Patients and samples

Follicular fluid samples were donated by 80 patients with PCOS (29.6 +/- 4.1 years old) and 80 controls (29.3 +/- 4.1 years old) at Hainan Women and Children’s Medical Center after the Ethics Committee of this hospital approved this study. All participants signed the informed consent, who underwent their first round of in-vitro fertilization treatment. Sample collection was performed during in vitro fertilization (IVF) through intracytoplasmic injection. Controls in the present study received IVF treatment due to male factors. Rotterdam revised criteria were followed to diagnose PCOS [19]. Participants older than 40 years old and patients with a body mass index higher than 35 were excluded.

Cells and cell culture method

Gene interactions and the functional roles of genes were assessed through in vitro cell assays employing human ovarian granulosa cell-like KGN cells (ATCC). Subculture was carried out in a 10:1 volume ratio when cell confluent reached approximately 70 and 80%. Cells were cultivated in 6 cm plates using DMEM supplemented with 10% FBS. In instances of cell contamination, the affected cell culture plates were discarded, and the cell culture process was repeated.

Cell transfection

By means of transfection with BBOX1-AS1 vector, BBOX1-AS1 siRNA, and miR-19b mimic, we investigated their interactions and roles in modulating cellular behaviors. To outline the process, cells were harvested, rinsed with ice-cold PBS, and then quantified. Then cells were transferred to a tube containing transfection mixture (Lipofectamine™ 3000 and vector, siRNA or miRNA). After incubation for 6 h, cells were washed for three times with PBS and transferred to 6 cm cell culture plates to reduce cytotoxicity.

RNA isolation

For RNA isolation, Trizol (Invitrogen) was combined with samples in a 10:1 volume ratio. After incubating at room temperature for 30 min, centrifugation (12,000 g) was carried out for 12 min to eliminate cellular debris. Subsequently, the supernatant was collected, and chloroform was mixed in a 1:4 volume ratio to eliminate protein contamination. After mixing, another centrifugation step (12,000 g) was conducted for 12 min. The supernatant was then collected and combined with methanol at a 1:1 volume ratio, followed by centrifugation (12,000 g) to precipitate the RNA samples.


RNase-free water was used to dissolve RNA samples. RNA concentration, purity, and integrity were determined using a 2100 bioanalyzer. After cDNA preparation, PCR amplification of GAPDH was performed on each cDNA sample to determine cDNA quality. Samples with satisfactory quality were used to perform qPCRs with 18S rRNA as the internal control to determine the expression levels of BBOX1-AS1 and miR-19b. Poly (A) addition was performed before the detection of miR-19 expression. In cases of the failure of GAPDH amplification, cDNA preparation and/or RNA preparation were repeated. PCR reactions were carried out on a Bio-Rad CFX96 qPCR Real-Time PCR Module (Bio-Rad). The 2−ΔΔCT method was used for normalization of Ct values. Primer sequences were: 5’-TGTGTGTTTCCTGAGGCCTC-3’ (forward) and 5’-CGCCTCTCTTGGAACACCTT-3’ (reverse) for BBOX1-AS1; 5’-GTAACCCGTTGAACCCCATT-3’ (forward) and 5’-CCATCCAATCGGTAGTAGCG-3’ (reverse) for 18S rRNA; 5’-TGTGCAAATCCATGCAAAAC-3’ (forward) and Oligo d (T) for miR-19b.

RNA-RNA pull-down

RNA-RNA pull-down was performed as previously described [20]. Briefly, Biotin-labeled RNAs, including NC (Bio-NC), BBOX1-AS1 wild type (Bio-BBOX1-AS1-wt), and BBOX1-AS1 mutant (Bio-BBOX1-AS1-mut) were prepared by performing in vitro transcriptions with T7 in vitro transcriptase, followed by biotin labeling. After transfecting the cells with labeled RNAs, cell culture was carried out for 48 h, then cells were collected, and cell lysis was performed for 30 min on ice. RNA pulldown was performed by incubating cell lysates with streptavidin agarose resin beads. After the collection of beads, RNA complexes were purified and miR-19b accumulation was determined by performing RT-PCR.

Subcellular fractionation assay

Nuclear and cytoplasmic fractions were prepared from KGN cells using the NE-PER Nucleus and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). All operations were completed following the manufacturers’ instructions. Briefly, cells were used to prepare cell lysate, and cytoplasmic fraction was isolated through centrifugation. Nuclear fraction, which was the pellet, was subjected to nucleus lysis. Two fractions were then used to isolate RNA, which was subjected to RT-PCR to determine the expression of BBOX1-AS1.

BrdU assay

BrdU assay was performed as previously described [21]. Briefly, cells were collected, washed with PBS, and counted. Subsequently, cell culture was carried out in a 24-well plate, and BrdU (10 µg/mL) was introduced 46 h after initiating the cell culture. Following a 2-h incubation period, a peroxidase-coupled BrdU antibody was added and incubated for an additional 2 h. Then, tetramethylbenzidine, the substrate of the antibody was added, followed by incubation for 30 min. Finally, OD values at 450 nm were measured to determine cell proliferation.

Statistical analyses

Statistical power was calculated using GraphPad Prism 6 software and a statistical power > 0.85 was achieved in all cases. Student’s t-test was used to explore the differences between the 2 groups. Multiple groups were compared by ANOVA Tukey’s test. Data comparisons and image preparations were performed with SPSS software 21 (SPSS Inc). A p-value smaller than 0.05 indicated a difference with statistical significance.

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