Mechanistic role of the KRTAP5-AS1/miR-199b-5p/CYP19A1 axis in polycystic ovary syndrome pathogenesis

Abstract

Background

Polycystic ovary syndrome (PCOS) pathogenesis involves dysregulated granulosa cell function, but molecular mechanisms remain unclear.

Methods

High-throughput RNA sequencing was performed on ovarian granulosa cells from 6 PCOS patients and 3 controls to identify differentially expressed mRNAs. Bioinformatics analyses including ceRNA network construction predicted the KRTAP5-AS1/miR-199b-5p/CYP19A1 regulatory axis, which was experimentally validated through dual-luciferase reporter assays. qRT-PCR confirmed the expression patterns of these molecules in expanded clinical cohorts (38 PCOS vs. 30 controls), with Pearson correlation analysis examining relationships between gene expression and clinical parameters. Using the KGN granulosa cell line, functional studies included: (1) ELISA quantification of estradiol production; (2) proliferation assessment via CCK-8 and colony formation assays; and (3) apoptosis evaluation by flow cytometry and Bax/Bcl-2 protein analysis. These experiments were performed following both gain-of-function (overexpression) and loss-of-function (shRNA knockdown) manipulations of KRTAP5-AS1 and miR-199b-5p.

Results

Through RNA sequencing of ovarian granulosa cells from 6 PCOS patients and 3 controls, we identified CYP19A1 as significantly upregulated in PCOS. Expanded validation in 38 PCOS vs. 30 controls confirmed elevated CYP19A1 and reduced miR-199b-5p in PCOS, with KRTAP5-AS1 showing negative correlation to miR-199b-5p and positive to CYP19A1. Clinically, CYP19A1 upregulation correlated with poor embryo quality, elevated testosterone, AMH, BMI, and infertility duration, while miR-199b-5p levels associated positively with embryo quality. In KGN granulosa cells, miR-199b-5p overexpression suppressed CYP19A1 expression and estradiol synthesis, whereas KRTAP5-AS1 overexpression alleviated this suppression via competitive miR-199b-5p binding. Functional studies demonstrated that miR-199b-5p overexpression combined with KRTAP5-AS1 knockdown inhibited proliferation, promoted apoptosis, and reduced estradiol production, while opposite manipulations reversed these effects.

Conclusions

Our findings reveal that KRTAP5-AS1 modulates granulosa cell dysfunction in PCOS through the miR-199b-5p/CYP19A1 axis, highlighting miR-199b-5p as a potential therapeutic target for PCOS-related ovarian dysfunction and endocrine abnormalities.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01746-8.

Introduction

Polycystic ovary syndrome (PCOS) represents a complex endocrine disorder whose etiology remains incompletely understood. While numerous studies have investigated PCOS pathogenesis, consensus regarding its underlying mechanisms has yet to be established. Current evidence suggests that PCOS develops through the interplay of genetic predisposition and environmental influences [1]. A key hallmark of PCOS is abnormal follicular development driven by endocrine dysfunction, which manifests as excessive recruitment of preantral and antral follicles, disrupted follicular dominance, and impaired ovulation [2, 3]. The hormonal imbalance in PCOS involves dysregulated steroidogenesis, where luteinizing hormone (LH)-stimulated theca cells produce androgens that are subsequently converted to estrogens by granulosa cell aromatase [4]. This rate-limiting enzyme, encoded by CYP19A1, belongs to the cytochrome P450 superfamily and plays a pivotal role in estrogen biosynthesis [5]. Granulosa cells, which envelop developing oocytes, critically regulate folliculogenesis through endocrine and paracrine signaling. These specialized cells synthesize steroid hormones and growth factors, express corresponding receptors, and coordinate theca cell and oocyte development via gap junction-mediated communication [6]. Emerging evidence implicates non-coding RNAs in the regulation of granulosa cell function. MicroRNAs (miRNAs) have been shown to modulate various aspects of reproductive physiology, including hormone metabolism, granulosa cell proliferation, and apoptosis [7, 8]. Among these, miR-199b-5p, located on chromosome 9q34, has been associated with several malignancies including breast [9, 10] cervical carcinoma [11] and thyroid carcinoma [12]. Similarly, the long non-coding RNA KRTAP5-AS1 (chromosome 11p15.5) has been identified as differentially expressed in papillary thyroid carcinoma [13]. Despite extensive investigation, the expression pattern of CYP19A1 in PCOS remains controversial, with studies reporting both decreased and enhanced expression [14–16]. Furthermore, the functional roles of KRTAP5-AS1 and miR-199b-5p in PCOS-associated follicular development have not been elucidated. This study therefore aims to: (1) characterize the regulatory relationships between miR-199b-5p, KRTAP5-AS1, and CYP19A1; and (2) determine how dysregulation of this axis impacts granulosa cell function, potentially contributing to the follicular abnormalities characteristic of PCOS.

Materials and methods

Ethics statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the First Affiliated Hospital of Xiamen University (Approval No. 2020042).

Study subjects

This prospective study enrolled 77 women undergoing in vitro fertilization (IVF) treatment at the Reproductive Medicine Center of the First Affiliated Hospital of Xiamen University, comprising 44 PCOS patients and 33 non-PCOS controls (with tubal factor or male factor infertility). All participants were aged < 35 years. PCOS diagnosis was established according to the 2003 Rotterdam criteria [17]: requiring: (1) oligo/anovulation, plus (2) clinical and/or biochemical hyperandrogenism and/or (3) polycystic ovarian morphology on ultrasound. Exclusion criteria included other causes of hyperandrogenism (e.g., congenital adrenal hyperplasia, androgen-secreting tumors) and thyroid dysfunction-related menstrual disorders.

Discovery Cohort (Hypothesis Generation): An initial cohort underwent RNA sequencing to identify potential differentially expressed genes (DEGs) and pathways (n = 6 PCOS patients; n = 3 control subjects). This cohort served an exploratory role for hypothesis generation.

Independent Validation Cohort (Primary Analysis): A significantly larger, independent cohort (n = 38 PCOS patients; n = 30 control subjects), distinct from the discovery cohort and processed separately, was used for targeted validation and forms the primary basis for the differential gene expression and clinical findings reported in this study.

Collection and processing of follicular fluid and granulosa cells

Following oocyte retrieval, follicular fluid (FF) containing granulosa cells was centrifuged at 1,500 × g for 10 min at room temperature. The supernatant was carefully collected and stored at − 80 °C for subsequent analysis. The remaining cell pellet was resuspended in phosphate-buffered saline (PBS) and gently layered onto Ficoll separation solution (Solarbio, China) at a 1:1 (v/v) ratio. The mixture was centrifuged at 2,000 × g for 20 min to isolate the granulosa cell layer.

The intermediate granulosa cell band was carefully aspirated and transferred to a sterile centrifuge tube. To remove residual erythrocytes, red blood cell lysis buffer (Solarbio, China) was added at a 1:3 (v/v) ratio, followed by thorough mixing and incubation on ice for 10 min. After centrifugation at 1,500 × g for 5 min, the supernatant was discarded, and the cell pellet was washed twice with PBS. Finally, the purified granulosa cells were lysed in 500–1,000 µL of TRIzol™ reagent (Invitrogen, Thermo Fisher Scientific, USA) and stored at − 80 °C until RNA extraction.

Electrochemiluminescence immunoassay (ECLIA) for neutral hormone detection in FF

The frozen FF samples were thawed at room temperature and equilibrated for 1 h prior to analysis. Hormone concentrations were determined using an electrochemiluminescence immunoassay (ECLIA) system (Architect i2000SR, Abbott Laboratories, USA) following the manufacturer’s protocol. Briefly, standard calibrators were prepared to establish a calibration curve. Samples were then incubated with the assay reagents, followed by addition of the chemiluminescent substrate. The relative light units (RLUs) were measured by the automated analyzer, which calculated hormone concentrations based on the standard curve.

Cell culture

The human ovarian granulosa-like tumor cell line KGN was obtained commercially from Ybscience (Shanghai, China). Cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Australia origin) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin). All cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

High throughput transcriptome sequencing

Total RNA was isolated from granulosa cells using TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol. RNA quality and concentration were assessed by 1% agarose gel electrophoresis, NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). For mRNA library construction, ribosomal RNA was removed from total RNA using the Ribo-Zero Gold rRNA Removal Kit (Illumina, USA). First-strand cDNA synthesis was performed using random hexamer primers and M-MuLV Reverse Transcriptase (New England Biolabs, USA), followed by second-strand synthesis with DNA Polymerase I (Thermo Fisher Scientific, USA). cDNA fragments were purified using Dynabeads™ mRNA Purification Kit (Thermo Fisher Scientific, USA), followed by end repair, poly(A) tailing, and adapter ligation. After second-strand cDNA digestion and size selection via agarose gel electrophoresis, PCR amplification was conducted. The final library quality and quantity were verified using the Agilent 2100 Bioanalyzer before sequencing on the Illumina HiSeq 4000 platform (Illumina, USA).

RNA microarray data analysis

Differential gene expression analysis was conducted using both DESeq2 and edgeR algorithms to ensure robust identification of differentially expressed mRNAs (DEmRNAs). Transcripts meeting the threshold criteria of p-value < 0.05 with a fold change > 1.5 were considered statistically significant. Subsequent functional annotation of these DEmRNAs was performed through comprehensive Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to elucidate their biological roles and potential involvement in key molecular pathways.

Grouping and treatment of KGN cells

KGN cells were divided into two experimental series to examine the functional relationship between miR-199b-5p and KRTAP5-AS1. The first series consisted of untreated control cells, negative control for miR-199b-5p mimic (mimics NC), miR-199b-5p mimic-transfected cells, co-transfection with both miR-199b-5p mimic and KRTAP5-AS1 overexpression plasmid, KRTAP5-AS1 overexpression alone, and empty plasmid control for KRTAP5-AS1 overexpression. The second series included untreated control cells, negative control for miR-199b-5p inhibitor (inhibitor NC), miR-199b-5p inhibitor-transfected cells, co-transfection with miR-199b-5p inhibitor and KRTAP5-AS1 shRNA plasmid, KRTAP5-AS1 knockdown alone, and scrambled shRNA control for KRTAP5-AS1 knockdown.

Cell transfections were carried out using Lipo6000™ transfection reagent following the manufacturer’s protocol. After 6 h of incubation, the medium was replaced with fresh complete culture medium, and cells were maintained at 37 °C with 5% CO₂ for 48 h prior to further experimentation. This experimental design allowed for comprehensive evaluation of the interaction between miR-199b-5p and KRTAP5-AS1 in KGN cells.

Enzyme-linked immunosorbent assay (ELISA)

The concentration of estradiol (E2) in KGN cell culture medium was quantified using a commercial E2 ELISA Kit (Fine Test, China) following the manufacturer’s protocol. Briefly, cell culture supernatants were collected from each experimental group post-transfection. Aliquots of 50 µL from both samples and standards were loaded into designated wells, followed by sequential addition of assay reagents. After completing the reaction protocol, absorbance was measured at 450 nm using a microplate reader. A standard curve was generated by plotting optical density values against known E2 concentrations, with curve fitting performed using a quadratic polynomial regression model. Sample E2 concentrations were subsequently determined by interpolation from the standard curve.

Western blot analysis

Protein extraction and western blot analysis were performed according to standard protocols. Briefly, harvested cells were lysed using RIPA buffer (Thermo Fisher Scientific, USA) and protein concentrations were determined by BCA assay (Beyotime, China). Protein samples were normalized to equal concentrations using 5×SDS loading buffer (Beyotime), denatured at 100 °C for 10 min, and separated by SDS-PAGE electrophoresis. Subsequently, proteins were transferred onto PVDF membranes and blocked with 5% skim milk. Membranes were incubated with primary antibodies at 4 °C overnight, followed by TBST (Solarbio, China) washes and incubation with appropriate secondary antibodies for 1–2 h at room temperature. Protein bands were visualized using ECL substrate (Thermo Scientific, USA) and quantified by ImageJ software with GAPDH orβ-actin serving as the loading control.

Luciferase reporter constructs and dual-luciferase reporter gene assay

To investigate the potential regulatory relationship between miR-199b-5p and target genes, we performed dual-luciferase reporter assays. Wild-type and mutant 3’UTR sequences of CYP19A1 and KRTAP5-AS1 were cloned into the pmirGLO dual-luciferase reporter vector (Promega, USA). Positive clones were identified through colony PCR screening and validated by restriction enzyme digestion and DNA sequencing. For functional validation, KGN cells were co-transfected with the constructed reporter vectors along with either miR-199b-5p mimics or negative control (mimics NC). After 48 h of transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

Cell counting kit-8 (CCK-8) assay

For proliferation analysis, KGN cells were seeded in 96-well plates at a density of 5,000 cells per well (n = 5 replicates). Following 48 h of transfection, cell viability was assessed using the enhanced CCK-8 assay (Meilunbio, China) according to manufacturer’s protocol. Briefly, 10 µL of CCK-8 reagent was added to each well and absorbance at 450 nm was measured at 0, 24, 48, and 72-hour time points to generate proliferation curves.

Apoptosis was evaluated by flow cytometry using Annexin V-FITC/PI double staining. Transfected cells were harvested by trypsinization, washed with PBS, and resuspended at 1 × 10^6 cells/mL in binding buffer. Cell suspensions were stained with 5 µL Annexin V-FITC and 10 µL propidium iodide (PI) (Biotime, China), followed by 15-minute incubation in darkness at room temperature. Samples were immediately analyzed by flow cytometry to quantify apoptotic populations.

qRT-PCR

Total RNA was extracted from follicular fluid and cellular samples using TRIzol™ Reagent (Thermo Fisher Scientific, USA). cDNA synthesis was performed with the Evo M-MLV Reverse Transcription Kit II (Agbio, China) following manufacturer protocols. Sequence-specific primers for PCR amplification (Qingke Biotechnology, China) are detailed in Supplementary Table 1. Quantitative real-time PCR (qRT-PCR) was conducted on a QuantStudio™ 5 system (Applied Biosystems, USA) with U6 snRNA and β-actin serving as endogenous controls for miRNA and lncRNA/mRNA quantification, respectively. Technical triplicates were analyzed for each sample, with data normalized using the 2 − ΔΔCT method for comparative quantification.

RNA-fluorescence in situ hybridization (FISH)

KGN cells were seeded in 24-well plates pre-coated with chambered glass slides at a density of 2 × 10⁴ cells/well and cultured overnight. Cells were fixed with 4% paraformaldehyde (Solarbio, China) and processed using a fluorescence in situ hybridization (FISH) kit (GenePharma, China) according to manufacturer specifications. Target-specific probes were hybridized with samples in a humidified, light-protected chamber at 37 °C for 16 h. Nuclei were counterstained with DAPI for 10 min, followed by PBS washes and mounting with anti-fade medium on microscope slides. Cellular localization was visualized using a confocal laser scanning microscope (LSM900, Carl Zeiss AG, Germany) with sequential channel imaging to avoid spectral overlap.

Colony formation assay

Log-phase cells from experimental groups were harvested via trypsinization and centrifugation, followed by resuspension and counting. Cells (1 × 10³/well) were seeded into 24-well plates and cultured for 14 days under standard conditions (37 °C, 5% CO2). Colonies were fixed with methanol (2 mL/well, 20 min), washed twice with ice-cold PBS, and stained with 0.1% crystal violet for 10 min. Visible colonies (> 50 cells) were quantified across triplicate wells, with the clonogenic rate calculated as (number of colonies / initial cell count) × 100%.

Statistical analysis

Data are presented as mean ± SD and analyzed using SPSS 16.0. Intergroup differences were assessed via unpaired t-tests (two groups) or one-way ANOVA with Tukey’s post hoc test (multiple groups). Pearson’s correlation analysis evaluated linear relationships between variables. Statistical significance was defined at P < 0.05.Results.

CYP19A1 upregulation in PCOS granulosa cells correlates with metabolic and reproductive dysfunction

A total of 20,284 mRNAs were detected in granulosa cells (GCs) from control (n = 3) and PCOS (n = 6) patients. Based on the screening criteria (fold change > 1.5, p < 0.05), RNA sequencing revealed 2,611 upregulated and 897 downregulated differentially expressed mRNAs. KEGG pathway analysis identified signaling pathways potentially contributing to GC dysfunction in PCOS (Fig. 1B). Volcano plots and heat maps illustrated the global differential mRNA expression profiles (Fig. 1A, D). Notably, quantitative analysis revealed significantly higher CYP19A1 expression levels in granulosa cells (GCs) from PCOS patients compared to control subjects (p < 0.001). Bioinformatics pathway analysis identified CYP19A1 as a central regulator in two critical metabolic networks: (1) the endocrine system pathway, which showed strong interactions with gonadotropin-releasing hormone (GNRH) signaling, insulin signaling, and ovarian steroidogenesis pathways; and (2) the lipid metabolism pathway, which demonstrated close associations with steroid hormone biosynthesis and fatty acid metabolism (Fig. 1C). These interconnected pathways collectively contribute to the characteristic metabolic and reproductive abnormalities observed in PCOS pathogenesis.

Fig. 1.

Differential gene expression profiles in granulosa cells (GCs) from PCOS patients and controls. (A) Hierarchical clustering analysis of all differentially expressed genes (DEGs) between PCOS (n = 6) and control (n = 3) groups. (B) KEGG pathway enrichment analysis of DEGs. (C) Pathway interaction network of biological pathways associated with CYP19A1. (D) Volcano plot displaying DEGs between groups, with red dots indicating upregulated genes and blue dots indicating downregulated genes (fold change > 2, p < 0.05). PCOS, polycystic ovary syndrome; KEGG, Kyoto Encyclopedia of Genes and Genomes

To validate these findings, we analyzed GCs from 68 patients (30 controls and 38 PCOS) using qRT-PCR and measured FF testosterone (T) and E2 levels. As shown in Table 1, PCOS patients demonstrated significantly lower basal follicle-stimulating hormone (FSH) (p < 0.001) but higher testosterone (T) (p = 0.006) and anti-Müllerian hormone (AMH) levels (p < 0.001) compared to controls, while other clinical parameters including maternal age, BMI, infertility duration, luteinizing hormone (LH), basal E2, fasting plasma glucose (FPG), and progesterone showed no significant differences. qRT-PCR analysis confirmed significant upregulation of CYP19A1 in PCOS GCs (Fig. 2). Further analysis revealed higher FF T levels (4.60 vs. 3.72, p = 0.011) and a significantly reduced E2/T ratio (5.92 vs. 7.02, p = 0.023) in PCOS patients compared to controls, although FF E2 levels were comparable between groups (Table 2). Pearson correlation analysis demonstrated significant positive associations between CYP19A1 expression and multiple metabolic and reproductive parameters, including FPG (r = 0.313, p = 0.009), infertility duration (r = 0.269, p = 0.027), BMI (r = 0.251, p = 0.039), AMH (r = 0.327, p = 0.007), basal T (r = 0.348, p = 0.004), and FF T levels (r = 0.378, p = 0.001), while showing a negative correlation with day 3 available embryo rate during IVF cycles (Fig. 3D-I) (Tables 3 and 4).

Table 1.

Baseline characteristics of the cycles for qRT-PCR

	PCOS	Control	P value
TOTAL: n = 68	n = 38	n = 30
Maternal age (years)	30.74 ± 4.14	31.13 ± 3.62	0.68
BMI (kg/m2)	23.64 ± 4.48	21.53 ± 2.04	0.012
Infertility years	2.66 ± 2.63	2.83 ± 2.76	0.79
Total dose of Gn (IU)	2432.11 ± 1017.29	1924.58 ± 345.95	0.022
Basal FSH (mIU/ml)	5.37 ± 1.32	6.87 ± 1.79	< 0.001
Basal LH (mIU/ml)	5.87 ± 3.77	5.54 ± 3.11	0.701
Basal T (ng/ml)	0.47 ± 0.18	0.32 ± 0.11	< 0.001
Basal E2 (pg/ml)	35.40 ± 12.41	37.44 ± 15.57	0.549
AMH (ng/ml)	7.17 ± 5.72	3.00 ± 1.46	< 0.001
FPG (mmol/l)	5.15 ± 0.66	5.14 ± 0.66	0.493
P4 (ng/ml)	0.35 ± 0.29	0.39 ± 0.34	0.536

Continuous variables are expressed as mean ± SD; categorical variables are expressed as numbers (%)

Fig. 2.

Regulatory relationship between CYP19A1 and miR-199b-5p in PCOS granulosa cells. (A) CYP19A1 mRNA expression levels in PCOS versus control GCs (**p < 0.01). (B) miR-199b-5p expression levels in PCOS versus control GCs (*p < 0.05). (C) Negative correlation between miR-199b-5p and CYP19A1 expression in GCs. (D) Predicted miR-199b-5p binding sites in the CYP19A1 3’UTR. (E) CYP19A1 expression in KGN cells following transfection with miR-199b-5p mimics or inhibitor (***p < 0.001 vs. inhibitor NC; *p < 0.05 vs. mimics NC). (F) Dual-luciferase reporter assay validating miR-199b-5p targeting of CYP19A1 (***p < 0.001 vs. mimics NC). NC, negative control

Table 2.

Comparative analysis of sex hormone concentrations in follicular fluid (FF) from the two study cohorts

Parameters	Control	PCOS	P
T(µg/L)	3.72 ± 0.91	4.60 ± 1.91	0.011
E2(ng/L)	25011.36 ± 8943.78	23677.48 ± 9144.57	0.524
E2/T ratio	7.02 ± 2.7	5.92 ± 2.82	0.023

Continuous variables are expressed as mean ± SD; categorical variables are expressed as numbers (%)

Fig. 3.

Associations of CYP19A1 and miR-199b-5p with metabolic and reproductive parameters in PCOS patients. (A–C) Correlation analysis of miR-199b-5p expression with (A) gonadotropin (GN) dosage, (B) blastocyst development rate, and (C) high-quality blastocyst rate. (D–I) Correlation analysis of CYP19A1 expression with (D) fasting blood glucose (FBG), (E) duration of infertility, (F) body mass index (BMI), (G) anti-Müllerian hormone (AMH) levels, (H) testosterone (T), and (I) IVF D3 available embryos rate. Data are presented as Pearson/Spearman correlation coefficients with p-values

Table 3.

miR-199b-5p & CYP19A1 expression in GCs and their correlation with patient baseline parameters

Parameters	miR-199b-5p		CYP19A1
	r (pearson)	P	r (pearson)	P
Infertility years	-0.018	0.882	0.269	0.027
BMI (kg/m2)	-0.163	0.185	0.251	0.039
Maternal age (years)	-0.036	0.774	-0.171	0.163
Basal FSH (mIU/ml)	0.12	0.33	-0.143	0.246
Basal LH (mIU/ml)	-0.048	0.699	-0.006	0.959
Basal T (ng/ml)	-0.127	0.301	0.348	0.004
T in FF	-0.097	0.429	0.378	0.001
Basal E2 (pg/ml)	0.225	0.064	-0.15	0.222
E2 in FF	0.127	0.303	-0.137	0.266
AMH (ng/ml)	0.01	0.933	0.327	0.007
FPG (mmol/l)	-0.029	0.817	0.313	0.009

Table 4.

The expression of miR-199b-5p &. CYP19A1 in GCs and associations with embryo quality

Parameters	miR-199b-5p		CYP19A1
	r (pearson)	P	r (pearson)	P
Total dose of Gn	-0.251	0.039	0.195	0.112
Mature oocytes rate	0.118	0.339	-0.07	0.57
2PN rate (%)	0.096	0.438	0.012	0.921
Available embryos rate (%)	0.156	0.204	0.011	0.929
Available embryos rate (%) (IVF)	0.276	0.048	-0.285	0.04
Blastocyst development rate (%)	0.235	0.05	-0.037	0.765
Good quality blastocyst rate	0.285	0.019	-0.155	0.207

Downregulation of miR-199b-5p in PCOS granulosa cells and its positive correlation with embryo quality

Bioinformatic prediction identified miR-199b-5p as a potential regulator of CYP19A1, with a binding site in the 3’ untranslated region (UTR) (Fig. 2D). qRT-PCR analysis demonstrated significantly reduced miR-199b-5p expression in PCOS GCs compared with controls (p < 0.05) (Fig. 2B). Moreover, miR-199b-5p levels were inversely correlated with CYP19A1 expression (r=-0.276, p = 0.022) (Fig. 2C). Correlation analysis further demonstrated that miR-199b-5p expression exhibited a significant negative correlation with the total gonadotropin (Gn) dose (r = -0.251, p = 0.039). Notably, positive correlations were observed between miR-199b-5p levels and key embryological outcomes, including blastocyst development rate (r = 0.235, p = 0.05), good-quality blastocyst rate (r = 0.285, p = 0.019), and day 3 available embryo rate (r = 0.276, p = 0.048) (Fig. 3A-C) (Table 4). However, no significant associations were detected between miR-199b-5p expression and baseline patient characteristics (Table 3).

miR-199b-5p directly regulates CYP19A1 expression through 3’UTR binding

Functional validation experiments confirmed that miR-199b-5p negatively regulates CYP19A1 expression. Transfection of miR-199b-5p mimics into KGN cells significantly reduced CYP19A1 mRNA levels (p < 0.05), while miR-199b-5p inhibitor treatment increased CYP19A1 expression (Fig. 2E), demonstrating its inhibitory effect on CYP19A1.

To further investigate this regulatory relationship, we performed dual-luciferase reporter assays. Wild-type (wt) and mutant (mut) 3’UTR sequences of CYP19A1 containing the predicted miR-199b-5p binding site were cloned into reporter vectors (Fig. 2D). Co-transfection of miR-199b-5p mimics with the CYP19A1 3’UTR-wt construct significantly reduced luciferase activity compared to the negative control (NC) mimics (p < 0.01). In contrast, no significant difference was observed when using the mutant 3’UTR construct (Fig. 2F). These results demonstrate that miR-199b-5p specifically binds to the 3’UTR of CYP19A1 to suppress its expression, confirming CYP19A1 as a direct target of miR-199b-5p.

KRTAP5-AS1 functions as a competing endogenous RNA to regulate the miR-199b-5p/CYP19A1 Axis

Bioinformatic analysis using miRNet, Starbase, and LncBase databases predicted KRTAP5-AS1 as a potential regulator of miR-199b-5p (Fig. 4G). qPCR analysis revealed a downregulation of KRTAP5-AS1 in granulosa cells from polycystic ovary syndrome (PCOS) patients (Fig. 4A). However, KRTAP5-AS1 expression exhibited a positive correlation with CYP19A1 mRNA levels but a negative correlation with miR-199b-5p expression, suggesting a potential regulatory role in PCOS pathogenesis (Fig. 4B, C). To validate the proposed KRTAP5-AS1/miR-199b-5p/CYP19A1 regulatory axis, we performed gain- and loss-of-function experiments in KGN cells. qRT-PCR analysis revealed that KRTAP5-AS1 knockdown significantly increased miR-199b-5p expression (p < 0.05), while KRTAP5-AS1 overexpression decreased miR-199b-5p levels (Fig. 4D). Conversely, CYP19A1 expression showed an inverse correlation, being downregulated upon KRTAP5-AS1 knockdown and upregulated following KRTAP5-AS1 overexpression (Fig. 4E, all p < 0.05).

Fig. 4.

KRTAP5-AS1 modulates the miR-199b-5p/CYP19A1 axis in PCOS granulosa cells. (A) KRTAP5-AS1 expression levels in GCs from PCOS patients compared to controls (*p < 0.05). (B, C) Correlation analysis of KRTAP5-AS1 expression with (B) CYP19A1 and (C) miR-199b-5p levels. (D, E) Relative expression of (D) miR-199b-5p and (E) CYP19A1 in KGN cells following KRTAP5-AS1 overexpression or knockdown. (F) Fluorescence in situ hybridization (FISH) confirming the subcellular localization of KRTAP5-AS1 in KGN cells. (G) Predicted binding sites between KRTAP5-AS1 and miR-199b-5p. (H) Dual-luciferase reporter assay validating the regulatory interaction between KRTAP5-AS1 and miR-199b-5p (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control)

Fluorescence in situ hybridization (FISH) demonstrated that KRTAP5-AS1 exhibits both nuclear and cytoplasmic localization in KGN cells (Fig. 4F), suggesting its potential role as a competing endogenous RNA (ceRNA). To further investigate the molecular interaction between KRTAP5-AS1 and miR-199b-5p, we performed dual-luciferase reporter assays. Wild-type (wt) and mutant (mut) KRTAP5-AS1 3’UTR sequences containing the predicted miR-199b-5p binding sites were cloned into pmirGLO vectors (Fig. 4G). Co-transfection experiments showed that miR-199b-5p mimics significantly reduced the luciferase activity of wt-KRTAP5-AS1 compared to negative control mimics (p < 0.05), while no significant effect was observed on mut-KRTAP5-AS1 (Fig. 4H). These results confirm that KRTAP5-AS1 directly binds miR-199b-5p through specific 3’UTR sequences, functioning as a molecular sponge to regulate CYP19A1 expression.

The CeRNA mechanism of KRTAP5-AS1/miR-199b-5p in regulating aromatase expression and Estrogen production

Systematic functional analyses in KGN cells revealed a critical regulatory axis involving KRTAP5-AS1, miR-199b-5p, and CYP19A1. Overexpression of miR-199b-5p significantly downregulated CYP19A1 protein levels compared to control and negative control groups (p < 0.05). This suppressive effect was partially reversed when cells were co-transfected with KRTAP5-AS1 overexpression plasmid, while independent KRTAP5-AS1 overexpression further enhanced CYP19A1 expression beyond control levels (Fig. 5C, D). Complementary experiments demonstrated that miR-199b-5p inhibition upregulated CYP19A1 expression, an effect that was abolished by concurrent KRTAP5-AS1 knockdown. Notably, independent shRNA-mediated KRTAP5-AS1 silencing significantly reduced CYP19A1 levels compared to both combinatorial treatment and control groups (p < 0.05) (Fig. 5C, E). These findings strongly support a ceRNA mechanism whereby KRTAP5-AS1 sequesters miR-199b-5p to derepress CYP19A1 expression.

Fig. 5.

KRTAP5-AS1 competitively binds miR-199b-5p to regulate CYP19A1 protein expression and E2 secretion in KGN cells. (A, B) Estradiol (E2) secretion levels across experimental groups. (C-E) Western blot analysis of CYP19A1 protein expression under different treatment conditions. Statistical significance: ^ap < 0.05 versus mimics-NC group; ^bp < 0.05 versus mimics group; ^cp < 0.05 versus mimic + oe group; ^dp < 0.05 versus oe group; ^ep < 0.05 versus inhibitor-NC group; ^fp < 0.05 versus inhibitor group; ^gp < 0.05 versus inhibitor + sh group; ^hp < 0.05 versus sh group

The functional consequences of this regulatory interaction were further demonstrated through measurements of E2 production. KRTAP5-AS1 overexpression significantly increased E2 levels compared to controls (p < 0.05), while miR-199b-5p mimics substantially suppressed estrogen biosynthesis. Co-transfection experiments revealed bidirectional modulation, with the combination of miR-199b-5p mimics and KRTAP5-AS1 overexpression partially rescuing the inhibitory effect of miR-199b-5p while attenuating the stimulatory impact of KRTAP5-AS1 (p < 0.05) (Fig. 5A). Parallel experiments using miR-199b-5p inhibitor and KRTAP5-AS1 knockdown confirmed these reciprocal regulatory patterns. While miR-199b-5p inhibition enhanced E2 production, this effect was counteracted by simultaneous KRTAP5-AS1 silencing. Independent KRTAP5-AS1 knockdown significantly reduced E2 levels, further establishing its role as a positive regulator of estrogen synthesis (p < 0.05) (Fig. 5B).

Overexpression of KRTAP5-AS1 and inhibited miR-199b-5p promote the proliferation of KGN cells

To investigate the functional impact of KRTAP5-AS1 and miR-199b-5p on granulosa cell proliferation, clonogenic assays and CCK-8 analyses were conducted alongside cyclin D1 protein quantification. Colony formation and cell viability assays demonstrated that miR-199b-5p overexpression significantly inhibited KGN cell proliferation, while KRTAP5-AS1 overexpression enhanced proliferative capacity. The suppressive effect of miR-199b-5p mimics was partially rescued by concurrent KRTAP5-AS1 overexpression, as evidenced by intermediate colony formation and viability in co-transfected cells. Conversely, miR-199b-5p inhibition promoted proliferation, whereas KRTAP5-AS1 knockdown suppressed growth, with combined treatment again showing intermediate effects (all p < 0.05, Fig. 6A-C).

Fig. 6.

KRTAP5-AS1 overexpression and miR-199b-5p inhibition promote KGN cell proliferation. (A) Representative images and quantification of colony formation assays. (B) Cell viability assessed by CCK-8 assay. (D-F) Cyclin-D1 protein expression analyzed by western blot. Statistical annotations follow the same scheme as Fig. 5

Western blot analysis of cyclin D1, a key cell cycle regulator, revealed parallel changes in protein expression. miR-199b-5p overexpression decreased cyclin D1 levels, while KRTAP5-AS1 overexpression increased its expression. Co-transfection experiments demonstrated reciprocal regulation, with the combined treatment mitigating the individual effects of either manipulation alone. Similarly, the increased cyclin D1 expression observed with miR-199b-5p inhibition was attenuated by simultaneous KRTAP5-AS1 knockdown, while the decreased expression resulting from KRTAP5-AS1 knockdown was partially rescued by miR-199b-5p inhibition (Fig. 6D-E).

Overexpression of KRTAP5-AS1 and inhibited miR-199b-5p suppress the apoptosis of KGN cells

The regulatory effects of KRTAP5-AS1 overexpression and miR-199b-5p inhibition on KGN cell apoptosis were investigated through flow cytometry and Western blot analysis. Flow cytometry results demonstrated that miR-199b-5p mimics significantly increased the apoptosis rate of KGN cells, whereas KRTAP5-AS1 overexpression (oe-KRTAP5-AS1) reduced cellular apoptosis. Notably, the pro-apoptotic effect of miR-199b-5p mimics and the anti-apoptotic effect of oe-KRTAP5-AS1 were partially reversed by co-transfection with both constructs (all p < 0.05). Conversely, inhibition of miR-199b-5p decreased KGN cell apoptosis, while KRTAP5-AS1 knockdown (sh-KRTAP5-AS1) promoted apoptosis. The anti-apoptotic effect of miR-199b-5p inhibitor and the pro-apoptotic effect of sh-KRTAP5-AS1 were attenuated when both treatments were combined (all p < 0.05) (Fig. 7A, B).

Fig. 7.

KRTAP5-AS1 overexpression and miR-199b-5p inhibition suppress KGN cell apoptosis. (A) Flow cytometry analysis of apoptotic cells. (B) Quantitative representation of apoptosis rates. (C-E) Protein expression levels of apoptosis markers Bax and Bcl-2. Statistical annotations follow the same scheme as Fig. 5

Western blot analysis of apoptosis-related proteins revealed consistent findings. miR-199b-5p mimics upregulated the expression of pro-apoptotic Bax while downregulating anti-apoptotic Bcl-2, whereas oe-KRTAP5-AS1 produced opposite effects on these proteins. Co-transfection with miR-199b-5p mimics and oe-KRTAP5-AS1 neutralized these regulatory effects (all p < 0.05). Similarly, miR-199b-5p inhibitor decreased Bax and increased Bcl-2 expression, while sh-KRTAP5-AS1 increased Bax and decreased Bcl-2 levels. The combined treatment with miR-199b-5p inhibitor and sh-KRTAP5-AS1 mitigated these individual effects (all p < 0.05) (Fig. 7C-E). These results collectively demonstrate that KRTAP5-AS1 and miR-199b-5p exert opposing regulatory effects on KGN cell apoptosis through modulation of Bax and Bcl-2 expression.

Discussion

PCOS, a prevalent endocrine disorder, involves reproductive dysfunction due to hormonal imbalances and impaired follicular development [18]. Non-coding RNAs (e.g., miRNAs, lncRNAs) are emerging as key regulators of PCOS pathogenesis through modulation of critical genes [19]. In our study, we elucidated the functional role of the KRTAP5-AS1/miR-199b-5p/CYP19A1 axis in KGN cells. We demonstrated that KRTAP5-AS1 interacts with miR-199b-5p to modulate CYP19A1 expression, E2 levels, and cellular proliferation/apoptosis. Importantly, CYP19A1-mediated suppression of miR-199b-5p appears to contribute to PCOS pathogenesis. Given its role in regulating granulosa cell apoptosis, miR-199b-5p represents a potential therapeutic target for PCOS.

The expression pattern of CYP19A1 in PCOS remains controversial across studies, with reports of both downregulation [14, 20] and upregulation [21], potentially reflecting phenotypic heterogeneity among PCOS patients. Notably, both aberrantly high and low CYP19A1 expression can disrupt steroid synthesis homeostasis. Rad et al. demonstrated distinct CYP enzyme profiles in PCOS subtypes, with elevated CYP11 and CYP17 in hyperandrogenic PCOS versus increased CYP19A1 in hypoandrogenic cases [22]. However, the limited sample size in that study (total n = 19 across three subgroups) may introduce substantial bias. Similarly, our initial discovery RNA-seq cohort (n = 6 PCOS vs. n = 3 controls) was underpowered for definitive conclusions—a limitation inherent to exploratory omics screening. To overcome these constraints and ensure robust statistical inference, we established an independent validation cohort with adequate power. In this cohort comprising 38 PCOS patients, the majority exhibited non-hyperandrogenic phenotypes (n = 33), with only 5 cases classified as hyperandrogenic PCOS. Importantly, CYP19A1 expression demonstrated consistent upregulation trends in both phenotypes compared to controls, with fold changes (FC) of 1.86 (p = 0.012) in non-hyperandrogenic PCOS and 2.69 (p = 0.054) in hyperandrogenic PCOS. Our study reveals significant endocrine alterations in PCOS patients, characterized by markedly elevated serum anti-Müllerian hormone (AMH) and testosterone (T) levels compared to healthy controls. Quantitative analysis demonstrated a strong positive correlation between ovarian CYP19A1 expression and both AMH and T concentrations. Notably, although follicular fluid E2 and basal E2 levels did not differ significantly between groups, the PCOS cohort showed substantially higher T concentrations. This indicates that increased CYP19A1 expression fails to enhance E2 biosynthesis in PCOS. Consequently, the reduced E2/T ratio reflects decreased aromatase activity [23]. These findings suggest a potential pathophysiological mechanism wherein testosterone accumulation in PCOS triggers compensatory upregulation of CYP19A1 in granulosa cells. However, the concomitant reduction in functional aromatase activity prevents effective conversion of excess T to E2. This endocrine dysfunction may subsequently impair granulosa cell apoptosis [24]. The accumulation of preantral follicles likely explains the elevated AMH production and follicular arrest characteristic of PCOS. Furthermore, CYP19A1 expression showed positive correlations with metabolic parameters (fasting blood glucose and body mass index), which aligns with our KEGG pathway analysis and reinforces the known link between endocrine dysfunction and metabolic dysregulation in PCOS pathogenesis [25]. Additionally, the inverse correlation between CYP19A1 expression and IVF embryo quality suggests that elevated CYP19A1 levels mechanistically contribute to compromised embryogenesis in obesity-associated PCOS. This finding aligns with our prior observations: embryo quality remains preserved in simple obesity but is significantly impaired when obesity coexists with PCOS. These results indicate a synergistic pathophysiological interaction between PCOS and obesity [26, 27].

The expanding literature on non-coding RNAs in PCOS reveals their extensive involvement in regulating endocrine function and reproductive processes through modulation of hormone metabolism and granulosa cell dynamics [28]. For instance, miR-9 and miR-155 overexpression in PCOS enhances proliferating cell nuclear antigen (PCNA) expression while suppressing testosterone secretion [29], whereas miR-335-5p impairs granulosa cell proliferation and promotes follicular atresia via serum/glucocorticoid-regulated kinase 3 (SGK3) inhibition [30]. miR-199b-5p exhibits context-dependent functions across pathologies. It demonstrates tumor-suppressive effects in breast cancer (via targeting activin receptor-like kinase 1) and papillary thyroid carcinoma (via Stonin 2 interaction) [31, 32], but paradoxically promotes malignancy in cervical cancer [33]. Notably, miR-199b-5p downregulation has been consistently observed in estrogen-related malignancies including breast cancer, papillary thyroid carcinoma, and prostate cancer [31, 32, 34, 35]. Our findings establish that miR-199b-5p directly targets CYP19A1 through 3’-UTR binding, suggesting its potential role in maintaining steroidogenic homeostasis in granulosa cells.

LncRNAs represent a critical class of non-coding RNAs that participate in diverse biological processes including epigenetic modulation, cell cycle control, and differentiation through various mechanisms, with the ceRNA network being one of the most well-characterized regulatory paradigms [36]. In the current study, while KRTAP5-AS1 expression was significantly reduced in PCOS patients compared to controls, our correlation analysis revealed a positive association with CYP19A1 expression (r = 0.296, p = 0.014) and an inverse relationship with miR-199b-5p levels (r=-0.256, p = 0.035). Subsequent mechanistic investigations confirmed a direct targeting interaction between KRTAP5-AS1 and miR-199b-5p. Functional studies demonstrated that KRTAP5-AS1 overexpression coupled with miR-199b-5p knockdown significantly upregulated CYP19A1 expression and enhanced E2 secretion in KGN cells. These findings align with previous work by Zhu et al. showing that lncRNA ZFAS1 downregulation or miR-129 overexpression elevates E2 production via HMGB1 regulation in PCOS granulosa cells [37].

Further investigation revealed that miR-199b-5p inhibition and KRTAP5-AS1 upregulation collectively promote KGN cell proliferation while suppressing apoptosis. Notably, the pro-proliferative and anti-apoptotic effects of miR-199b-5p inhibition could be counteracted by KRTAP5-AS1 knockdown, and vice versa. This regulatory pattern corroborates existing literature documenting miR-199b-5p’s tumor-suppressive roles in breast cancer, papillary thyroid carcinoma (PTC), and prostate cancer [32, 34, 35]. In contrast, KRTAP5-AS1 has been reported as an oncogenic factor in PTC with prognostic significance [13]. Clinical analyses revealed a significant negative correlation between miR-199b-5p expression levels and Gn requirements, while demonstrating a positive association with embryo quality. In PCOS patients, elevated CYP19A1 likely functions as a negative regulator of miR-199b-5p through direct molecular interaction, leading to suppression of granulosa cell apoptosis. This anti-apoptotic mechanism may promote pathological accumulation of antral follicles, consequently contributing to ovarian follicular dysfunction and PCOS progression.

KRTAP5-AS1 was lower expressed in PCOS GCs than in control samples. Considering the protracted 6-month developmental timeline required for primordial follicles to reach the preovulatory stage [38], apart from individual differences, we hypothesize that this phenomenon may be attributed to the intricate intrafollicular milieu, wherein dynamic microenvironmental alterations during folliculogenesis potentially drive stage-specific fluctuations in KRTAP5-5p expression, thereby contributing to the pathogenesis of PCOS. Moreover, KRTAP5-AS1 is potentially targeted by other miRNAs, such as miR-186 and miR-132, whose aberrant expression may further suppress KRTAP5-AS1 levels [22, 39–42].

In conclusion, our study demonstrates that granulosa cells from PCOS patients exhibit significantly elevated CYP19A1 expression concomitant with reduced miR-199b-5p levels. Mechanistically, we identified that KRTAP5-AS1 knockdown enhances miR-199b-5p expression, leading to subsequent downregulation of CYP19A1, and ultimately promotes apoptosis and suppresses proliferation in KGN cells. These findings establish the KRTAP5-AS1/miR-199b-5p/CYP19A1 axis as a potential regulatory pathway in PCOS pathogenesis. However, several limitations should be acknowledged. First, the findings were generated using the KGN immortalized cell line, which, despite retaining key granulosa cell characteristics, is ultimately a tumor-derived model system. Second, the in vitro monolayer culture cannot fully recapitulate the complex 3D architecture and dynamic endocrine physiology of intact ovarian follicles in vivo. To address these limitations, future investigations should focus on: (1) comprehensive mechanistic studies employing both advanced in vitro models (e.g., organoid cultures) and in vivo models to validate the regulatory mechanisms; (2) large-scale clinical validation to assess the therapeutic potential of targeting this axis; and (3) systematic exploration of crosstalk between this molecular pathway and metabolic factors, such as insulin resistance. Furthermore, given the well-established link between metabolic dysfunction and PCOS pathogenesis, subsequent research should specifically elucidate how systemic metabolic disturbances may modulate this newly identified molecular pathway.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

ZW and PT conceived the idea and conception. PT contributed to acquisition, statistical analysis and interpretation of data; drafted the manuscript. XY analyzed part of the data. All authors have read and approved the final version of the manuscript.

Funding

Supported by the Natural Science Foundation of Fujian Province (grant no. 2022J011378).

Data availability

The data and materials are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

The studies involving human participants were reviewed and approved by the ethics committee of First Affiliated Hospital of Xiamen University. The patients/participants provided their written informed consent to participate in this study.

Competing interests

The authors declare no competing interests.