., Yang L., Zhang Z., Pen H., Chen J., Xiong X., Xia B., Jiang X., Du H., Li Q., Zhu S., Liu S., Yang C., Liu Y. Whole transcriptome analysis reveals the key genes and noncoding RNAs related to follicular atresia in broilers. Anim. Biotechnol. 2023;34:3144–3153. doi: 10.1080/10495398.2022.2136680. [DOI] [PubMed] [Google Scholar]
  34. Yu J., Liu Y., Lan X., Wu H., Wen Y., Zhou Z., Hu Z., Sha J., Guo X., Tong C. CHES-1-like, the ortholog of a non-obstructive azoospermia-associated gene, blocks germline stem cell differentiation by upregulating Dpp expression in Drosophila testis. Oncotarget. 2016;7:42303–42313. doi: 10.18632/oncotarget.9789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yuan H., Lu J., Xiao S.Y., Han X.Y., Song X.T., Qi M.Y., Liu G.S., Yang C.X., Yao Y.C. miRNA expression analysis of the sheep follicle during the prerecruitment, dominant, and mature stages of development under FSH stimulation. Theriogenology. 2022;181:161–169. doi: 10.1016/j.theriogenology.2022.01.001. [DOI] [PubMed] [Google Scholar]
  36. Zhang J., Jiang Q., Liu T., Li H., Liang Y., Lu X., Pang H., Li S., Li Y., Zhang R. Crosstalk between FOXN3 and E2F5 reveals a novel tumor suppressive pathway in acute myeloid leukemia via MAPK signaling: implications for potential future targeted therapy. Blood Lymphat Cancer. 2026;16:1–16. doi: 10.2147/blctt.S571966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhang M., Wang Y., Shan S., Liu S. miRNA-182-5p promotes myogenic differentiation of C2C12 cells via the suppression of ZBTB7A. Front. Vet. Sci. 2025;12 doi: 10.3389/fvets.2025.1637277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhao C., Mo L., Li C., Han S., Zhao W., Liu L. FOXN3 suppresses the growth and invasion of papillary thyroid cancer through the inactivation of Wnt/β-catenin pathway. Mol. Cell. Endocrinol. 2020;515 doi: 10.1016/j.mce.2020.110925. [DOI] [PubMed] [Google Scholar]
  39. Zhou Z.Y., Qin Q., Dong C., Liu Y., Cao C., Teng L. The dual guardians of cellular stability: exploring nesprin and lamin in senescence. Cell Death Dis. 2025;16:757. doi: 10.1038/s41419-025-08087-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu X., Huang B., Zhao F., Lian J., He L., Zhang Y., Ji L., Zhang J., Yan X., Zeng T., Ma C., Liang Y., Zhang C., Lin J. p38-mediated FOXN3 phosphorylation modulates lung inflammation and injury through the NF-κB signaling pathway. Nucleic Acids Res. 2023;51:2195–2214. doi: 10.1093/nar/gkad057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zirkin B.R., Papadopoulos V. Leydig cells: formation, function, and regulation. Biol. Reprod. 2018;99:101–111. doi: 10.1093/biolre/ioy059. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

mmc1.pdf (1.4MB, pdf)
mmc2.pdf (73.4KB, pdf)

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