Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is characterized by follicular dysplasia. An insufficient glycolysis-derived energy supply of granulosa cells (GCs) is an important cause of follicular dysplasia in PCOS. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been proven to regulate the function of GCs. In this study, exosomes extracted from clinical FF samples were used for transcriptome sequencing (RNA-seq) analysis, and a human ovarian granulocyte tumour cell line (KGN cells) was used for in vitro mechanistic studies. In FF exosomal RNA-seq analysis, a decrease in glycolysis-related pathways was identified as an important feature of the PCOS group, and the differentially expressed miR-143-3p and miR-155-5p may be regulatory factors of glycolysis. By determining the effects of miR-143-3p and miR-155-5p on hexokinase (HK) 2, pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate, lactate and apoptosis in KGN cells, we found that upregulated miR-143-3p expression in exosomes from the PCOS group inhibited glycolysis in KGN cells; knockdown of miR-143-3p significantly alleviated the decrease in glycolysis in KGN cells in PCOS. MiR-155-5p silencing attenuated glycolytic activation in KGN cells; overexpression of miR-155-5p significantly promoted glycolysis in KGN cells in PCOS. In this study, HK2 was found to be the mediator of miR-143-3p and miR-155-5p in FF-derived exosome-mediated regulation of glycolysis in KGN cells. Reduced glycolysis accelerated apoptosis of KGN cells, which mediated follicular dysplasia through ATP, lactate and apoptotic pathways. In conclusion, these results indicate that miR-143-3p and miR-155-5p in FF-derived exosomes antagonistically regulate glycolytic-mediated follicular dysplasia of GCs in PCOS. Video Abstract.

Cao J ，Huo P ，Cui K ，Wei H ，Cao J ，Wang J ，Liu Q ，Lei X ，Zhang S ... -  《Cell Communication and Signaling》

Follicular Fluid-Derived Exosomal MicroRNA-18b-5p Regulates PTEN-Mediated PI3K/Akt/mTOR Signaling Pathway to Inhibit Polycystic Ovary Syndrome Development.

Small RNA sequences in follicular fluid (FF)-derived exosomes (extracellular vesicles contain proteins, DNA, and RNA) vitally function in the development of polycystic ovary syndrome (PCOS). It has been identified that microRNA (miR)-18b-5p is one of miRs that differ between control and PCOS women that passed the false discovery rate, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important modifier of biological functions of ovarian granulosa cells (GCs) in PCOS. However, whether miR-18b-5p could functionally mediate the progression of PCOS via PTEN was not clarified completely, which was the issue we wanted to solve in our research. FF-derived exosomes were isolated using an extraction kit. KGN cells were co-cultured with miR-18b-5p-modified exosomes or transfected with a PTEN-related vector. After treatment, cell proliferation and apoptosis were observed. A rat model of PCOS was established by letrozole and then injected with miR-18b-5p-modified exosomes. Then, serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and estradiol (E2) levels in PCOS rats were measured. miR-18b-5p, PTEN, and phosphatidylinositol 3 kinases/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway-related genes were tested. In PCOS patients, miR-18b-5p was downregulated, and PTEN was highly expressed in FF and GCs. PTEN knockdown increased KGN cell proliferation and limited apoptosis. FF-derived exosomes stimulated proliferation and suppressed apoptosis of KGN cells; decreased FSH, LH, and testosterone; and increased E2 in PCOS rats. Upregulating miR-18b-5p further enhanced the inhibitory effects of exosomes on suppressing the progression of PCOS. miR-18b-5p targeted PTEN and could activate PI3K/Akt/mTOR pathway. miR-18b-5p produced by FF-derived exosomes reduces PTEN expression and promotes the activation of the PI3K/Akt/mTOR signaling pathway to improve PCOS. Based on that, circulating miR-18b-5p levels can contribute to the progression of PCOS complications.

Zhou Z ，Tu Z ，Zhang J ，Tan C ，Shen X ，Wan B ，Li Y ，Wang A ，Zhao L ，Hu J ，Ma N ，Zhou J ，Chen L ，Song Y ，Lu W ... -  《-》

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression.

Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease, characterized by increased ovarian androgen biosynthesis, chronic anovulation and polycystic ovaries. The objective of this study was to identify the altered miRNA expression profiles in follicular fluid derived exosomes isolated from PCOS patients and to investigate the molecular functions of exosomal miR-424-5p. Herein, small RNA sequencing showed that twenty-five miRNAs were differentially expressed between control and PCOS group. The alterations in the miRNA profile were related to the endocrine resistance, cell growth and proliferation, cellular senescence and insulin signaling pathway. Among these differentially expressed miRNAs, we found that the expression of miR-424-5p was significantly decreased in PCOS exosomes and primary granulosa cells (GCs). Exosome-enriched miR-424-5p significantly promoted GCs senescence and suppressed cell proliferation. Similar to the results obtained in the cells transfected with miR-424-5p mimic, miR-424-5p mimic significantly decreased cell proliferation ability and induced senescence, but treatment with miR-424-5p inhibitor got the opposite results. In addition, cell division cycle associated 4 (CDCA4) gene displayed an inverse expression pattern to those of miR-424-5p, was identified as the direct target of miR-424-5p. Overexpression of CDCA4 reversed the effects of exosomal miR-424-5p on GCs via activation of Rb/E2F1 signaling pathway. These results demonstrate that exosomal miR-424-5p inhibits GCs proliferation and induces cellular senescence in PCOS through blocking CDCA4-mediated Rb/E2F1 signaling. Our findings provide new information on abnormal follicular development in PCOS.

Yuan D ，Luo J ，Sun Y ，Hao L ，Zheng J ，Yang Z ... -  《-》

Exosomal miR-143-3p derived from follicular fluid promotes granulosa cell apoptosis by targeting BMPR1A in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder that occurs in women of reproductive age. Anovulation caused by abnormal follicular development is still the main characteristic of PCOS patients with infertile. Granulosa cell (GC) is an important part of the follicular microenvironment, the dysfunction of which can affect follicular development. Increasing evidence indicates that exosomal miRNAs derived from the follicular fluid (FF) of patients play critical roles during PCOS. However, which follicular fluid-derived exosomal miRNAs play a pivotal role in controlling granulosa cell function and consequently follicular development remain largely unknown, as does the underlying mechanism. Herein, we showed that miR-143-3p is highly expressed in the follicular fluid exosomes of patients with PCOS and can be delivered into granulosa cells. Furthermore, functional experiments showed that translocated miR-143-3p promoted granulosa cell apoptosis, which is important in follicle development. Mechanistically, BMPR1A was identified as a direct target of miR-143-3p. Overexpression of BMPR1A reversed the effects of exosomal miR-143-3p on GC apoptosis and proliferation by activating the Smad1/5/8 signaling pathway. These results demonstrate that miR-143-3p-containing exosomes derived from PCOS follicular fluid promoted granulosa cell apoptosis by targeting BMPR1A and blocking the Smad1/5/8 signaling pathway. Our findings provide a novel mechanism underlying the roles of exosomal-miRNAs in the follicular fluid of PCOS patients and facilitate the development of therapeutic strategies for PCOS.

Zhao Y ，Pan S ，Li Y ，Wu X ... -  《Scientific Reports》

Follicular fluid derived exosomal miR-4449 regulates cell proliferation and oxidative stress by targeting KEAP1 in human granulosa cell lines KGN and COV434.

Polycystic ovary syndrome (PCOS) is characterized by ovulatory dysfunction, hyperandrogenism, and polycystic ovary morphology, affecting more and more women of reproductive age. Our study aimed to explore the molecular mechanism and effect of exosomal miR-4449 on granulosa cells (GCs). Two immortalized human ovarian granulosa cells (KGN and COV434 cells) were used for in vitro functional studies. Our study found that follicular fluid (FF) derived exosomal miR-4449 was significantly decreased in women with PCOS compared with the control patients. And exosomal miR-4449 could alleviate GCs oxidative stress (OS) and promote GCs proliferation, while the opposite trend was observed after inhibiting the expression of miR-4449. In addition, we demonstrated that Kelch-like ECH-associated protein 1(KEAP1) was a direct target of miR-4449 through dual-luciferase reporter assay, and the expression patterns of KEAP1 and miR-4449 in PCOS FF-derived exosomes were exactly opposite. In addition, KEAP1/NRF2 signaling pathway may play an important role in GCs proliferation and OS. Our results demonstrated that the decreased FF-derived exosomal miR-4449 expression in PCOS might aggravate the OS of GCs and inhibit GCs proliferation via KEAP1/NRF2 signaling pathway. Exosomal miR-4449 might be a potential biomarker for the diagnosis of PCOS. Our study contributes to a new understanding of the pathogenesis of PCOS.

Wang M ，Sun Y ，Yuan D ，Yue S ，Yang Z ... -  《-》