Background
Polycystic ovary syndrome (PCOS) is a common endocrinopathy in women of reproductive age [
1] and accounts for approximately 75% of anovulatory infertility disorders [
2]. The phenotype of PCOS is variable and includes hyperandrogenism, menstrual irregularity and polycystic ovarian morphology [
3]. Patients suffering from PCOS are often diagnosed with obesity, hirsutism, insulin resistance, increased risk of endometrial cancer, metabolic syndrome [
4], type 2 diabetes (T2D) and cardiovascular diseases [
5,
6]. Although the aetiology of PCOS remains unclear, most researchers regard PCOS as multifactorial and suggest that genetic factors play a pivotal role in its development and maintenance [
7,
8]. Many studies have reported gene expression profiles based on tissues (i.e. theca cells [
9], ovaries [
10,
11], oocytes [
12] and cumulus cells [
13]) from controls and patients with PCOS. Genes associated with PCOS are involved in the insulin receptor signalling pathway, steroid biosynthesis and regulation of gonadotropin secretion [
14]. However, the mechanism by which these genes are regulated has not been thoroughly elucidated.
Long noncoding RNAs (lncRNAs) are defined as noncoding RNAs with greater than 200 base pairs [
15]. LncRNAs were previously regarded as transcriptional ‘noise’ without biological functions [
16]; however, increasing lines of evidence indicate that lncRNAs play key roles in normal development and diseases [
17]. To date, several reports have demonstrated that lncRNAs may function in PCOS-related diseases, including T2D, obesity and cardiac diseases. For instance, a β-cell-specific lncRNA (
HI-LNC25) is dysregulated in T2D by down-regulating the mRNA expression levels of
GLIS3 (Kruppel-like zinc finger transcription factor) [
18].
In our previous research [
19], we used microarrays [Agilent human lncRNA+mRNA Array v2.0 (4 × 180 K format)] to describe lncRNA profiles in cumulus cells isolated from 10 patients (five patients with PCOS and five normal women). A total of 623 lncRNAs were differentially expressed in PCOS and may contribute to its occurrence [
19].Among these lncRNAs, Prader-Willi region nonprotein coding RNA 2 (
PWRN2) (transcript ID: ENST00000567246.1), which is expressed in the testes and is up-regulated after meiosis during spermatogenesis [
20], was found to be up-regulated (3.11-fold) in the cumulus cells of patients with PCOS. Oocyte nuclear maturation has two meiosis resumption processes at the MI (the first meiosis resumption) and MII (the second meiosis resumption) stages. Hence,
PWRN2 may be associated with oocyte nuclear maturation in PCOS. In addition, abnormal folliculogenesis is regarded as a common characteristic of PCOS although its clinical and biochemical signs are typically heterogeneous [
21,
22]. Thus, studying the abnormal regulatory mechanisms in oocyte development of PCOS is important.
Increasing lines of evidence suggest that lncRNAs function as miRNA sponges or competing endogenous RNAs (ceRNAs) to reduce the availability of miRNAs for mRNA target binding [
23,
24]. In the present study, we confirmed the potential roles of
PWRN2 in oocyte nuclear maturation of PCOS. We then constructed a
PWRN2- mediated ceRNA network by analysing three microarray datasets (lncRNA+mRNA microarray in PCOS cumulus cells [
19], miRNAs microarray in PCOS cumulus cells [
25] and lncRNA+mRNA microarray in KGN/shPWRN2 in this study) to investigate the mechanism of
PWRN2. Results revealed the potential roles of ceRNA in oocyte maturation in PCOS. This work highlights a novel mechanism of oocyte nuclear maturation in PCOS and provides new targets for PCOS treatment.
Methods
Patients and IVF treatment
The inclusion criteria for the recruited patients (PCOS and normal) and the methods for collecting CCs were based on our previous reports [
19,
26]. This study was approved by the Institutional Ethical Review Board of Tongji University School of Medicine. Sixty participants (30 patients with PCOS and 30 normal) who were referred to our centre for IVF were included in this study after obtaining written informed consent. All patients had no history of taking drugs that affect glucose and lipid metabolism and did not have any known medical conditions or diseases, such as Cushing’s syndrome, congenital adrenal hyperplasia, androgen-secreting tumours and endometriosis. Patients with PCOS were diagnosed according to the revised Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine Criteria [
3]. The patients were required to present at least two of the following criteria: chronic oligo-ovulation or anovulation, androgen excess and polycystic ovaries. The inclusion criteria for the recruited patients in this study were as follows: age < 36 years, BMI ranging between 20 and 26 kg/m
2, basal serum LH/FSH more than 2.0, serum testosterone more than 0.5 ng/mL, antral follicle count ranging between 18 and 35 and number of obtained oocytes ranging between 12 and 28 per cycle. Control patients had regular menstrual cycles, normal ovary sonographs and normal ovulation with bilateral tube occlusion, were nondiabetic and showed no clinical signs of hyperandrogenism and anovulation. The clinical characteristics of control patients and those with PCOS are summarised in Table
1.
Table 1
Clinical characteristics of patients
Age (years) | 33.6 ± 2.2 | 32.6 ± 3.1 | NS |
BMI (kg/m2) | 21.4 ± 1.6 | 21.6 ± 1.5 | NS |
FSH (mIU/ml) | 6.51 ± 1.2 | 5.41 ± 1.3 | < 0.05 |
LH (mIU/ml) | 4.30 ± 1.4 | 11.91 ± 2.6 | < 0.001 |
Basal LH/FSH | 0.65 ± 0.2 | 2.52 ± 0.61 | < 0.001 |
E2 (pg/ml) | 38.5 ± 4.3 | 41.3 ± 9.5 | NS |
Testosterone (ng/ml) | 0.12 ± 0.05 | 0.68 ± 0.05 | < 0.001 |
Progesterone (ng/ml) | 0.55 ± 0.22 | 0.70 ± 0.23 | NS |
Antral follicle count | 10.2 ± 1.1 | 24.1 ± 3.7 | < 0.001 |
Oocytes obtained | 8.5 ± 3.0 | 17.8 ± 5.2 | < 0.001 |
No of MII oocytes | 6.3 ± 1.8 | 14.5 ± 4.6 | < 0.001 |
Patients in both groups received an agonist protocol as described previously [
27]. All patients received the GnRH agonist triptorelin acetate (0.05 mg/day, Diphereline; Ipsen Pharma Biotech, Paris, France) subcutaneously starting at the mid-luteal phase. Once adequate pituitary down-regulation was confirmed [serum LH levels < 3.0 ng/mL and serum estradiol (E
2) levels < 30 pg/mL], the patients received recombinant FSH (150–187.5 IU; Gonal-f, Follitropin Alfa, Serono) subcutaneously for COS. When two or more follicles were at least 18 mm in diameter and the serum E
2 levels were at least 300 pg/mL per dominant follicle, all patients received 250 μg of hCG (Profasi, Serono).
Retrieval of cumulus cells
Collection of CCs and assessment of oocytes were conducted as previously described [
27,
28]. Cumulus-oocyte complex (COC) retrieval was performed by vaginal puncture under ultrasound echo-guidance 36 h after hCG administration. After COC retrieval, a portion of CCs surrounding a single oocyte was removed using a sharp needle. For RNA extraction, the cumulus cells were lysed in 80 μL of lysis buffer (mirVana miRNA Isolation Kit; Ambion, Austin, TX, USA) and stored at − 80 °C. For vector transfection and luciferase activity assay, the cumulus cells were firstly digested with trypsin and then cultured directly. Oocytes were further inseminated by ICSI and cultured in sequential media of SAGE (CooperSurgical, Leisegang Medical, Berlin) individually in 20 μL of droplets covered with mineral oil. The embryos were transferred or vitrified on day 3, and the other embryos were cultured to blastula stage on days 5–6.
Assessment of oocyte and division of the groups of cumulus cells
The morphological characteristics of the oocytes were individually recorded. The oocytes were denudated to assess the maturation stage before ICSI. Few of germinal vesicle (GV)-stage COCs (12 in patients with PCOS and only 3 in normal patients) were retrieved. We classified the COCs into two categories based on nuclear status: (i) MI/GV group: immature MI oocytes exhibiting no polar bodies (PB) or immature oocytes at the GV stage, and (ii) MII group: mature MII oocytes that extruded a clearly visible PB. The corresponding cumulus cells were divided into CCMI/GV and CCMII groups. Each group had ≥ three replicates. Each subgroup, containing at least 15 cumulus cells, represented a biological replicate. Each CCMI/GV subgroup has one CCGV.
Total RNA was isolated using a Qiagen RNeasy Mini Kit (Qiagen, Hilder, Germany) according to the manufacturer’s instructions. This RNA isolation kit significantly reduced contamination from genomic DNA and proteins. The purity and concentration of RNA were determined from OD260/280 readings using a spectrophotometer (NanoDrop ND-1000). RNA integrity was determined using 1% formaldehyde denaturing gel electrophoresis.
qRT-PCR
The expression levels of PWRN2 in the CCMI/GV and CCMII groups of normal patients and those with PCOS were tested by qRT-PCR analysis to evaluate the correlation of changes in PWRN2 with oocyte maturation. The potential ceRNA network was constructed to investigate the action mechanism of PWRN2.The co-expression characteristics of the candidate genes of the ceRNA network were also tested in the CCs corresponding to oocytes at different nuclear maturity stages (MI/GV and MII) of patients with PCOS through qRT-PCR analysis.
Total RNA was reverse transcribed into cDNA by using a miScript Reverse Transcription Kit (Qiagen). qRT-PCR analysis was performed using SYBR green assay (Takara Bio, Inc., Dalian, China) according to the manufacturer’s protocols. PCR was performed in a total reaction volume of 20 μL containing 10 μL of 2× QuantiTest SYBR Green PCR Master Mix, 1 μL of cDNA template, 1 μL of each primer and RNase-free water. The primers used in this study are listed in Additional file
1: Table S1. All reactions were performed using the ABI PRISM 7300 system. The amplification conditions were as follows: 10 min at 98 °C; 40 cycles of 15 s at 95 °C, 1 min at 60 °C; and a final extension for 5 min at 72 °C. Amplification efficiency was evaluated by standard curve analysis.
PWRN2 and mRNA expression data were normalised to those of
GAPDH. The miRNA expression data were normalised to U6. Each set of qRT-PCR reactions was repeated at least three times, and fold change in the expression of each gene was analysed by the ΔΔCt method [
29].
KGN cell culture
PWRN2-regulated genes were identified using RNA interference technology to inhibit the expression of PWRN2 in KGN cell lines and eliminate differences in the genetic backgrounds of different patients with PCOS. KGN (RCB1154; RIKEN, Wako, Japan) is a steroidogenic human ovarian granulosa tumour cell line. KGN cells were cultured in 1:1 Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/ F12; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% foetal bovine serum (FBS) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin). On the day before lentivirus transfection, KGN cells were placed into the medium without serum and incubated overnight.
Construction of lentivirus shRNA and cell transfections
Lentivirus shRNA construction and cell transfection were conducted using previously described methods [
30,
31]. We selected three target sequences to construct lentiviral shRNAs (LV-
PWRN2-homo-502, LV-
PWRN2-homo-1574 and LV-
PWRN2-homo-1261) and included a negative control (LV-NC) (Table
2). The target sequences were used to design two complementary oligonucleotides, which were synthesised and cloned into pGLV3/H1/GFP + Puro Vector (GenePharma, China). The positive purified lentiviral shRNA-expressing plasmids were transfected with packaging plasmids into 293 T cells for lentivirus generation (GenePharma, China). The vectors described above were used to infect KGN cells. Stable KGN cell lines were selected using 3 μg/mL bulk puromycin-resistance culture (puromycin, Sigma, St Louis, MO, USA) for 5 days. Afterwards, the cells were examined microscopically for lentiviral GFP expression. The expression levels of
PWRN2 in KGN/sh
PWRN2 cells and the corresponding negative-control KGN cells were tested by qRT-PCR to validate the effects of RNA interference.
Table 2
Target sequences of lentiviral shRNAs for interfering PWRN2
PWRN2-homo-502 | 502–522 | 5’-GCCATTCGGTTACCATCTACT-3’ |
PWRN2-homo-1574 | 1574–1594 | 5’-GCAAAGGAATTACCGTTTACA-3’ |
PWRN2-homo-1261 | 1261–1281 | 5’-GGCAGAAAGCAATGAAGAAGA-3’ |
NC | Nonsense | 5’-TTCTCCGAACGTGTCACGT-3’ |
Microarray hybridisation and data analysis
For microarray analysis, three KGN/shPWRN2 cell lines with down-regulated (FC < 0.5)
PWRN2 mRNA levels were selected as shPWRN2 groups. The corresponding three KGN cell lines with negative control vectors were used as the control groups. The methods for RNA labelling, array hybridisation and data analysis were described in our previous report [
19]. The purified RNA extracted from KGN/sh
PWRN2 samples or normal KGN cells was amplified and transcribed into fluorescent cDNA for hybridisation to the Agilent human lncRNA+ mRNA Array v4.0 (4 × 180 K format) with each array containing probes interrogating approximately 41,000 human lncRNAs and approximately 34,000 human mRNAs. The lncRNA+mRNA array data were analysed to summarise, normalise and assess the quality of the data using GeneSpring software V11.5 (Agilent). To select differentially expressed genes, we used threshold values of ≥2.0- and ≤ − 2.0-fold changes and a Benjamini–Hochberg corrected
P value of 0.05. The data were log2 transformed and median centred by genes using the Adjust Data function of the CLUSTER 3.0 software. The data were further analysed using hierarchical clustering with average linkages. Finally, we visualised the tree using Java TreeView (Stanford University School of Medicine, Stanford, CA, USA). Microarray hybridisation and data analysis were performed by CapitalBio Corporation, Beijing, P. R. China.
Gene ontology (GO) and KEGG pathway analyses
GO analysis was performed to describe genes and gene product attributes in any organism (
http://www.geneontology.org). This ontology covers three domains: biological processes, cellular components and molecular functions. The
P value denotes the significance of the GO term enrichment among differentially expressed genes (P value ≤0.05 is recommended). For pathway analysis, we used the free web-based Molecular Annotation System 3.0 (MAS 3.0;
http://bioinfo.capitalbio.com/mas3/), which integrates three different open-source pathway resources: KEGG, BioCarta and GenMAPP. The significantly altered pathways were selected using the threshold of the P value and FDR (corrected P value) < 0.05 derived from the hypergenomic test. GO and KEGG pathway analyses were performed by CapitalBio Corporation, Beijing, P. R. China.
Construction of the PWRN2-mediated ceRNA network based on microarray data
A potential
PWRN2-mediated ceRNA network was constructed based on three microarray datasets to explain whether
PWRN2 functions as miRNA sponges or ceRNAs. The putative miRNAs and mRNAs included in the construction of the
PWRN2-mediated ceRNA network are as follows: (i) miRNA selection: miRNAs were predicted to possess
PWRN2 binding sites by using miRanda v3.3a software and were compared with previous miRNA microarray data of PCOS; putative miRNAs with MREs in
PWRN2 and identified from previous microarray data of PCOS were selected; and (ii) mRNA selection: the target genes of the miRNAs were predicted using miRbase (
http://www.mirbase.org/) and compared with the lncRNA+mRNA microarray data from KGN cells with
PWRN interference. The selected mRNAs met two criteria: 1) differentially expressed mRNAs identified from our lncRNA+mRNA microarray data; and 2) putative target genes of miRNAs with MREs in
PWRN2.
Luciferase reporter constructs and luciferase activity assay
The direct interaction between the candidate genes of
PWRN2-mediated ceRNA network was evaluated by luciferase activity assay. A luciferase reporter vector (pmirGLO Dual-Luciferase miRNA Target Expression Vector; Promega) was used for luciferase constructs.
PWRN2 and the 3’UTR of
TMEM120B were cloned by RT-PCR.
PWRN2-WT,
PWRN2-mutant and
TMEM120B- 3′UTR (WT and mutant) were constructed as previously reported [
26,
32]. Cumulus cells of patients with PCOS were digested with 0.25% trypsin for 5 min. The digestion was inhibited by adding 2.5 mL of DMEM: F12 containing 10% FBS and by incubating at room temperature for 5–10 min. The cumulus cells were pooled in a sterile 15 mL conical tube on ice, centrifuged at 200 g for 10 min, washed once with sterile saline, centrifuged again and resuspended in 3 mL of culture media. The number of viable cells was determined by trypan blue exclusion and ranged between 35 and 45%. The cumulus cells were plated onto 24-well plates and allowed to grow for ~ 24 h before transfection. The constructed reporter vectors (300 ng) were transfected into cells together with the miRNA and control mimics (100 nM) in Lipofectamine 2000 (2 μL). The cells were lysed after ~ 24 h of transfection. Luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activities were normalised to Renilla luciferase activities. The experiments were performed independently in triplicate.
Statistical analysis
All statistical analyses were performed using SPSS 17.0 software (SPSS Inc.) unless otherwise noted. Differences in the expression levels of the candidate genes of the PWRN2–mediated ceRNA network in cumulus cell samples were evaluated by two-tailed t-test. Differences were considered statistically significant at P < 0.05.
Discussion
PWRN2 was up-regulated in PCOS cumulus cells and could be involved in oocyte development [
19]. In the present study, we proved that
PWRN2 is associated with oocyte nuclear maturation in patients with PCOS, in contrast to that in normal patients. Hence,
PWRN2 plays important roles during the oocyte development in PCOS.
To elucidate the roles of
PWRN2 in oocyte development, we identified
PWRN2-regulated genes in KGN cells by using RNA interference technology. A total of 176 lncRNAs and 131 mRNAs were identified and determined to be regulated by
PWRN2.The GO and KEGG pathway analyses of these DEGs showed that the differentially expressed genes were mostly involved in various catabolic processes (e.g. nuclear-transcribed mRNA and RNA catabolic processes) and metabolism pathways (e.g. carbon and nitrogen metabolism). These results indicated that
PWRN2 may play an important role in metabolic processes. Metabolic abnormalities in cumulus cells in PCOS change the follicular microenvironment during oocyte development and subsequently affect oocyte quality [
35,
36]. This deduction is consistent with PCOS because it is a metabolic disease often accompanied by poor oocyte developmental potential.
Although potential target genes of
PWRN2 were identified, the
molecular mechanism of
PWRN2 remains largely unknown. Several recent reports provided support for the ceRNA hypothesis, which accounts for the function of a substantial proportion of uncharacterised lncRNAs [
37‐
40]. To examine if
PWRN2 functions as a ceRNA, we developed a new method for elucidating specific lncRNA-mediated ceRNA networks. The most widely used methods for exploring potential lncRNA-miRNA-mRNA networks largely depend on shared MREs that are predicted by miRNA target discovery algorithms [
41,
42]. We predicted miRNAs that possess
PWRN2 binding sites and potential miRNA-mRNA pairs. We then obtained
PWRN2-mediated miRNAs by combining the analyses of predicted miRNAs and miRNA microarray data from previous report [
25]. Meanwhile,
PWRN2-regulated mRNAs were obtained by comparing potential miRNA-target mRNA with the microarray data from KGN/sh
PWRN2 in the present study. Finally, we incorporated a bioinformatic prediction tool to construct a
PWRN2-miRNA-mRNA ceRNA network. Based on the dual-luciferase activity assay, the ceRNA network is reliable because it is based on datasets from three microarrays.
Furthermore, we investigated
PWRN2 and miR-92b-
TMEM120B pair and validated their expression levels in cumulus cells according to oocyte nuclear maturity (CC
MI/GV and CC
MII) of patients with PCOS. The results were in accordance with the ceRNA hypothesis. In this regard,
PWRN2 functions as ceRNA to reduce the availability of miR-92b-3p for
TMEM120B target binding.
TMEM120B is a fat-specific nuclear envelope transmembrane protein that may play a contributory role in adipogenesis [
43]. In PCOS, up-regulated
TMEM120B will promote adipocyte differentiation/metabolism and induce obesity. Severe obesity has been shown to be associated with a high prevalence of spindle anomalies and non-aligned chromosomes in failed fertilised oocytes [
44]. Thus, the ceRNA network of
PWRN2-miR-92b-
TMEM120B provides a basis for explaining the poor quality of oocyte in patients with PCOS.
Our study presents limitations. Firstly, we cannot identify large lncRNA-miRNA-mRNA ceRNA networks by combining microarray datasets. Moreover, the molecular roles of the PWRN2-miR-92b-TMEM120B network in oocyte development in PCOS require further investigation. Secondly, a large number of samples must be analysed to validate the ceRNA network in PCOS. Our future work will further validate the PWRN2-miR-92b-TMEM120B network and elucidate its role in the pathogenesis of PCOS, especially in oocyte development.
In conclusion, our results proved that lncRNA (PWRN2) is associated with oocyte nuclear maturation in PCOS. The constructed PWRN2-miR-92b-TMEM120B ceRNA network based on three microarray datasets indicated that PWRN2 functions as ceRNA to reduce the availability of miR-92b-3p for TMEM120B target binding during oocyte nuclear maturation in PCOS. This ceRNA network provides new information and helps clarify the metabolic disorder that induces abnormal oocyte development in PCOS.