Clinical sample collection
Twelve patients who underwent assisted reproductive technology (ART)-based assisted conception using an antagonist regimen at the Department of Reproduction and Genetics (Affiliated Hospital of Shandong University of Traditional Chinese Medicine) were included in the study. These included six patients with advanced DOR (age ≥ 35 years) and six young women with normal ovarian reserve (NOR). The diagnosis of DOR was based on the fulfillment of at least two of the following criteria [17]: (i) bilateral anterior follicle count (AFC) < 6, (ii) anti-Müllerian hormone (AMH) < 1.10 ng/mL; and (iii) basal follicle-stimulating hormone (FSH) of 10–40 mIU/mL. The NOR group included women with normal ovarian reserve but infertility due to male or tubal factors. Patients with structural malformations of the reproductive organs, endometriosis, other endocrine disorders (such as thyroid dysfunction and hyperprolactinemia), or chromosomal abnormalities were excluded from the study. Follicular fluid discarded after oocyte retrieval was collected, and GCs were extracted, purified via density gradient centrifugation [18], and immediately stored in a -80°C refrigerator. This study was approved by the ethics committee of the Department of Reproductive Medicine at the hospital and was registered in the China Clinical Trial Registry on October 30, 2021 (registration number: ChiCTR2100052522). Informed consent was obtained from all the patients.
Cell culture
The human ovarian GC tumor cell line KGN was purchased from Procell Life Science and Technology Co. (Wuhan, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium (BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; BasalMedia) and 1% penicillin-streptomycin solution (Biosharp, Hefei, China) in an incubator at 37°C with 5% CO2.
Cell transfection
NEAT1 overexpression plasmid (OE-NEAT1), NEAT1 small interfering RNA (siRNA; si-NEAT1), ESR1 siRNA (si-ESR1), miR-204-5p mimics (mimics-miR-204-5p), miR-204-5p inhibitor (inhibitor-miR-204-5p), and the corresponding negative controls were obtained from Tsingke Biotech Co. Ltd. (Beijing, China). The plasmids, siRNA, and miR-204-5p mimics/inhibitors were transfected into KGN cells using Lipofectamine® 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The transfection efficiency was determined using quantitative reverse transcription PCR (RT-qPCR).
Fluorescence in situ hybridization (FISH)
A FISH Kit (RiboBio, Guangzhou, China) was used to analyze the subcellular localization of NEAT1. KGN cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Then, pre-hybridization buffer was added to the cells and incubated for 30 min, followed by overnight incubation in a hybridization buffer containing a fluorescein isothiocyanate (FITC)-labeled NEAT1 probe (Sangon Biotech, Shanghai, China). The cells were counterstained with DAPI (incubation for 8 min protected from light) and imaged under a laser confocal microscope (Leica, Germany).
Dual-luciferase reporter assay
Bioinformatic analysis predicted the downstream targets and complementary pairing sites of NEAT1 and miR-204-5p. Wild-type (NEAT1-WT or ESR1-WT) and mutant plasmids (NEAT1-MUT or ESR1-MUT) containing miR-204-5p binding sites were constructed and cloned into a pGL4 vector (Promega, Madison, WI, USA). KGN cells were co-transfected with the luciferase reporter plasmids, mimics-miR-204-5p, inhibitor-miR-204-5p, and the corresponding negative controls using Lipofectamine® 3000. After 48 h, the luciferase activity in the cells was measured using a Dual-Luciferase Reporter Gene Assay Kit (Promega, USA).
Cell counting kit-8 (CCK-8) assay
A CCK-8 Kit (Vazyme Biotech Co. Ltd., Nanjing, China) was used to analyze cellular activity. KGN cells were plated in 96-well plates at 4 × 103 cells/well (100 µL/well). After cell adherence, 10 µL CCK-8 solution was added to each well and incubated at 37°C for 2 h in the incubator. Absorbance was measured at 450 nm using a multifunctional enzyme marker (Bio-Rad, Hercules, CA, USA).
EdU proliferation assay
Cell proliferation was assessed by measuring DNA synthesis using the EdU Cell Proliferation Kit (Beyotime, Shanghai, China). KGN cells were incubated with 500 µL EdU working solution for 2 h. The cells were then fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton-100 for 10 min, and the nuclei were stained with Hoechst 33342 for 10 min in the dark. Images were captured using a fluorescence microscope (Nikon, Tokyo, Japan).
Flow cytometric assay
Apoptosis was detected using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (Vazyme). KGN cells were collected, washed with phosphate-buffered saline (PBS), and resuspended in a binding buffer. Subsequently, the cells were stained with Annexin V-FITC and PI by incubating for 15 min in the dark. The cells were analyzed using a flow cytometer (BD, Franklin Lakes, NJ, USA).
The cell cycle was analyzed using a Cell Cycle and Apoptosis Detection Kit (Beyotime). KGN cells were fixed with pre-cooled PBS and 70% ethanol at 4 ºC overnight. The cells were then resuspended in PI staining solution for 30 min at 37 ºC protected from light and detected using a flow cytometer.
RT-qPCR
Total RNA was extracted from clinical GC samples and KGN cells using the Total RNA Extraction Reagent RNA Isolator (Vazyme). The concentration and purity of the extracted RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was reverse transcribed to cDNA using HiScript II Q RT SuperMix for qPCR (Vazyme). RT-qPCR was performed using a fluorescent quantitative PCR instrument (Bio-Rad) and the ChamQ SYBR® qPCR Master Mix (Vazyme). GAPDH or U6 were used as the reference genes, and the 2−ΔΔCt method was used to quantify the expression level of RNAs. The specific primers used in this study were obtained from Sangon Biotech, and their sequences are listed in Table 1.
Table 1
Primer sequences for gene expression
Gene | Primer sequence (5'-3') |
NEAT1 | Forward: AGTGTGAGTCCTAGCATTG |
Reverse: GAACTTCCTCCTCCTAAGC |
hsa-miR-204-5p | Forward: CGCGTTCCCTTTGTCATCCT |
Reverse: AGTGCAGGGTCCGAGGTATT |
hsa-miR-125a-5p | Forward: TCGGCAGGTCCCTGAGACCCTT |
Reverse: CTCAACTGGTGTCGTGGA |
StAR | Forward: GCCACATTTGCCAGGAAACAATG |
Reverse: CTCCTGGTCACTGTAGAGAGTCT |
CYP19A1 | Forward: GCAAAGCACCCTAATGTTGAAGA |
Reverse: CGAGTCTGTGCATCCTTCCAATA |
ESR1 | Forward: CTCTAACCTCGGGCTGTGC |
Reverse: AGATGCTTTGGTGTGGAGGG |
NOTCH2 | Forward: ATCCCACAAAGCCTAGCACC |
Reverse: CCTTGTCCCTGAGCAACCAT |
IGFBP5 | Forward: GAAGCAGATGTGTCTCTGCCC |
Reverse: TTCCCTCTGCTTCCTGGTTC |
GAPDH | Forward: TGCACCACCAACTGCTTAGC |
Reverse: GGCATGGACTGTGGTCATGAG |
U6 | Forward: CAGCACATATACTAAAATTGGAACG |
Reverse: ACGAATTTGCGTGTCATCC |
CYP19A1, cytochrome P450 family 19 subfamily A member 1; ESR1, estrogen receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGFBP5, insulin-like growth factor binding protein 5; NEAT1, nucleo-enriched abundant transcript 1; NOTCH2, recombinant notch homolog 2; StAR, steroidogenic acute regulatory protein; U6, RNU6.
Western blotting
Proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Servicebio, Wuhan, China), and the protein concentration was determined using the Bicinconinic acid (BCA) Protein Assay Kit (CWBIO, Taizhou, China). Equal amounts of proteins were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to polyvinylidene difluoride membranes (Bio-Rad). The non-specific sites on the membranes were blocked by incubating with 5% skimmed milk for 2 h at 22°C. The membranes were then incubated with the primary antibody overnight at 4°C. The membranes were washed with TBST buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. After washing with TBST buffer, the protein bands on the membrane were visualized using an enhanced chemiluminescence Detection Kit (Vazyme) and scanned using a gel imaging system. GAPDH was used as an internal reference. All the antibodies used in the western blotting, including ESR1 (20698-1-AP), phospho-ERK1/2 (80031-1-RR), ERK1/2 (11257-1-AP), phospho-CREB1 (28792-1-AP), CREB1 (12208-1-AP), StAR (12225-1-AP), CYP19A1 (16554-1-AP), and GAPDH (10494-1-AP) were purchased from Proteintech Group Inc. (Wuhan, China).
Enzyme-linked immunosorbent assay (ELISA)
KGN cells were collected, and androstenedione (10 µg/mL) was added to the culture medium. The cells were then cultured for 24 h. The estradiol level in the supernatant was measured using the Human Estrogen, E ELISA Kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions.
Statistical analysis
The experimental data were analyzed using SPSS software (version 23.0; IBM, Armonk, NY, USA). All results are expressed as mean ± standard deviation (SD). The normality of data distribution was assessed using the Shapiro–Wilk test. Student's t-test or Wilcoxon test was used to assess the significance of the difference between the two groups of data. Pearson's correlation test was used for the correlation analysis between variables. The graphs were generated using GraphPad Prism (version 8.0; San Diego, CA, USA) software. Statistical significance was set at p < 0.05.