Functional Characterization of MicroRNA-27a-3p Expression in Human Polycystic Ovary Syndrome.

The goal of this study was to characterize the function of microRNA-27a-3p (miR-27a-3p) in polycystic ovary syndrome (PCOS). miR-27a-3p expression was analyzed in excised granulosa cells (GCs) from 21 patients with PCOS and 12 normal patients undergoing in vitro fertilization cycle treatments and in 17 nontreated cuneiform ovarian resection PCOS samples and 13 control ovarian samples from patients without PCOS. We found that the expression of miR-27a-3p was significantly increased in both excised GCs and the ovaries of patients with PCOS compared with the controls. Insulin treatment of the human granulosa-like tumor cell line (KGN) resulted in decreased downregulated expression of miR-27a-3p, and this effect appeared to be mediated by signal transducer and activator of transcription STAT1 and STAT3. The overexpression of miR-27a-3p in KGN cells inhibited SMAD5, which in turn decreased cell proliferation and promoted cell apoptosis. After KGN cells were stimulated with insulin for 48 hours, there was increased expression of SMAD5 protein and decreased apoptosis. Additionally, knockdown/overexpression of SMAD5 in KGN cells reduced/increased cell number and promoted/inhibited cell apoptosis. Insulin-stimulated primary GCs isolated from patients with PCOS, in contrast to normal GCs or KGN cells, did not exhibit decreased miR-27a-3p expression. The differences in the expression levels in KGN cells and human PCOS GCs are likely explained by increased miR-27a-3p expression in the GCs caused by insulin resistance in PCOS. Taken together, our data provided evidence for a functional role of miR-27a-3p in the GCs' dysfunction that occurs in patients with PCOS.

Wang M ，Sun J ，Xu B ，Chrusciel M ，Gao J ，Bazert M ，Stelmaszewska J ，Xu Y ，Zhang H ，Pawelczyk L ，Sun F ，Tsang SY ，Rahman N ，Wolczynski S ，Li X ... -  《-》

LncRNA HCP5 promotes cell proliferation and inhibits apoptosis via miR-27a-3p/IGF-1 axis in human granulosa-like tumor cell line KGN.

This study aimed to reveal the potential roles of long non-coding RNA HCP5 (lncRNA HCP5) and its potential molecular mechanism in polycystic ovarian syndrome (PCOS). The human granulosa-like tumor cell line KGN was used for assessing the effects of HCP5 in the proliferation and apoptosis of granulosa cells (GCs). The results showed that downregulation of HCP5 suppressed cell proliferation through arresting cell cycle progression at G1 phase, and induced the apoptosis via activating mitochondrial pathway, while overexpression of HCP5 played the opposite effects in KGN cells. We predicted and confirmed miR-27a-3p was a directly target to HCP5 and it could directly bind with insulin-like growth factor-1 (IGF-1). Next, we performed gain- and loss-of-functions approaches by transfecting miR-27a-3p inhibitor into HCP5 knocking down cells and transfecting miR-27a-3p mimics into HCP5 overexpressing cells. The results demonstrated that downregulation and upregulation of miR-27a-3p could block the effects on the proliferation and apoptosis mediated by silencing and overexpressing HCP5 in KGN cells. Additionally, miR-27a-3p inhibitor remarkably reversed the IGF-1 decrease regulated by knocking down HCP5 and miR-27a-3p mimics inhibited the IGF-1 increase modulated by overexpressing HCP5 in KGN cells. Furthermore, we observed that the promoted cell vitality and reduced apoptosis mediated by enforced expression of HCP5 could be alleviated when the KGN cells transfected with IGF-1 siRNA. Our findings indicate that HCP5 might be a potential regulatory factor for development of PCOS through regulating the miR-27a-3p/IGF-1 axis.

Chen Y ，Zhang X ，An Y ，Liu B ，Lu M ... -  《-》

MicroRNA-27a-3p affects estradiol and androgen imbalance by targeting Creb1 in the granulosa cells in mouse polycytic ovary syndrome model.

Polycystic ovary syndrome (PCOS) is a common endocrine abnormality in women characterized by a menstrual disturbance with chronic anovulation and hyperandrogenism, polycystic ovaries, and insulin resistance. MicroRNAs (miRNAs) are important fine-tune regulators involved in various physiological and pathological processes, but their actions are not fully understood. In this study, we observed the increased expression of miR-27a-3p in the ovaries of mice with PCOS and explored its functions in primary mouse granulosa cells (mGCs) and the mouse granulosa-like tumor cell line, KK-1, using several approaches. QPCR results showed that miR-27a-3p expression was significantly higher in mGCs at the preantral follicle (PrF) stage. Using flow cytometry and hormone analysis, we found that overexpression of miR-27a-3p promoted apoptosis and inhibited estradiol (E2) production in KK-1 cells. Moreover using a luciferase assay and Western blotting analysis, we verified that the gene of cyclic AMP response element (CRE)-binding protein 1 (Creb1) was a potential target of miR-27a-3p, which in effect hindered the expression of its downstream factor cytochrome P450 family 19 subfamily A polypeptide 1 (Cyp19a1). With the decrease of aromatase activity, testosterone (T) is reduced to dihydrotestosterone (DHT) and this exerts its effect of upregulation of the miR-27a-3p expression. The imbalance of androgen and E2 levels affected by miR-27a-3p and its function of promoting GC apoptosis could be involved in the pathophysiology of PCOS. Our results indicate that miR-27a-3p in PCOS GCs may play an important role in ovarian follicular development and provide new insights into GC dysfunction in PCOS.

Wang M ，Liu M ，Sun J ，Jia L ，Ma S ，Gao J ，Xu Y ，Zhang H ，Tsang SY ，Li X ... -  《-》

miR-130b-3p is high-expressed in polycystic ovarian syndrome and promotes granulosa cell proliferation by targeting SMAD4.

Being one of the most prevalent metabolic and endocrine disorders, Polycystic Ovary Syndrome (PCOS) has been proven to be associated with microRNA-130b-3p (miR-130b-3p). However, the exact role played by miR-130b-3p in the pathogenesis and progression of PCOS remains unknown. Thus, this article is focused on elucidating the function of miR-130b-3p in the pathogenesis of PCOS. The expression levels of miR-130b-3p and SMAD4 in tissues and cells responsible for the development of PCOS were determined by RT-qPCR and western blot. A miR-130b-3p mimic/inhibitor or si-SMAD4 were transfected into KGN cells. The cell viability was detected by CCK-8 and EDU methods. The activity of caspase-3 was measured by caspase-3 analysis. Subsequently, apoptosis and the cell cycle were measured via flow cytometry. The correlation between SMAD4 and miR-130b-3p was confirmed using an RNA pull-down assay and a dual luciferase reporter system assay. MiR-130b-3p was upregulated in the KGN cells and ovarian granulosa cells (GCs) of PCOS patients. It was found that miR-130b-3p overexpression or SMAD4 silencing can promote KGN cell proliferation and positive EDU rates, induce S phase arrest, inhibit apoptosis and caspase-3 activity. On the other hand, miR-130b-3p inhibitors reduce KGN cell proliferation, inhibit apoptosis and reverse the effect of si-SMAD4. MiR-130b-3p directly interacts with SMAD4 to induce KGN cell proliferation, inhibit apoptosis, suggesting that miR-130b-3p expression is positively correlated with the development of PCOS. This may serve as new evidence for the abnormal proliferation of GCs in PCOS.

Bao D ，Li M ，Zhou D ，Zhuang C ，Ge Z ，Wei Q ，Zhang L ... -  《-》

microRNA-194 is increased in polycystic ovary syndrome granulosa cell and induce KGN cells apoptosis by direct targeting heparin-binding EGF-like growth factor.

Polycystic ovary syndrome (PCOS) is an endocrine-related follicular developmental disorder that affects 50 %-70 % of reproductive-aged women diagnosed with ovulation-related infertility. Abnormal proliferation and apoptosis of granulosa cells (GCs) are thought to be the critical factors leading to abnormal maturation of follicles. It has been shown that microRNAs (miRNAs) exert a significant influence in the pathogenesis of PCOS; however, the relationship between miRNA, PCOS, and GC apoptosis is not entirely understood. To clarify the effect of miR-194 in PCOS, CCK-8, Ki67 staining, AO/EB, and flow cytometry assays were used to assess cell growth, proliferation, and apoptosis in KGN cells, which were artificially stimulated to overexpress miR-194. Luciferase reporter assays and rescue experiments were used to elucidate the mechanism underlying miR-194 in PCOS. miR-194 expression was significantly up-regulated in rat models of PCOS and the ovarian GCs of PCOS patients. miR-194 suppression promoted KGN cell growth and proliferation. miR-194 overexpression also induced cell apoptosis, while miR-194 downregulation had an opposite effect. Furthermore, up-regulating heparin-binding EGF-like growth factor (HB-EGF) expression rescued the pro-apoptotic effects of miR-194 upregulation on KGN cells. miR-194 is increased in PCOS granulosa cell and may function as a novel biomarker and therapeutic target for KGN cells via HB-EGF regulation.

Wu YX ，Lin YS ，Li SC ，Yao X ，Cheng M ，Zhu L ，Liu HY ... -  《Reproductive Biology and Endocrinology》