The rat ovary granulosa cells used in the present study were primary cells prepared by Procell Life Science & Technology Co., Ltd. (cat. no. CP-R050) via mechanical separation, collagenase digestion and differential adhesion. The cells were cultured in DMEM/F12 (HyClone; GE Healthcare Life Sciences) with 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), epidermal growth factor (10 ng/ml; cat. no. E5036; Sigma-Aldrich; Merck KGaA), and penicillin and streptomycin (100 U/ml) at 37°C and 5% CO₂. miR-28-5p mimics (50 nM), inhibitor (50 nM) and NC (50 nM) were synthesized by Shanghai GenePharma Co., Ltd., and pcDNA3.0-PROK1 plasmids (4 µg) were transfected into cells using Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according the manufacturer's protocols. Briefly, cells (1.5×10⁵ cells/well) were seeded in six-well plates containing complete medium without antibiotics for 24 h prior to transfection. Cell transfection was conducted in six-well plates, in which each well contains a total of 2 ml medium with 5 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and 10 µl mimics. Assays were performed 24 h following cell transfection. Empty pcDNA3.0 vector was used as a negative control (NC) for PROK1 overexpression. Nonsense sequences were used as the NC for miR-28-5p mimics and inhibitors. The sequences of mimics, inhibitor and NC are the as follows: miR-28-5p mimics, 5′-AAGGAGCTCACAGTCTATTGAG-3′; miR-28-5p inhibitor 5′-UUCCUCGAGUGUCAGAUAACUC-3′; NC, 5′-UUCUCCGAACGUGUCACGUTT-3′.

Female Sprague Dawley rats (age, 21 days; weight, 50±10 g; n=12) had free access to food and water under a controlled temperature of 23±2°C and humidity (55–65%) with a 12-h light/dark cycle. A PCOS model was established as previously described (22).

Total RNA was extracted using TRIzol reagent (Takara Bio, Inc.) from ovary granulosa cells (cat. no. CP-R050; Procell Life Science & Technology Co., Ltd.) according to the manufacturer's protocols. Reverse transcription reaction was performed using a Bester™ qPCR RT Kit (cat. no. DBI-2220; DBI Bioscience). The reverse transcription protocol was as follows: RNA denaturation at 65°C for 5 min; genomic DNA removal at 37°C for 5 min; reverse transcription at 37°C for 15 min; reverse transcriptase inactivation at 98°C for 5 min. The vector pcDNA3.0 (Guangzhou Vipotion Biotechnology, Co., Ltd.) was used for the overexpression of PROK1. The coding sequence of PROK1 was cloned using the following primers: Forward 5′-CGGGATCCATGAGAGGTGCTGTGCAAGTCT-3′ and reverse, 5′-CCGCTCGAGCTAAAAGTTGACATTCTTCAAGTCC-3′. The PCR mixture contained: 0,25 µl PrimeSTARRHS DNA Polymerase (Clontech Laboratories, Inc.), 5 µl 5X PrimeSTAR buffer (Clontech Laboratories, Inc.), 2 µl dNTP Mix (Clontech Laboratories, Inc.); 0.5 µl primers (Sangon Biotech Co., Ltd.), 1 µl cDNA and 15.75 µl ddH2O, (Takara Bio, Inc.). The thermocycling conditions as follows: Initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec; 58°C for 30 sec; 72°C for 30 sec; 72°C 5 min. Then, amplification products were ligated into the vector using BamHI and XhoI restriction sites and identified via double enzyme digestion. Empty vector was used as a NC. Animal experiments were performed in compliance with the ARRIVE guidelines (23), once ethical approval from the Nanfang Hospital Animal Ethics Committee had been obtained.

Total RNAs were extracted using TRIzol^® reagent (Takara Bio, Inc.) according to the manufacturer's protocols. Total RNA (~400 ng) was reverse transcribed into cDNA using a Bestar qPCR RT kit (cat. no. 2200; Kay Biological Technology Co., Ltd.). Samples were incubated at 37°C for 15 min and at 85°C for 5 min. U6 small nuclear RNA was used as an internal reference for miR-28-5p expression; primers were obtained from Takara Bio, Inc. GAPDH was used as internal reference; primers were synthesized by Sangon Biotech Co., Ltd. A SYBR Green qPCR kit (Kay Biological Technology Co., Ltd.) was used to quantify miRNA or mRNA expression using a Stratagene Real time PCR system (Mx3000P; Agilent Technologies, Inc.). The amplification reactions were performed under the following conditions: Initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec, 58°C for 20 sec, and 72°C for 30 sec. All qPCR reactions were repeated at least three times. Relative mRNA or miRNA expressions were calculated by 2^−ΔΔCq method (24). The primers are listed in Table I.

Cells (~10⁶) were lysed using protein lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and total protein was then extracted. Subsequently, a Pierce BCA Protein Assay kit (cat. no. 23227; Pierce; Thermo Fisher Scientific, Inc.) was used to determine the total protein concentration. Equal amounts of total protein (10 µg) were separated by 12% SDS-PAGE. The PVDF membrane was placed in a 5% BSA blocking solution for 1.5 h at room temperature. The membranes were incubated at 4°C for 12 h with primary antibodies against PROK1 (1:500; cat. no. ab72807; Abcam), Bcl-2 (1:1,000; cat. no. ab196495; Abcam), Bax (1:1,000; cat. no. ab32503; Abcam), caspase-3 (1:500; cat. no. ab13847; Abcam), p62 (1:1,000; cat. no. 16177; Cell Signaling Technology), cyclin D1 (1:200; cat. no. ab16663; Abcam), PI3K (1:1,000; cat. no. 4255S; Cell Signaling Technology), phosphorylated (p-)PI3K (1:1,000; cat. no. 4228T; Cell Signaling Technology), AKT (1:1,000; cat. no. 4691S; Cell Signaling Technology), p-AKT (1:2000; cat. no. 4060S; Cell Signaling Technology), mTOR (1:1,000; cat. no. 2983S; Cell Signaling Technology), p-mTOR (1:1,000; cat. no. 5536S; Cell Signaling Technology) and GAPDH (1:1,000; cat. no. ab8245; Abcam). The PVDF membrane was placed in a diluted horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G secondary antibody (1:10,000; cat. no. ab6728; Abcam) solution and incubated for 1 h at room temperature. The intensities of labeled proteins were visualized using X-film (cat. no. 4741023951; Yestar Healthcare Holdings Co., Ltd.; http://www.yestarcorp.com/) with an ECL chemiluminescence detection kit (BeyoECL Plus; cat. no. P0018M; Beyotime Institute of Biotechnology) and ImageJ (version 1.8.0; National Institutes of Health) was used to quantify expression by densitometry.

Cells (2×10⁴) transfected with exogenous sequences were suspended in 200 µl culture medium and seeded into 96-well plates. The CCK-8 assay was conducted following culture under standard conditions (37°C) for a further 48 h. Then, 10 µl CCK-8 reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, followed by incubation for a further 1 h at 37°C. Finally, the absorbance at 450 nm was detected for quantification of cell proliferation rates using a plate reader (BioTek Instruments, Inc.).

Cells (10⁴) transfected with exogenous sequences were plated into 96-well plates and cultured for 48 h. Then, 100 µl EdU reagent (cat. no. B23151; Invitrogen; Thermo Fisher Scientific, Inc.) was added to each well after washing with PBS. The cells were incubated at 37°C for 2 h. 100 µl glycerol was added to each well for neutralization after cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Subsequently, 0.5% Triton X-100 was added into each well, and cells were incubated for 10 min at room temperature, followed by washing with PBS. Apollo reagents and Hoechst 33342 staining reagent included in an EdU kit (cat. no. CA1170; Beijing Solarbio Science & Technology Co., Ltd.) were used according to the manufacturer protocols. Then, the cells were incubated for 30 min at room temperature. Cells were analyzed via flow cytometry and the software used for the analysis was Cytomics FC500 Flow Cytometry CXP Software v2.0 (CytoFLEX; Beckman Coulter, Inc.).

Following transfection, 10⁴ cells were cultured for 48 h, and the apoptosis of cells was then analyzed using the Annexin V-FITC/propidium iodide (PI) cell apoptosis detection kit (BD Biosciences) containing according to the manufacturer's protocols, and cells were incubated at 4°C for 30 min. Cells with positive signals for Annexin V-FITC and PI were in the late stage of apoptosis, whereas cells with positive signals for Annexin V-FITC only were in the early apoptosis stage. The total apoptosis ratio was defined by the summation of late-apoptotic and early-apoptotic cells.

Following transfection of cells with exogenous sequences, cells were stained with PI dye was incubated at 4°C for 30 min to determine the cell cycle distribution. A flow cytometer was used to analyze the cells and the software used for the analysis was Cytomics FC500 Flow Cytometry CXP Software v2.0 (Beckman Coulter, Inc.).

TargetScan 7.2 (http://www.targetscan.org) was used to identify the putative targets of miR-28-5p, and PROK1 was found to be one of the targets of miR-28-5p. The software was used according to previous report (25). The 3′-UTR sequences of the PROK1 gene were obtained using primers (forward, 5′-CCGCTCGAGTGGGTGACCTCTTGTGTTACATCTG-3′ and reverse, 5′-ATTTGCGGCCGCCCCAGCCCACCTCACACTCATA-3′; Sangon Biotech Co., Ltd.). Mutated (MUT) 3′-UTR sequences were amplified using paired primers with a mismatched nucleotide introduced in the upstream primer (MUT; forward, 5′-TTTCTTTCATTGGCTCCGATGATGTTTTAGGAGTAAG-3′ and reverse, 5′-CTTACTCCTAAAACATCATCGGAGCCAATGAAAGAAA-3′; Sangon Biotech Co., Ltd.). Amplification products including the wild-type (WT) and MUT 3′-UTRs of PROK1 were then separately ligated into Psi-CHECK2 plasmids (cat. no. C8021; Promega Corporation). Psi-CHECK2 plasmids were co-transfected with miR-28-5p mimics or NC into cells using Lipofectamine 2000™ and incubated for 48 h at 37°C. The luciferase activities of firefly and Renilla were measured with the Dual-Luciferase Reporter Assay system (cat. no. C8021; Promega Corporation) according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity. Cell lysates were collected using a GloMax machine (Promega Corporation).

Data from >3 biological replicates for each assay were presented as the mean ± standard deviation, and data were analyzed using SPSS 16.0 software (SPSS, Inc.). Student's t-test was used to analyze differences between two groups, and one-way analysis of variance followed by Tukey's test was performed to compare differences between three or more groups.

To investigate the relationship between miR-28-5p and PROK1, the mRNA expression levels of PROK1 were analyzed by qPCR, and the protein expression was evaluated via western blotting following transfection of ovary granule cells with miR-28-5p mimics. It was demonstrated that mRNA expression of PROK1 was significantly suppressed in miR-28-5p overexpressing cells compared with the NC (Fig. 1A). Similarly, the protein expression of PROK1 was significantly downregulated following the transfection of ovary granule cells with miR-28-5p mimics compared with the NC (Fig. 1B). Conversely, transfection with miR-28-5p inhibitor induced the opposite results in PROK1 expression in ovary granule cells (Fig. 1A and B).

To determine whether miR-28-5p binds to PROK1, the 3′-UTR of PROK1 was cloned and inserted into psi-CHECK2 plasmids. For comparison, the binding site predicted by the online database TargetScan was mutated, and then also inserted into psi-CHECK2 plasmids (Fig. 1C). Cells co-transfected with WT 3′-UTR and miR-28-5p exhibited significantly reduced luciferase activity compared with cells transfected with NC or MUT 3′-UTR (Fig. 1D). The results indicated that miR-28-5p negatively regulated PROK1 expression via direct binding to its 3′-UTR.

PCOS is a common endocrine-reproductive disease in females and it has been demonstrated that ovary granular cells in PCOS patients showed increased apoptotic rates (17,26). To verify whether PROK1 promotes PCOS in rat ovary granule cells, a PROK1 overexpression plasmid was constructed. Additionally, a rescue experiment series was designed to further demonstrate the influence of the interaction between miR-28-5p and PROK1 on rat ovary granule cell survival. As presented in Fig. 2A, a western blot assay verified the overexpression of the PROK1 plasmid, revealing that PROK1 was significantly upregulated in cells transfected with the PROK1 overexpression plasmid compared with empty vector. The overexpression of PROK1 protein was accompanied by significantly increased cell proliferation rates in rat ovary granule cells (Fig. 2B and C); however, the effects of PROK1 upregulation on proliferation were attenuated by co-expression of PROK1 and miR-28-5p mimics, compared with cells transfected with pcDNA-PROK1 alone.

Additionally, apoptosis was significantly suppressed in rat ovary granule cells overexpressing PROK1 compared with empty vector (Fig. 3A and B), and a significantly increased percentage of these cells were in S phase (Fig. 3C and D). These tendencies were attenuated by co-transfection with miR-28-5p mimics, which elevated cell apoptosis, and redistributed cells to the G0/G1 phase compared with pcDNA-PROK1 alone. The expression of apoptosis- and cell cycle-associated proteins was investigated via western blotting (Fig. 3E). Compared with empty pcDNA3.0 vector, transfection with pcDNA3-PROK1 significantly upregulated Bcl-2 and cyclin D1 expression, whereas caspase-3, Bax and p62 expression levels were downregulated; however, co-transfection with miR-28-5p mimics attenuated these effects. Additionally, it was revealed via western blotting that PI3K/AKT/mTOR activation was also potentiated in PROK1-overexpressing cells compared with the control (Fig. 3F). Conversely, this activation was reversed by co-transfection with miR-28-5p mimics. These results indicated that PROK1 promoted the proliferation and suppressed the apoptosis of rat ovary granule cells via the PI3K/AKT/mTOR signaling pathway.