Polycystic Ovary Syndrome Granulosa Cells Research Articles

Circular RNA circ_RANBP9 exacerbates polycystic ovary syndrome via microRNA-136-5p/XIAP axis

ABSTRACT Polycystic ovary syndrome (PCOS) is an endocrine disease that affects the health of many women. Circular RNAs (circRNAs) are associated with the occurrence and progression of PCOS. This study aimed to explore the function of circ_RANBP9 in PCOS. First, the circ_RANBP9 level was found to be increased in the plasma of patients with PCOS and ovarian granulosa cells (GCs) using Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR). In GCs, loss of circ_RANBP9 decelerated proliferation and accelerated apoptosis of KGN and COV434 cells, as determined by MTT assay, colony formation assay, and flow cytometry. Furthermore, bioinformatics analysis showed that circ_RANBP9 and XIAP can be targeted by the microRNA, miR-136-5p. Luciferase reporter assay and RNA pull-down assay further verified the interaction between miR-136-5p and circ_RANBP9 or XIAP. Importantly, knockdown of circ_RANBP9 suppressed proliferation and promoted apoptosis of KGN and COV434 cells, whereas inhibition of miR-136-5p reversed these effects. Additionally, XIAP abolished the repression of proliferation and acceleration of apoptosis induced by miR-136-5p. The promotion of apoptosis was accompanied by upregulation of caspase-3 and Bax, and downregulation of Bcl-2, as estimated by western blotting. In conclusion, silencing of circ_RANBP9 inhibited GC proliferation and facilitated apoptosis by mediating the miR-136-5p/XIAP pathway. These findings provide a new theoretical basis for screening and treatment of PCOS.

Cangfudaotan Decoction Alleviates Insulin Resistance and Improves Follicular Development in Rats with Polycystic Ovary Syndrome via IGF-1-PI3K/Akt-Bax/Bcl-2 Pathway.

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder prevalent in females of reproductive age; insulin resistance (IR) is the major pathogenic driver. Pharmacology is a basic option for PCOS therapy; traditional Chinese medicine (TCM), as a significant part of complementary and alternative medicine, has a long history in the clinical management of PCOS. Cangfudaotan decoction (CFD) has been used clinically for gynaecological diseases especially PCOS. In this study, first, chemical components in CFD were clarified using UPLC-Q/TOF-MS analysis. Then, an animal model of PCOS was established, granular cells were also isolated from the rats with PCOS, and CFD was administrated at different dosages in PCOS rats and granular cells, to investigate the therapeutic effect and mechanisms of CFD for PCOS treatment. The result showed that CFD treatment is effective in PCOS rats and granulosa cells. CFD was able to improve IR, restore the serum hormone levels, inhibit the inflammatory cytokines in PCOS rat, and alleviate ovary morphological injury and apoptosis in PCOS rats. In granulosa cells of PCOS, the result showed that the cell viability was improved, and cell apoptosis was inhibited after CFD administration. Further experiments suggested that CDF improves IR, follicular development, cell apoptosis, and inflammatory microenvironment, and this was associated to the regulation of IGF-1-PI3K/Akt-Bax/Bcl-2 pathway-mediated gene expression. Given that CFD sufficiently suppresses insulin resistance and improves follicular development in this study, exploring these mechanisms might help to optimize the therapeutic treatment of CFD in PCOS patients.

Identification of Potential Biomarkers of Polycystic Ovary Syndrome via Integrated Bioinformatics Analysis.

Polycystic ovary syndrome (PCOS) is a life-long reproductive, endocrine, and metabolic disorder that affects up to 17% of women of reproductive age. However, the effect of granulosa cells (GCs) transcriptome changes on oocyte capacity and follicular development in patients with PCOS has not been elucidated. This study aims to analyze transcriptome changes in GCs of PCOS from different perspectives and explore potential biomarkers for the diagnosis and treatment of PCOS. The gene expression profiles of GSE34526 and GSE107746 were obtained from the GEO database. Differentially expressed genes (DEGs) and key signaling pathways were identified. Gene Set Enrichment Analysis (GSEA) revealed that Toll-like receptors, NOD-like receptors, and NOTCH signaling pathways were obviously enriched in GCs of PCOS. We further analyzed DEGs from three aspects: transcription factors (TFs), secreted proteins, and follicular development. Compared with normal GCs, the DEGs encoding TFs and secretory proteins in GCs of PCOS remarkably changed. Besides, HAS2 and CBLN1, which are highly expressed in preovulatory follicular GCs and may trigger ovulation, were significantly decreased in GCs of PCOS. This study found candidate genes and signaling pathways in PCOS, providing new insights and foundations for the etiology of PCOS. Besides, HSA2 and CBLN1 may be potential therapeutic biomarkers for ovulation disorders in PCOS.

Protein Lysine Acetylation in Ovarian Granulosa Cells Affects Metabolic Homeostasis and Clinical Presentations of Women With Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is one of the most common reproductive endocrine disorders accompanied by obvious metabolic abnormalities. Lower-quality oocytes and embryos are often found in PCOS women during assisted reproductive technology treatment. However, there is still no clarity about the mechanism of ovarian metabolic disorders and the impact on oocyte maturation in PCOS. The aim of this study was to understand the potential effect of the posttranslational modification on ovarian metabolic homeostasis and oocyte development potential in women with PCOS. A quantitative analysis of acetylated proteomics in ovarian granulosa cells of PCOS and control groups was carried out by mass spectrometry. There was widespread lysine acetylation of proteins, of which 265 proteins had increased levels of acetylation and 68 proteins had decreased levels of acetylation in the PCOS group. Most notably, differentially acetylated proteins were significantly enriched in the metabolic pathways of glycolysis, fatty acid degradation, TCA cycle, tryptophan metabolism, and branched-chain amino acid degradation. Acetyl-CoA acetyltransferase 1 (ACAT1) was an enzyme central to these metabolic pathways with increased acetylation level in the PCOS group, and there was a negative correlation of ACAT1 acetylation levels in PCOS granulosa cells with oocyte quality and embryo development efficiency in the clinic. Lysine acetylation changes of key enzymes in PCOS granulosa cells might attenuate their activities and alter metabolic homeostasis of follicular microenvironment for oocyte maturation and embryo development.

MiR-130b regulates gap junctional intercellular communication through connexin 43 in granulosa cells from patients with polycystic ovary syndrome.

MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression post-transcriptionally. We explored whether connexin 43 (Cx43) was differently expressed in luteinized granulosa cells from women with polycystic ovary syndrome (PCOS) compared with luteinized granulosa cells from women with a normal menstrual cycle, and whether certain miRNAs regulate the Cx43 level and gap junctional intercellular communication (GJIC). The miRNA profile was investigated in ovarian cortex tissues from five women with PCOS and five women without PCOS using a miRNA microarray. The levels of miR-130b and Cx43 mRNA were measured using real-time PCR in human luteinized granulosa cells from 20 women with PCOS and 25 women without PCOS. Protein and mRNA expression analysis and luciferase assays were conducted to confirm the substrate of miR-130b. PCOS ovarian cortex showed differential expression of miRNAs compared with non-PCOS ovarian cortex. Furthermore, miR-130b levels were increased in PCOS ovarian cortex and in luteinized granulosa cells compared with those in women with normal menstrual cycles, whereas the level of Cx43 mRNA, the identified target of miR-130b, was decreased in granulosa cells from patients with PCOS. Overexpression of miR-130b in a granulosa cell line resulted in reduced Cx43 protein levels and inhibited GJIC using scrape loading and dye transfer assay. Meanwhile, inhibition of miR-130b increased the Cx43 level. In conclusion, miR-130b was increased in PCOS granulosa cells, where it targets Cx43 to affect GJIC. The results of the present study suggested that miR-130b, via post-transcriptional regulation of Cx43, is involved in the pathophysiology of PCOS, which provides new insight into the pathological mechanism of PCOS.

Long non-coding RNA TUG1 and its molecular mechanisms in polycystic ovary syndrome

ABSTRACT Polycystic ovary syndrome (PCOS) causes anovulatory infertility in women of reproductive age, but etiopathogenesis of PCOS remains undetermined. Taurine up-regulated 1 (TUG1), an evolutionarily conserved long non-coding RNA, performs various biological functions; however, the role of TUG1 in PCOS remains unclear. Herein, TUG1 expression was assayed in granulosa cells (GCs) of 100 patients with PCOS and 100 control participants. Receiver operating characteristic (ROC) curve analysis was conducted to determine the diagnostic value of TUG1 in PCOS. TUG1 expression was also silenced in KGN cells to explore the role of TUG1 in cellular proliferation, apoptosis, cell-cycle progression, autophagy, and steroidogenesis. We found that TUG1 levels were dramatically increased in the PCOS group compared with those of the control group; this increased expression was related to a rising antral follicle count (R = 0.209, P < 0.001 versus control). The ROC curve indicated a significant separation between PCOS group and the control group (AUC: 0.702; 95% CI: 0.630–0.773; P < 0.001). TUG1 showed a predominantly nuclear localization in human GCs. TUG1 knockdown reduced cellular proliferation, and promoted MAPKs pathway-dependent apoptosis and P21-dependent autophagy, but may not affect cell-cycle progression. TUG1 knockdown increased aromatase expression and oestradiol biosynthesis. Our results indicate that increased TUG1 expression in PCOS GCs may contribute to excessive follicular activation and growth, and may disrupt the selection of dominant follicle. Our study shows that TUG1 can be used as a diagnostic biomarker for PCOS.

Role of Androgen Receptor for Reconsidering the \u201cTrue\u201d Polycystic Ovarian Morphology in PCOS

Purpose: Polycystic ovarian morphology (PCOM) is one of the key features of polycystic ovary syndrome (PCOS). The diagnosis of PCOM according to the Rotterdam criteria (≥12 antral follicles per ovary) is debated because of the high prevalence of PCOM in the general population. Androgen receptor (AR) is associated with the PCOS phenotype and might as well play a role during folliculogenesis. This study is aimed to investigate the expression of the AR in PCOS granulosa cells (GCs) and its relationship with the PCOM phenotype. Methods: 106 PCOS cases and 63 controls were included from the Center for Reproductive Medicine, Shandong University. The diagnosis of PCOS was following the Rotterdam criteria (2003). Total RNA was extracted from GCs retrieved from ovarian stimulation. The expression of AR was amplified by means of quantitative real-time polymerase chain reaction. Results: The AR expression was significantly decreased in PCOS cases, especially in the tPCOM subgroup (≥20 antral follicles per ovary). Correlation analyses showed that AR expression was significantly correlated with serum FSH levels in controls and non-tPCOM. In the tPCOM subgroup, the AR expression was significantly correlated with serum LH levels. Interestingly, the significance of these correlations gradually disappeared as the threshold of antral follicles increased above 24 for PCOM. Conclusions:AR was differently expressed in PCOS and especially in the tPCOM subtype. The correlation of AR expression with serum FSH and LH might be associated with the number of follicles in PCOM.

HMGB1-induced aberrant autophagy contributes to insulin resistance in granulosa cells in PCOS.

Insulin resistance (IR) disrupts ovarian functions in polycystic ovary syndrome (PCOS). The contributing factors remains elusive. High mobility group box 1 (HMGB1), a damage-associated molecular pattern molecule, has been shown to be related to IR and autophagy, respectively, in peripheral tissues. Here, we investigated whether increased HMGB1 contributes to IR in granulosa cells of PCOS patients via induction of aberrant autophagy. Results showed that HMGB1 abundance in the follicular fluid was significantly increased with enhanced autophagy in granulosa cells in PCOS patients with IR. HMGB1 exacerbated autophagy in granulosa cells as evinced by increased LC3B II/I ratio and ATG7 as well as decreased p62, the markers for autophagy. Concurrently, HMGB1 impaired insulin sensitivities by attenuating the abundance of insulin receptor substrate-1, Akt phosphorylation, GLUT4 translocation, and glucose uptake in granulosa cells, which were reversed by blocking autophagy pathways with siRNA-mediated knockdown of ATG7 or with chloroquine and bafilomycin A1, the lysosome inhibitors. In conclusion, our results indicate that increased HMGB1 contributes to IR development in granulosa cells of PCOS patients, which is associated with exacerbation of autophagy by HMGB1. Control of HMGB1 production may be benefical for the improvement of insulin sensitivity in granulosa cells in PCOS.

Melatonin ameliorates excessive PINK1/Parkin-mediated mitophagy by enhancing SIRT1 expression in granulosa cells of PCOS.

Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against mitochondrial injury in the granulosa cells of PCOS remain unclear. In this study, decreased mitochondrial membrane potential and mtDNA content, increased number of autophagosomes were found in the granulosa cells of PCOS patients and the dihydrotestosterone (DHT)-treated KGN cells, with decreased protein level of the autophagy substrate p62 and increased levels of the cellular autophagy markers Beclin 1 and LC3B-II, while the protein levels of PTEN-induced kinase-1 (PINK1) and Parkin were increased and the level of sirtuin 1 (SIRT1) was decreased. DHT-induced PCOS-like mice also showed enhanced mitophagy and decreased SIRT1 mRNA expression. Melatonin treatment significantly increased the protein level of SIRT1 and decreased the levels of PINK1/Parkin, whereas it ameliorated the mitochondrial dysfunction and PCOS phenotype in vitro and in vivo. However, when the KGN cells were treated with SIRT1 siRNA to knock down SIRT1 expression, melatonin treatment failed to repress the excessive mitophagy. In conclusion, melatonin protects against mitochondrial injury in granulosa cells of PCOS by enhancing SIRT1 expression to inhibit excessive PINK1/Parkin-mediated mitophagy.

Downregulation of lncRNA ZFAS1 and upregulation of microRNA-129 repress endocrine disturbance, increase proliferation and inhibit apoptosis of ovarian granulosa cells in polycystic ovarian syndrome by downregulating HMGB1

ObjectiveThe objective was to find the role of long-non-coding RNA zinc finger antisense 1 (lncRNA ZFAS1)/microRNA (miR)-129/high-mobility group box protein 1 (HMGB1) axis in polycystic ovary syndrome (PCOS). MethodsOvarian granulosa cells from non-PCOS patients and PCOS patients were collected, and HMGB1, miR-129 and lncRNA ZFAS1 expression were detected. Ovarian granulosa cells were transfected with si-ZFAS1 or miR-129 mimics to verify their roles in P4 and E2 secretion, and the biological functions of ovarian granulosa cells. ResultsLncRNA ZFAS1 and HMGB1 were elevated, while miR-129 was down-regulated in ovarian granulosa cells of PCOS patients. Down-regulated lncRNA ZFAS1 or overexpressed miR-129 could decrease HMGB1 expression, increase P4 and E2 secretion, promote proliferation activity while inhibit apoptosis of ovarian granulosa cells in PCOS. ConclusionLncRNA ZFAS1 could bind to miR-129 to promote HMGB1 expression, thereby affecting the endocrine disturbance, proliferation and apoptosis of ovarian granulosa cells in PCOS.

Loss of hsa_circ_0118530 inhibits human granulosa-like tumor cell line KGN cell injury by sponging miR-136.

Polycystic ovary syndrome (PCOS) is a kind of endocrine disease among women across the global. Recently, many researches have reported circular RNAs can act as significant molecular biomarkers for diseases, especially in tumors. Several Circular RNAs are reported to be aberrantly expressed in PCOS patients. Here, we investigated the biological effects of hsa_circ_0118530 on human granulosa cells, KGN. We observed that hsa_circ_0118530 was greatly elevated in PCOS patients and granulosa cells (including KGN and COV434 cells) compared to normal IOSE80 cells. hsa_circ_0118530 siRNA was transfected into KGN cells. We found that KGN cell viability was repressed, cell apoptosis was induced while cell migration was greatly inhibited. TGF-β1 was utilized to induce EMT process. As shown, loss of hsa_circ_0118530 significantly enhanced E-cadherin mRNA and protein levels while depressed N-cadherin expression. Furthermore, we indicated that decrease of hsa_circ_0118530 was able to inhibit ROS accumulation, MDA levels while induced SOD activity. Next, it was demonstrated that releases of inflammatory cytokine were suppressed by hsa_circ_0118530 down-regulation. Additionally, miR-136 was predicted and confirmed as the target of hsa_circ_0118530. For another, the functions of hsa_circ_0118530 on KGN cell progression, oxidative stress and inflammation releases were obviously reversed by miR-136 suppression. In conclusion, knockdown of hsa_circ_0118530 repressed PCOS progression via sponging miR-136.

RETRACTED: lncRNA PVT1/MicroRNA-17-5p/PTEN Axis Regulates Secretion of E2 and P4, Proliferation, and Apoptosis of Ovarian Granulosa Cells in PCOS

Recently, the roles of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) were identified in polycystic ovary syndrome (PCOS). In the present study, we investigated the role of the lncRNA PVT1/miR-17-5p/PTEN axis in PCOS ovarian granulosa cells. Expression of PVT1, miR-17-5p and PTEN in PCOS ovarian granulosa cells and follicular fluid was detected, and homeostatic model assessment of insulin resistance (HOMA-IR) and the levels of fasting plasma glucose (FPG), fasting insulin (FINS), and sex hormones were assessed. Then, the proliferation, apoptosis, and colony formation ability of ovarian granulosa cells were evaluated. The binding relationship between PVT1 and miR-17-5p as well as the target relationship between miR-17-5p and PTEN were determined by bioinformatics analysis, luciferase activity assay, RNA-induced silencing complex assay, and RNA pull-down assay. The levels of sex hormone-binding globulin and follicle-stimulating hormone were abated and the levels of luteinizing hormone, testosterone, FINS, FPG, and HOMA-IR were increased in PCOS serum. PVT1 and PTEN were overexpressed and miR-17-5p was reduced in PCOS ovarian granulosa cells and follicular fluid. Overexpressed miR-17-5p and inhibited PVT1 could decelerate apoptosis while accelerating colony formation ability and proliferation of ovarian granulosa cells in PCOS. Moreover, overexpression of PVT1 and reduced miR-17-5p could reverse these results. There existed target relation among PVT1, miR-17-5p, and PTEN, and PVT1 could inhibit miR-17-5p, thereby elevating PTEN. Our study suggests that inhibited PVT1 and overexpressed miR-17-5p result in downregulation of PTEN and promotion of cell proliferation, as well as inhibition of apoptosis of ovarian granulosa cells in PCOS.

RETRACTED ARTICLE: MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells

BackgroundWe elucidated the role of specific MicroRNAs (miRNAs) in the development of polycystic ovary syndrome (PCOS) and explained the changes in the proliferation of granulosa cells. Excised ovarian cortex specimens were collected for miRNA profiling analysis (n = 20 PCOS females and 5 non-PCOS females). Insulin-treated ovarian granulosa cells isolated from mice were used for mechanical studies.ResultsHigh miR-140 expression was observed in PCOS samples and insulin-treated granulosa cells compared to that in non-PCOS and unstimulated cells, respectively. However, the Ras-related protein Rap-2a precursor (RAP2A) was downregulated in in PCOS. MTT assay and EdU staining showed that an miR-140 inhibitor attenuated viability in insulin-treated granulosa cells; cell viability increased with miR-140 overexpression. Reduced expression of miR-140 and the expression of the miR-140 mimic resulted in marked cell apoptosis, as evidenced by the results of PI flow cytometry and Annexin V-FITC; miR-140 overexpression results in downregulated RAP2A expression, and the miR-140 mimic directly bound to the RAP2A 3′-UTR, causing increase in RAP2A levels in insulin-treated granulosa cells; RNA-mediated silencing of RAP2A in insulin-treated granulosa cells restored cell proliferation and apoptosis to normal levels. Phosphorylated AKT was found to be negatively regulated through cross-talk between miR-140 and RAP2A.ConclusionsIn conclusion, PCOS ovarian cortex specimens and insulin-treated granulosa cells showed elevated expression of miR-140, which could lead to increased proliferation and reduced apoptosis of cells by targeting RAP2A. This study may pave the way for future research on the properties of granulosa cells in PCOS.

Long Noncoding RNA HUPCOS Promotes Follicular Fluid Androgen Excess in PCOS Patients via Aromatase Inhibition.

Androgen excess is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear. To determine the role and mechanism of novel long noncoding RNA (lncRNA) highly up-regulated in PCOS (HUPCOS) in the androgen excess of PCOS patients. The lncRNA expression profile in granulosa cells derived from PCOS and non-PCOS women were analyzed by using microarray assay. Human granulosa cell line KGN was used for mechanism investigation. This was a university-based study. Thirty-eight PCOS and 38 control patients were recruited: 8 PCOS and 8 control samples used for microarray discovery, the remaining 30 PCOS cases and 30 controls for quantitative RT-PCR validation. The aberrant expression lncRNA profile of PCOS patients was measured using microarray. The relationship of HUPCOS and follicular fluid testosterone was measured. Aromatase expression were analyzed after HUPCOS downregulation. HUPCOS interaction protein was confirmed by RNA pull-down. The significantly elevated lncRNA in PCOS granulosa cells was named HUPCOS, which was positively correlated with follicular fluid testosterone of PCOS patients. HUPCOS downregulation increased aromatase expression and promoted conversion of androgen to estrogen. RNA-binding protein with multiple splicing (RBPMS) was the most likely protein that combined with HUPCOS. Our findings suggested that HUPCOS mediated androgen excess in follicular fluid of PCOS patients by suppressing aromatase expression via interaction with RBPMS.

MicroRNA-135a Regulates VEGFC Expression and Promotes Luteinized Granulosa Cell Apoptosis in Polycystic Ovary Syndrome.

Androgen is known to regulate microRNA-135a (miR-135a) and can be regulated by androgen, suggesting that it may contribute to polycystic ovary syndrome (PCOS) with hyperandrogenism. However, its roles and mechanisms of action in PCOS are unknown. In this study, the role and molecular mechanisms underlying miR-135a in granulosa cells (GCs) in PCOS were evaluated. miR-135a expression was upregulated in patients with PCOS and in GCs isolated from patients compared with that in the respective controls (P < 0.01), as determined by RT-qPCR. The overexpression of miR-135a inhibited GC proliferation and induced GC apoptosis, as observed by CCK-8 assay and apoptosis assay. Furthermore, miR-135a overexpression increased the expression of double-strand break maker, γH2AX, as confirmed by western blotting. Our results further suggest that these effects were mediated via downregulation of vascular endothelial growth factor C (VEGFC), which was identified as a direct target of miR-135a. Moreover, levels of VEGFC and miR-135a expression showed a negative correlation. These findings indicate that miR-135a promotes apoptosis and the DNA damage response in GCs in PCOS, likely via VEGFC signaling. This study provides novel insights into GC dysregulation in PCOS and suggests that miR-135a is a promising therapeutic target for PCOS treatment.

MicroRNA-9119 regulates cell viability of granulosa cells in polycystic ovarian syndrome via mediating Dicer expression.

Polycystic ovary syndrome (PCOS) is a hormonal disorder common among women of reproductive age. Although much is understood concerning the pathology of PCOS, further investigation into the influence of microribonucleic acids (miRNAs) on the proliferation of ovarian granulosa cells (GCs) is needed. This study investigated the role of specific miRNAs in ovarian dysfunction of PCOS and its effect on the proliferation of GCs. Initially, miRNA profiling was performed on the ovarian cortexes of 15 rats in which PCOS had been induced and 15 rats without PCOS (non-PCOS). This mechanical study was performed on ovarian GCs extracted from human chorionic gonadotrophin (hCG)-induced rats. Insulin was used to treat GCs to establish the PCOS cell model. Increased Equus caballusmir-9119 expression was observed and confirmed in the insulin-induced model of PCOS in GCs (GC-PCOS) as well as in the hCG-induced rats when compared to non-PCOS rats and cells. Observation and confirmation were carried out through both miRNA array and quantitative PCR. In contrast, downregulation of the nuclear factor kappa B (NFκB) p65 was observed in the PCOS cell model. Additionally, annexin V, FITC, and propidium iodide flow cytometry showed overexpression of miR-9119-induced apoptosis. In this study, we revealed that miR-9119 inhibition regulates p65 expression levels in insulin-treated GCs by binding to the 3'-untranslated of p65. Additionally, regulation of p65 expression was positively correlated with the expression of the double-stranded RNA endoribonuclease DICER. Moreover, RNA silencing/overexpression of p65 affected the functional role of miR-9119. In conclusion, GCs of PCOS, the expression of miR-9119, and targeted NFκB/p65-DICER axis are upregulated in order to maintain cell viability and prevent apoptosis, thereby promoting Anti-Müllerian hormone production in GCs. This study may provide a new understanding of the mechanism of GC dysfunction.

Effect of Vitamin D3 on Mitochondrial Biogenesis in Granulosa Cells Derived from Polycystic Ovary Syndrome.

BackgroundPolycystic ovary syndrome (PCOS) is an endocrine disorder diagnosed by anovulation hyperandro- genism. Hyperandrogenism increases apoptosis, which will eventually disturb follicular growth in PCOS patients. Since mitochondria regulate apoptosis, they might be affected by high incidence of follicular atresia. This may cause infertility. Since vitamin D3 has been shown to improve the PCOS symptoms, the aim of study was to investigate the effects vitamin D3 on mtDNA copy number, mitochondrial biogenesis, and membrane integrity of granulosa cells in a PCOS-induced mouse model.Materials and MethodsIn this experimental study, the PCOS mouse model was induced by dehydroepiandrosterone (DHEA). Granulosa cells after identification by follicle-stimulating hormone receptor (FSHR) were cultured in three groups: 1. granulosa cells treated with vitamin D3 (100 nM for 24 hours), 2. granulosa cells without any treatments, 3. Non-PCOS granulosa cells (control group). Mitochondrial biogenesis gene (TFAM) expression was compared between different groups using real-time PCR. mtDNA copy number was also investigated by qPCR. The mitochon- drial structure was evaluated by transmission electron microscopy (TEM). Hormonal levels were measured by an enzymelinked immunosorbent assay (ELISA) kit.ResultsThe numbers of pre-antral and antral follicles increased in PCOS group in comparison with the non-PCOS group. Mitochondrial biogenesis genes were downregulated in granulosa cells of PCOS mice when compared to the non-PCOS granulosa cells. However, treatment with vitamin D3 increased mtDNA expression levels of these genes compared to PCOS granulosa cells with no treatments. Most of the mitochondria in the PCOS group were spherical with almost no cristae. Our results showed that in the PCOS group treated with vitamin D3, the mtDNA copy number increased significantly in comparison to PCOS granulosa cells with no treatments.ConclusionAccording to this study, we can conclude, vitamin D3 improves mitochondrial biogenesis and membrane integrity, mtDNA copy number in granulosa cells of PCOS mice which might improve follicular development and subsequently oocyte quality.

Several critical genes and microRNAs associated with the development of polycystic ovary syndrome

BackgroundWe aimed to identify key genes and microRNAs (miRNAs) associated with the development of polycystic ovary syndrome (PCOS). MethodsGSE84376 mRNA microarray data (15 PCOS granulosa cells and 13 control granulosa cells) and GSE34526 mRNA microarray data (7 PCOS granulosa cells and 3 control granulosa cells) were downloaded from the Gene Expression Omnibus (GEO) database. First, differentially expressed gene (DEG) analysis, gene set enrichment analysis (GSEA) for differentially expressed mRNAs, and protein–protein interaction (PPI) network analysis were conducted. Next, miRNA-target genes were analyzed and functions predicted, and a competing endogenous RNA (ceRNA) network was constructed. Finally, the relationship between miR-486-5p and PRELID2 was experimentally validated. ResultsSpleen tyrosine kinase (SYK), major histocompatibility complex, class II, DR alpha (HLA-DRA), and interleukin 10 (IL-10) were important nodes in the PPI network. Interestingly, HLA-DRA was significantly enriched in phagosomes mediated by Staphylococcus aureus infection, and in IL-10 enriched during S. aureus infection. One miRNA (miR-486-5p) and a single target gene (PRELID2) were obtained from the ceRNA network. Further experiments showed that miR-486-5p is upregulated and PRELID2 is downregulated in PCOS patient granulosa cells, and that miR-486-5p targets the PRELID2 3′UTR. Topological property analysis showed that hsa-miR-4687-5p downregulation and hsa-miR-4651 upregulation determined the levels of most mRNAs. Levels of the hsa-miR-4651 target gene were significantly enriched in the leukocyte transendothelial migration pathway. ConclusionsOur results suggest that HLA-DRA and IL-10 may contribute to PCOS progression via phagosome enriched by S. aureus infection, while miR-486-5p may be implicated in follicular development in PCOS by targeting PRELID2. Besides, miR-4651 may be involved in inflammation via leukocyte transendothelial migration, by regulating its target gene. These findings may indicate new directions and constitute a breakthrough in studying the pathophysiology of PCOS.

Identification and potential value of candidate microRNAs in granulosa cells of polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is a major cause of anovulatory infertility. Some studies showed that miRNAs were used as diagnostic/prognostic biomarkers for various diseases. To identify candidate miRNAs in Granulosa Cells (GCs) of PCOS and evaluate their potential values for PCOS diagnosis. We screened differentially expressed miRNAs in GCs between PCOS and controls by the microarray data from the GEO database. GCs were collected from 21 controls and 24 PCOS. The candidate miRNAs were verified by qRT-PCR. The correlation was investigated between candidate miRNAs and clinical characteristics in participants. Diagnostic value of candidate miRNAs was analyzed by receiver operating characteristic (ROC) curve. Seven miRNAs were differentially expressed in PCOS compared with controls. Furthermore, the validation results demonstrated that hsa-miR-3188 and hsa-miR-3135b showed higher levels in GCs with PCOS patients (p< 0.05). In addition, the expressions of hsa-miR-3188 and hsa-miR-3135b were negative correlated with FSH and hsa-miR-3188 was positive correlated with BMI (p< 0.05). ROC analysis indicated that hsa-miR-3188 and hsa-miR-3135b could differentiate PCOS from controls, and the hsa-miR-3188/3135b improved the predictive accuracy for PCOS. The expressions of hsa-miR-3188 and hsa-miR-3135b in human GCs were significantly associated with PCOS. Moreover, the hsa-miR-3188/3135b has certain diagnostic value for distinguishing PCOS.

Dietary Glycotoxins, Advanced Glycation End Products, Inhibit Cell Proliferation and Progesterone Secretion in Ovarian Granulosa Cells and Mimic PCOS-like Symptoms.

Women with polycystic ovary syndrome (PCOS) have been reported to have an elevated serum advanced glycation end product (AGE) level. However, the effect of AGEs on the pathophysiological ovarian granulosa cells of PCOS is still unclear. In this study, five indented BSA-derived AGE products were used to evaluate their effect on the function of human granulosa cells. We found that the proliferation of both primary human ovarian granulosa (hGC) cells and human granulosa-like tumor (KGN) cells were inhibited by treatment with these five AGE products. The progesterone secretion level was also reduced in both hGC and KGN cells by treatment with these AGE products through downregulation of LH receptor/cAMP regulatory activity. The granulosa cell layer and serum progesterone level were reduced in rats by treatment with MG-BSA; moreover, an increased number of follicle cysts and an irregular estrous cycle were observed. MG-BSA treatment had a similar effect on the phenotypes of the DHEA-induced PCOS model. Additionally, the insulin resistance and hepatic lesions seen in the DHEA-induced PCOS model were observed in the MG-BSA treatment group. Taken together, we found that AGEs exert a toxic effect on ovarian granulosa cells, ovarian morphology, and the estrous cycle that mimics the DHEA-induced PCOS phenotypes.