The GSE124226 dataset in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) were analyzed for assessing TNC expression in PCOS patients and healthy people. To retrieve the signaling pathways regulated by TNC, the Gene Set Enrichment Analysis (GSEA) software (http://software.broadinstitute.org/gsea/index.jsp) was conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG) datasets (c2.cp.kegg.v2023.1.Hs.symbols.gmt, http://www.gsea-msigdb.org/gsea/msigdb/human/genesets.jsp?collection=CP:KEGG_MEDICUS).

A total of 20 female Sprague-Dawley rats (3 weeks old, 50–60 g) were supplied by Jinan Pengyue Experimental Animal Breeding Co., Ltd. All rats were housed at a controlled temperature of 25±2°C in standard cages with 55±15% humidity under a 12-h light/dark cycle and given ad libitum access to sterilized food and water for 1 week before the experiments. All animal procedures were overseen and approved by the Ethical Committee of The Second Affiliated Hospital of Shandong First Medical University (no. 2021-128; Taian, China). Rats were randomly assigned to four groups (n=5 each): Control, PCOS, PCOS + short hairpin RNA (shRNA/sh)-negative control (NC) and PCOS + sh-TNC. A rat PCOS model was induced using dehydroepiandrosterone (DHEA). Briefly, rats in the PCOS group were subcutaneously injected daily with 60 mg/kg DHEA (MilliporeSigma) diluted with 0.2 ml sesame oil for 21 consecutive days. At the same time, the rats in the control group were injected with an equal volume of sesame oil (20–23). Prior to DHEA injection to induce PCOS in rats, lentivirus carrying sh-TNC or sh-NC (10 µl; Shanghai GeneChem Co., Ltd.) was injected into the ovaries of rats. The shRNA sequences were as follows: sh-TNC, 5′-CGACATGTTTACTATCGAA-3′ and scrambled sh-NC, 5′-TTCTCCGAACGTGTCACGT-3′. At 3 weeks after the lentivirus injection, blood and ovaries of the rats were collected for the subsequent analyses. The humane endpoints of the present study were as follows: i) At least 10% weight loss with <40% food and water intake within 7 days following DHEA treatment; and ii) at least 20% weight loss without food or water intake with repeated or prolonged convulsions or ataxia at the end of 21 days of DHEA treatment. The rats were sacrificed using intraperitoneal injection of 250 mg/kg pentobarbital sodium. After respiratory and cardiac arrest, monitoring was continued for a further 5 min to confirm animal death. The whole experiment, including a week of acclimation, lasted 29 days. No rats succumbed during the present study. The animal health and behavior were monitored daily.

Rats were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital sodium (24–28), and then blood (2 ml) was collected from the abdominal aorta. After blood collection, the rats were euthanized immediately by intraperitoneal injection of 250 mg/kg pentobarbital sodium. After respiratory and cardiac arrest, monitoring was continued for a further 5 min to confirm animal death. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, estradiol and insulin, as well as the levels of pro-inflammatory cytokines in ovarian tissues, were assessed using their corresponding ELISA kits (LH ELISA kit; cat. no. SEKR-0091; Beijing Solarbio Science & Technology Co., Ltd.; FSH ELISA kit, SEKR-0090; Beijing Solarbio Science & Technology Co., Ltd.; testosterone ELISA kit; cat. no. SEKSM-0003; Beijing Solarbio Science & Technology Co., Ltd.; estradiol ELISA kit; cat. no. SEKR-0107; Beijing Solarbio Science & Technology Co., Ltd.; insulin ELISA kit; cat. no. SEKR-0033; Beijing Solarbio Science & Technology Co., Ltd.; TNF-α ELISA kit; cat. no. EK0526; Wuhan Boster Biological Technology, Ltd.; IL-1β ELISA kit; cat. no. EK0393; Wuhan Boster Biological Technology, Ltd.; IL-6 ELISA kit; cat. no. EK0412; Wuhan Boster Biological Technology, Ltd.; IL-18 ELISA kit, EK0592; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

The IR was assessed by the homeostasis model assessment of IR (HOMA-IR) (29), which was calculated as follows: Fasting blood glucose (FBG) × fasting serum insulin (FINS)/22.5. The rats were fasted for 12 h after the last DHEA administration and the blood glucose level was measured utilizing a Glucose Assay Kit (Beyotime Institute of Biotechnology).

After being harvested from rats, the ovarian tissues were fixed in 4% paraformaldehyde buffer for 24 h at room temperature, embedded in paraffin and cut into 4-µm-thick slices. The sections were stained using hematoxylin for 8 min and eosin for 5 min at room temperature. Morphological changes in ovarian tissues were observed under a light microscope.

For immunohistochemistry, the ovarian tissues were fixed in 4% paraformaldehyde buffer for 24 h at room temperature, embedded in paraffin and cut into 4-µm-thick slices. Following this, the slides were heated at 60°C, then dewaxed by xylene and rehydrated by fractionated ethanol. The sections were incubated in hydrogen peroxide (3%, vol/vol) in PBS for 20 min at 37°C to block the endogenous peroxidase activity. After blocking of nonspecific activity with 5% goat serum (Oumarsi Biotech) for 60 min at room temperature, the sections were incubated with primary antibodies, including TNC (1:50; cat. no. ab108930; Abcam), TLR4 (1:50; cat. no. 19811-1-AP; Proteintech Group, Inc.) and phosphorylated (p-)NF-κB (1:100; cat. no. SAB5700363; MilliporeSigma) antibodies, overnight at 4°C. Subsequently, the slices were incubated with a biotinylated secondary antibody (1:500; cat. no. ab7176; Abcam) for 1 h at room temperature. The sections were observed under a light microscope after staining with 3,3′-diaminobenzidine (MilliporeSigma) for 5 min at room temperature and counterstaining with hematoxylin for 60 sec at room temperature.

According to the manufacturer's protocol, the levels of MDA, GSH and SOD in ovarian tissues and cell culture supernatants were determined using MDA content assay kit (Beijing Solarbio Science & Technology Co., Ltd.), GSH content assay kit (Beijing Solarbio Science & Technology Co., Ltd.) and SOD activity assay kit (Beijing Solarbio Science & Technology Co., Ltd.), respectively.

RNA was extracted from ovarian tissues and KGN cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using the GoScript^™ Reverse Transcription System (Promega Corporation) for 60 min at 42°C. RT and qPCR were performed using the Hifair III One-Step RT-qPCR SYBR Green Kit (Shanghai Yeasen Biotechnology Co., Ltd.). β-actin was used as the endogenous control. The thermocycling conditions were: 95°C for 5 min, followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec. The following primers were used: Rat TNC forward, 5′-TCTCTGGAATTGCTCCCAGC-3′ and reverse, 5′-TTCCGGTTCAGCTTCTGTGG-3′; human TNC forward, 5′-GGGGGTGGATGGATTGTGTT-3′ and reverse, 5′-CTTCTCTGCGGTCCCCAAAT-3′; human TNF-α forward, 5′-CCCCAGGGACCTCTCTCTAA-3′ and reverse, 5′-GCTTGAGGGTTTGCTACAACA-3′; human IL-1β forward, 5′-GCCCTAAACAGATGAAGTGCTC-3′ and reverse, 5′-CCATGGCCACAACAACTGAC-3′; human IL-6 forward, 5′-CTCCTTCTCCACAAGCGCC-3′ and reverse, 5′-GATGCCGTCGAGGATGTACC-3′; human IL-18 forward, 5′-TTGACCAAGGAAATCGGCCT-3′ and reverse, 5′-GTCCGGGGTGCATTATCTCT-3′; rat β-actin forward, 5′-GCCTTCCTTCCTGGGTATGG-3′ and reverse, 5′-AATGCCTGGGTACATGGTGG-3′; and human β-actin forward, 5′-GATTCCTATGTGGGCGACGA-3′ and reverse, 5′-AGGTCTCAAACATGATCTGGGT-3′. The 2^−ΔΔCq method was used to calculate the mRNA expression level (30).

RIPA buffer (Thermo Fisher Scientific, Inc.) was applied to lyse the cells and ovarian tissues to obtain proteins. The protein was quantified using a BCA kit (Beyotime Institute of Biotechnology). Next, 10% SDS-PAGE was employed to separate the protein samples (50 µg). Subsequently, the protein bands were transferred onto a PVDF membrane (Bio-Rad Laboratories, Inc.). After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with the corresponding primary antibodies, including TNC (1:500; cat. no. ab108930; Abcam), Bcl-2 (1:1,000; cat. no. ab196495; Abcam), cleaved caspase-3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), TLR4 (1:1,000; cat. no. 19811-1-AP; Proteintech Group, Inc.), NF-κB (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-NF-κB (1:500; cat. no. SAB5700363; MilliporeSigma) and anti-GAPDH (1:2,000; cat. no. ab181602; Abcam) antibodies, at 4°C overnight. After washing with TBST (TBS + 0.1% Tween-20) three times for 5 min each, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. SA00001-2; Proteintech Group, Inc.) for 1 h at room temperature. Protein bands were visualized by incubation with an enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology). Finally, the bands were quantified using Quantity One 4.1.1 gel analysis software (Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control.

KGN human granulosa cells were obtained from Procell Life Science & Technology Co., Ltd. and incubated in DMEM/F12 (Procell Life Science & Technology Co., Ltd.) at 37°C with 5% CO₂. The medium contained 10% FBS (Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin/streptomycin (Thermo Fisher Scientific, Inc.). The PCOS cell model was established by treating cells with dihydrotestosterone (DHT) solution (500 nM) for 24 h. Next, 100 nM small interfering RNA (siRNA/si)-TNC and 100 nM si-NC (both TsingKe Biological Technology) were transfected into KGN cells for 48 h at 37°C using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The medium was replaced with fresh medium and the cells were cultured for another 24 h. The siRNA sequences were as follows: si-TNC, sense 5′-CGGUGUAUAUUAAGUGCUAGU-3′ and antisense 5′-UAGCACUUAAUAUACACCGGG-3′; and si-NC, sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′.

Proliferation was evaluated using an EdU Apollo DNA in vitro kit (Guangzhou RiboBio Co., Ltd.). Briefly, treated KGN cells (5×10³ cells/well) were plated in 24-well plates and incubated for 24 h and cultured with 50 µM EdU for 120 min at room temperature. Following fixing with 4% paraformaldehyde for 30 min at room temperature and permeabilizing with 0.3% Triton X-100 for 10 min, KGN cells were stained with DAPI at room temperature in the dark for 20 min. The positive cells were observed under a fluorescence microscope and captured images were analyzed using ImageJ software v1.51 (National Institutes of Health).

The treated KGN cells were resuspended in binding buffer, and Annexin V-FITC (5 µl) and propidium iodide (5 µl) staining solutions (BD Biosciences) were added to the cell suspension. Next, the cells were incubated for 15 min at room temperature in a dark room, followed immediately by flow cytometry using a FACScan flow cytometry system (Becton, Dickinson and Company). The data were analysed using FlowJo software (v7.6.5; FlowJo, LLC).

The treated KGN cells were washed using PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.3% Triton X-100 on ice. After washing three times with PBS, the cells were blocked with 3% bovine serum albumin (MilliporeSigma) for half an hour at room temperature. Next, KGN cells were incubated at 4°C overnight with primary antibodies, including TLR4 (1:50; cat. no. 19811-1-AP; Proteintech Group, Inc.) and p-NF-κB (1:100; cat. no. SAB5700363; MilliporeSigma) antibodies. The following day, KGN cells were incubated with the goat anti-rabbit IgG H&L (Alexa Fluor^® 488; cat. no. ab150077; 1:500; Abcam) for 2 h at room temperature, followed by washing with PBS. Finally, cells were counterstained with DAPI for 10 min at room temperature and images were captured under a fluorescence microscope.

SPSS 22.2 (IBM Corp.) and GraphPad Prism 8.0 (Dotmatics) were employed to perform statistical analyses. An unpaired Student's t-test or one-way or two-way ANOVA with Tukey's post hoc test was utilized for group comparisons. All experiments were repeated three times. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Based on analysis of the Gene Expression Omnibus dataset GSE124226, TNC mRNA expression was significantly upregulated in the patients with PCOS compared with the healthy controls (Fig. 1A). Additionally, the TNC mRNA levels were higher in PCOS rats than in control rats (Fig. 1B). Similarly, immunohistochemistry and western blotting further demonstrated that TNC protein expression was upregulated in the ovarian tissues of PCOS rats (Fig. 1C and D).

As shown in Fig. 2A, the TNC level was significantly increased in the PCOS group compared with the control group. Compared with that in the PCOS + sh-NC group, the TNC level was reduced in the PCOS + sh-TNC group (Fig. 2A). IR is a key indicator of PCOS (31). The concentrations of FBG and FINS, and the HOMA-IR index were elevated in the PCOS group compared with the control group (Fig. 2B-D). The elevated FBG and FINS concentrations, and HOMA-IR index were reduced by injection of sh-TNC (Fig. 2B-D). Additionally, knockdown of TNC markedly reversed the elevated LH, testosterone and estradiol levels induced by DHEA, but did not affect FSH (Fig. 2E-H). H&E staining was used to explore the morphological changes in the ovaries (Fig. 2I). Cystic dilatation of follicles, reduced corpus luteum, increased atretic follicles and a thin layer of granulosa cells were observed in the PCOS group compared with the control group (Fig. 2I). Notably, these phenotypes were improved in the group injected with sh-TNC (Fig. 2I).

Western blotting demonstrated that knockdown of TNC significantly counteracted the decrease in Bcl-2 expression and the increase in cleaved caspase-3 expression caused by DHEA (Fig. 3A). Furthermore, upregulation of MDA and knockdown of GSH and SOD in PCOS rats were reversed by the injection of sh-TNC (Fig. 3B). The results of ELISAs showed that the elevated levels of TNF-α, IL-1β, IL-6 and IL-18 in PCOS rats were neutralized by the administration of sh-TNC (Fig. 3C). Overall, these data indicated that TNC knockdown could effectively inhibit ovarian cell apoptosis, oxidative stress and inflammation in PCOS rats.

As shown in Fig. 4A, Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that TNC inhibited the TLR pathway. Compared with those of the control group rats, TLR4 and p-NF-κB levels were elevated in PCOS group rats (Fig. 4B). TLR4 and p-NF-κB levels in PCOS + sh-TNC group rats were lower than those in PCOS + sh-NC group rats (Fig. 4B). The results of immunohistochemistry further confirmed this observation (Fig. 4C). All results suggested that knockdown of TNC could inhibit the activation of the TLR4/NF-κB signaling pathway in PCOS rats.

As shown in Fig. 5A, TNC mRNA expression was significantly decreased in the control + si-TNC group compared with the control + si-NC group. The results in Fig. 5B demonstrated that the TNC level was higher in the DHT group than the control group. Compared with that in the DHT + si-NC group, the TNC level was significantly reduced in the DHT + si-TNC group (Fig. 5B). Additionally, the results of the EdU assay and flow cytometry demonstrated that TNC silencing notably reversed the DHT-induced reduction in cell proliferation and increase in apoptosis (Fig. 5C and D). In addition, TNC silencing significantly reversed the DHT-induced decrease in Bcl-2 expression and increase in cleaved caspase-3 expression in KGN cells (Fig. 5E).

Elevated MDA levels, and downregulated GSH and SOD levels were observed in the DHT group compared with the control group; these phenotypes were reversed by silencing of TNC (Fig. 6A). In addition, the silencing of TNC significantly attenuated the increased expression levels of TNF-α, IL-1β, IL-6 and IL-18 in KGN cells induced by DHT (Fig. 6B). Together, these data indicated that knockdown of TNC could alleviate the DHT-induced oxidative stress and inflammatory response in KGN cells.

As shown in Fig. 7A, the levels of TLR4 and p-NF-κB were significantly increased in the DHT group compared with the control group. When compared with those in the DHT + si-NC group, the levels of TLR4 and p-NF-κB were significantly decreased in the DHT + si-TNC group (Fig. 7A). This observation was also confirmed by immunofluorescence (Fig. 7B and C). Overall, knockdown of TNC could inhibit the activation of the TLR4/NF-κB signaling pathway in DHT-treated KGN cells.