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 ... -  《-》

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 ... -  《-》

The role of MiR-324-3p in polycystic ovary syndrome (PCOS) via targeting WNT2B.

Jiang YC ，Ma JX 《-》

MicroRNA-141-3p targets DAPK1 and inhibits apoptosis in rat ovarian granulosa cells.

The polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. MicroRNAs negatively regulate the expression of target genes at posttranscriptional level by binding to the 3' untranslated region of target genes. Our previous study showed that miR-141-3p was dramatically decreased in the ovaries of rat PCOS models. In this study, we aimed to characterize the target of miR-141-3p in rat ovarian granulosa cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay showed that cell viability was dramatically increased when miR-141-3p was overexpressed but was decreased when miR-141-3p was interfered. Flow cytometry showed that cell apoptotic rate was dramatically decreased when miR-141-3p was overexpressed but was increased when miR-141-3p was interfered. Bioinformatics analysis predicted that death-associated protein kinase 1 (DAPK1) might be the target gene of miR-141-3p because the 3' untranslated region of DAPK1 contains sequences complementary to microRNA-141-3p. Transfection with miR-141-3p mimics and inhibitor into granulosa cells showed that both DAPK1 mRNA and protein levels were negatively correlated with miR-141-3p level. Dual-luciferase reporter assay established that DAPK1 was the target of miR-141-3p. Taken together, our data indicate that miR-141-3p may inhibit ovarian granulosa cell apoptosis via targeting DAPK1 and is involved in the etiology of PCOS.

Li D ，Xu D ，Xu Y ，Chen L ，Li C ，Dai X ，Zhang L ，Zheng L ... -  《-》