Induction of miR-15a expression by tripterygium glycosides caused premature ovarian failure by suppressing the Hippo-YAP/TAZ signaling effector Lats1.

Tripterygium glycosides (TGs) are chemotherapeutic drugs and immunosuppressant agents for the treatment of cancer and autoimmune diseases. We have previously reported that TGs induces premature ovarian failure (POF) by inducing cytotoxicity in ovarian granulosa cells (OGCs). Hence, we report that TGs suppress the expression of the Hippo-YAP/TAZ pathway in murine OGCs in vitro and in vivo. We found that the expressions of miR-181b, miR-15a, and miR-30d, were elevated significantly in the POF. Luciferase reporter assays confirmed that miR-15a targets Lats1 through a miR-15a binding site in the Lats1 3'UTR. Overexpression of miR-15a in mOGCs not only inhibited proliferation and growth of mOGCs, but also induced aging of mOGCs. Western blot and qPCR analysis indicated that miR-15a suppresses the expression of the Hippo-YAP/TAZ pathway in mOGCs. When the exogenous miR-15a was expressed on mouse OGCs, it could elevate the cytotoxicity effect of TG on mOGCs. We conclude that tripterygium glycosides promote cytotoxicity, senescence, and apoptosis in ovarian granulosa cells by inducing endogenous miR-15a expression and inhibiting the Hippo-YAP/TAZ pathway.

Ai A ，Xiong Y ，Wu B ，Lin J ，Huang Y ，Cao Y ，Liu T ... -  《-》

miR-126-3p containing exosomes derived from human umbilical cord mesenchymal stem cells promote angiogenesis and attenuate ovarian granulosa cell apoptosis in a preclinical rat model of premature ovarian failure.

In our previous research, we found that overexpression of miR-126-3p in human umbilical cord MSCs (hucMSCs) promoted human umbilical vein endothelial cells angiogenic activities through exosome-mediated mechanisms. The present study aimed to investigate the role of miR-126-3p-modified hucMSCs derived exosomes (miR-126-3p-hucMSCs-exosomes) on the treatment of premature ovarian failure (POF). Primary hucMSCs were isolated from human umbilical cords and identified by differentiation experiments and flow cytometry. miR-126-3p-hucMSCs were obtained by miR-126-3p lentivirus infection. miR-126-3p-hucMSCs-exosomes were purified by ultracentrifugation method and characterized by transmission electron microscopy and western blot analysis. Primary rat ovarian granulosa cells (OGCs) were collected from ovarian tissues and identified by cell immunohistochemistry. The effects of miR-126-3p-hucMSCs-exosomes and miR-126-3p on OGCs function were determined by cell proliferation and apoptosis assays in a cisplatin induced POF cell model. The levels of suitable target genes were analyzed by PCR and Western blot analysis and subsequent Dual-Luciferase reporter assay. The signal pathway was also analyzed by western blot analysis. A cisplatin-induced POF rat model was used to validate the therapeutic effects of miR-126-3p-hucMSCs-exosomes to treat POF. Ovarian function was evaluated by physical, enzyme-linked immunosorbent assay, and histological examinations in chemotherapy-treated rats. The angiogenesis and apoptosis of ovarian tissues were assessed by immunohistochemical staining and Western blots. Primary hucMSCs and miR-126-3p-hucMSCs-exosomes and primary rat OGCs were successfully isolated and identified. The cellular uptake experiments indicated that miR-126-3p-hucMSC-exosomes can be internalized into OGCs in vitro. Annexin V-FITC/PI staining and EDU assays revealed that both miR-126-3p-hucMSCs-exosomes and miR-126-3p promoted proliferation and inhibited apoptosis of OGCs damaged by cisplatin. PCR and western blot analysis and subsequent dual-luciferase reporter assay verified that miR-126-3p targets the sequence in the 3' untranslated region of PIK3R2 in OGCs. Further analysis showed that PI3K/AKT/mTOR signaling pathway took part in miR-126-3p/PIK3R2 mediated proliferation and apoptosis in OGCs. In rat POF model, administration of miR-126-3p-hucMSCs-exosomes increased E2 and AMH levels, increased body and reproductive organ weights and follicle counts, and reduced FSH levels. But more importantly, immunohistochemistry results indicated miR-126-3p-hucMSCs-exosomes significantly promoted ovarian angiogenesis and inhabited apoptosis in POF rats. Additionally, the analysis of angiogenic-related factors and apoptosis-related factors showed miR-126-3p-hucMSCs-exosomes had pro-angiogenesis and anti-apoptosis effect in rat ovaries. Our findings revealed that hucMSCs-derived exosomes carrying miR-126-3p promote angiogenesis and attenuate OGCs apoptosis in POF, which highlighted the potential of exosomes containing miR-126-3p as an effective therapeutic strategy for POF treatment.

Qu Q ，Liu L ，Cui Y ，Liu H ，Yi J ，Bing W ，Liu C ，Jiang D ，Bi Y ... -  《Stem Cell Research & Therapy》

FGF2 promotes the proliferation of injured granulosa cells in premature ovarian failure via Hippo-YAP signaling pathway.

Young women undergoing anticancer treatment are at risk of premature ovarian failure (POF). Endometrial-derived stem cells (EnSCs) have demonstrated significant therapeutic potential for treating ovarian insufficiency, although the underlying mechanisms remain to be fully understood. This study aims to further investigate the therapeutic effects of EnSCs, particularly through the paracrine action of fibroblast growth factor 2 (FGF2), on POF. The findings show that exogenous FGF2 enhances the survival of ovarian granulosa cells damaged by cisplatin. FGF2 stimulates the proliferation of these damaged cells by suppressing the Hippo signaling pathway and activating YAP expression. In vivo experiments also revealed that FGF2 treatment significantly improves ovarian reserve and endocrine function in mice with POF. These results suggest that FGF2 can boost the proliferative capacity of damaged ovarian granulosa cells through the Hippo-YAP signaling pathway, providing a theoretical foundation for using EnSCs and FGF2 in clinical treatments for POF.

Cheng F ，Wang J ，Wang R ，Pan R ，Cui Z ，Wang L ，Wang L ，Yang X ... -  《-》

Cyclophosphamide promotes the proliferation inhibition of mouse ovarian granulosa cells and premature ovarian failure by activating the lncRNA-Meg3-p53-p66Shc pathway.

The dysfunction of ovarian granulosa cells (OGCs) directly affects the premature ovarian failure (POF). In vivo experiments showed that cyclophosphamide significantly induced mouse ovarian atrophy and proliferation inhibition of OGCs. The expressions of p53, p66Shc and p16 were significantly higher in OGCs of the cyclophosphamide treatment group. MTT assay showed that cyclophosphamide effectively inhibited the proliferation of OGCs in vitro. SA-β-Gal staining showed that the OGCs in the cyclophosphamide treatment group had many senescent cells. And, the expression of p53, p66Shc, p16 and cleaved caspase-3 in the OGCs of the cyclophosphamide treatment group significant increases. The Northern blot showed that the intensity of the lncRNA-Meg3 hybridization signal of the OGCs in the cyclophosphamide treatment group was significantly higher than that in the control group. ChIP results confirmed the significant increase in the obtained p66Shc promoter DNA fragment, which was enriched on p53 protein, in the OGCs treated with cyclophosphamide. When cyclophosphamide treatment was conducted after siRNA-Meg3 was used, the expression of endogenous lncRNA-Meg3, p53, p66Shc, p16 and cleaved caspase-3 was significantly lower than that in the siRNA-Mock control group. In summary, cyclophosphamide promotes the proliferation inhibition of mouse OGCs and premature ovarian failure by activating the lncRNA-Meg3-p53-p66Shc pathway.

Xiong Y ，Liu T ，Wang S ，Chi H ，Chen C ，Zheng J ... -  《-》

Lats1 and Lats2 are required for ovarian granulosa cell fate maintenance.

Tsoi M ，Morin M ，Rico C ，Johnson RL ，Paquet M ，Gévry N ，Boerboom D ... -  《-》