A total of 60 patients with spontaneous (not induced by chemotherapy, radiation or surgery) POF (age range, 21-39 years; mean age, 28.1±4.6 years) that were admitted at The Sixth Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) between December 2016 and December 2018 were enrolled in the present study. All patients were diagnosed with POF based on their FSH level. Patients with FSH level >30 IU/l (measured twice at 4 weeks interval) were included in the study. Patients with POF who had other clinical complications, including other types of ovarian disorders, such as endometriosis, ovarian cysts and ovarian cancers, were excluded from this study. No therapy was initiated prior to admission. The control group consisted of 60 healthy women (age range, 21-39 years; mean age, 28.2±4.4 years) who were also enrolled at the aforementioned hospital between December 2016 and December 2018. These healthy women had suspected ovarian lesions that were removed during ovarian biopsy. All participants provided their written informed consent and the study was approved by the Ethics Committee of The Sixth Affiliated Hospital of Sun Yat-sen University (Guangzhou, China).

Ovarian biopsy was performed on ovaries of all patients and healthy women to collect ovarian tissue samples (0.08-0.12 g) from each participant. The two normal Chinese hamster ovary cell lines Lec8 and CHO (American Type Culture Collection) were used in the present study. Cells were cultured in ovarian epithelial cell medium (OEpiCM; ScienCell Research Laboratories, Inc.) supplemented with antibiotic-antimycotic (10,000 U/ml penicillin, 10,000 µg/ml streptomycin and 25 µg/ml amphotericin B from Thermo Fisher Scientific, Inc. (cat. no. 15240112; 100X) at 37˚C in a humidified incubator containing 5% CO₂.

NEAT1 and p53 expression vectors were constructed using the pcDNA3 vector (Sangon, Biotech Co., Ltd.) as a backbone. Negative control small interfering (si)RNA (5'-UGGUACGAUGUGGACACGACC-3') and NEAT1 siRNA (5'-ACAAUGCCACCGUUAAUUUGAC-3') were provided by Shanghai GenePharma Co., Ltd. Lipofectamine 2000^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect 10 nM NEAT1 expression vector, 10 nM p53 expression vector, 10 nM empty pcDNA3 vector (negative control, NC), 45 nM NEAT1 siRNA or 45 nM negative control siRNA (NC) into 10⁵ cells per well Lec8 or CHO cells at 37˚C in a 6-well cell culture plate. Untransfected cells were used as control cells (C). Cells were harvested 24 h post-transfection for subsequent experimentation.

Total RNA was extracted from ovarian tissues (0.05 g) or Lec8 and CHO cells (10⁶) using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific Inc.). Following DNase I digestion, cDNA was synthesized using a QuantiTect reverse transcription kit (Qiagen China Co., Ltd.) at the following thermal conditions: 55˚C for 20 min and 80˚C for 20 min. KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems; Roche Diagnostics Co., Ltd.) was used to prepare all qPCR mixtures. 18S rRNA or GAPDH were used as endogenous controls to detect the expression of NEAT1 and p53 mRNA, respectively. PCR reaction conditions were: 95˚C for 1 min, followed by 95˚C for 10 sec and 58˚C for 50 sec. The 2^-ΔΔCq method (16) was used to normalize data. Three replicates were conducted for each experiment. Primer sequences were: 5'-CTTCCTCCCTTTAACTTATCCATTCAC-3' (forward) and 5'-CTCTTCCTCCACCATTACCAACAATAC-3' (reverse) for NEAT1; 5'-AGAGTCTATAGGCCCACCCC-3' (forward) and 5'-GCTCGACGCTAGGATCTGAC-3' (reverse) for p53; 5'-GTCTCCTCTGACTTCAACAGCG-3' (forward) and 5'-ACCACCCTGTTGCTGTAGCCAA-3' (reverse) for GAPDH; 5'-CTACCACATCCAAGGAAGCA-3' (forward) and 5'-TTTTTCGTCACTACCTCCCCG-3' (reverse) for 18S rRNA.

Lec8 and CHO cells were collected 24 h following transfection. Cells were cultured in fresh medium (OEpiCM) for a further 48 h at 37˚C. After washing with PBS, cells (10⁶) were mixed with 500 µl of Annexin binding buffer (5X; Thermo Fisher Scientific, Inc.). Subsequently, 5 µl FITC labeled Annexin-V and 5 µl propidium iodide solution was added to the cells and incubated for 15 min in the dark at room temperature. A FACSCalibur flow cytometer (BD Biosciences) was used to detect apoptotic cells. Data were analyzed using FCSalyzer version 0.9.17 (SourceForge; https://sourceforge.net/projects/fcsalyzer/files/Version%200.9.17-alpha/).

Lec8 and CHO cells (10⁶ cells) collected 24 h after transfection were mixed with 0.1 ml ice-cold RIPA buffer (Sangon Biotech Co., Ltd.) for total protein extraction. BCA assay (Invitrogen; Thermo Fisher Scientific, Inc.) was used to determine protein concentration. Proteins (40 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked using 5% non-fat milk at room temperature for 2 h. Membranes were incubated with primary antibodies against GAPDH (1:1,500; cat. no. ab9485; Abcam) and p53 (1:1,500; cat. no. ab31333; Abcam) at 4˚C overnight. Subsequently, the membranes were incubated with IgG-horseradish peroxidase secondary antibody (1:1,000; goat anti rabbit; cat. no. MBS435036; MyBioSource, Inc.) at room temperature for 2 h. Bands were detected using enhanced chemiluminescence substrate (Merck KGaA). Relative expression level was normalized to endogenous control GAPDH using ImageJ version 1.46 (National Institutes of Health).

Data are presented as the mean ± SD values of three independent experiments. Unpaired t-test was used to compare differences between two groups. ANOVA followed by the Tukey's post hoc test was used for multiple comparison. Associations were analyzed using linear regression. P<0.05 was considered to indicate a statistically significant difference.

NEAT1 expression in ovarian tissues of patients from both the POF and control groups was detected using RT-qPCR. The results demonstrated that expression level of NEAT1 was significantly decreased in patients with POF compared with healthy controls (Fig. 1).

RT-qPCR was used to detect the expression level of p53 in ovarian tissues of patients from both the POF and control groups. The mRNA expression level of p53 was significantly increased in the POF group compared with the control group (Fig. 2A). Furthermore, association between p53 mRNA and NEAT1 expression was analyzed using linear regression. The results demonstrated that p53 and NEAT1 expression levels were inversely associated in the POF group (Fig. 2B). However, p53 and NEAT1 expression levels were not associated in the control group (Fig. 2C).

NEAT1 expression vector and siRNA were transfected into Lec8 and CHO cells. At 24 h following transfection, expression of NEAT1 was significantly altered in both cell lines compared with the C and NC groups (Fig. 3A). Furthermore, cells overexpressing NEAT1 demonstrated significantly downregulated mRNA and protein levels of p53 (Fig. 3B). Cells that had NEAT 1 silenced demonstrated significantly upregulated mRNA and protein levels of p53 (Fig. 3C).

Overexpression of p53 was also achieved 24 h following transfection (Fig. S1). Compared with the C and NC controls, overexpression of NEAT1 inhibited Lec8 (Fig. 4A) and CHO (Fig. 4B) cell apoptosis. Overexpression of p53 promoted Lec8 and CHO cell apoptosis. In addition, overexpression of p53 attenuated the effects of overexpressing NEAT1 on cell apoptosis.