MicroRNA-22-3p in human umbilical cord mesenchymal stem cell-secreted exosomes inhibits granulosa cell apoptosis by targeting KLF6 and ATF4-ATF3-CHOP pathway in POF mice.

Human umbilical cord mesenchymal stem cells (hUCMSCs)-derived exosomes carrying microRNAs (miRNAs) have promising therapeutic potential in various disorders, including premature ovarian failure (POF). Previous evidence has revealed the low plasma level of miR-22-3p in POF patients. Nevertheless, exosomal miR-22-3p specific functions underlying POF progression are unclarified. A cisplatin induced POF mouse model and in vitro murine ovarian granulosa cell (mOGC) model were established. Exosomes derived from miR-22-3p-overexpressed hUCMSCs (Exos-miR-22-3p) were isolated. CCK-8 assay and flow cytometry were utilized for measuring mOGC cell viability and apoptosis. RT-qPCR and western blotting were utilized for determining RNA and protein levels. The binding ability between exosomal miR-22-3p and Kruppel-like factor 6 (KLF6) was verified using luciferase reporter assay. Hematoxylin-eosin staining, ELISA, and TUNEL staining were performed for examining the alteration of ovarian function in POF mice. Exos-miR-22-3p enhanced mOGC viability and attenuated mOGC apoptosis under cisplatin treatment. miR-22-3p targeted KLF6 in mOGCs. Overexpressing KLF6 reversed the above effects of Exos-miR-22-3p. Exos-miR-22-3p ameliorated cisplatin-triggered ovarian injury in POF mice. Exos-miR-22-3p repressed ATF4-ATF3-CHOP pathway in POF mice and cisplatin-treated mOGCs. Exosomal miR-22-3p from hUCMSCs alleviates OGC apoptosis and improves ovarian function in POF mouse models by targeting KLF6 and ATF4-ATF3-CHOP pathway.

Gao T ，Chen Y ，Hu M ，Cao Y ，Du Y ... -  《-》

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》

miR-644-5p carried by bone mesenchymal stem cell-derived exosomes targets regulation of p53 to inhibit ovarian granulosa cell apoptosis.

Sun B ，Ma Y ，Wang F ，Hu L ，Sun Y ... -  《Stem Cell Research & Therapy》

Improving Granulosa Cell Function in Premature Ovarian Failure with Umbilical Cord Mesenchymal Stromal Cell Exosome-Derived hsa_circ_0002021.

The therapeutic potential of exosomes from human umbilical cord mesenchymal stem cells (HUMSCs-Exo) for delivering specific circular RNAs (circRNAs) in treating premature ovarian failure (POF) is not well understood. This study aimed to explore the efficacy of HUMSCs-Exo in delivering hsa_circ_0002021 for POF treatment, focusing on its effects on granulosa cell (GC) senescence and ovarian function. Bioinformatic analysis was conducted on circRNA profiles using the GSE97193 dataset from GEO, targeting granulosa cells from varied age groups. To simulate granulosa cell senescence, KGN cells were treated with cyclophosphamide (CTX). HUMSCs were transfected with pcDNA 3.1 vectors to overexpress hsa_circ_0002021, and the HUMSCs-Exo secreted were isolated. These exosomes were characterized by transmission electron microscopy (TEM) and Western blotting to confirm exosomal markers CD9 and CD63. Co-culture of these exosomes with CTX-treated KGN cells was performed to assess β-galactosidase activity, oxidative stress markers, ROS levels, and apoptosis via flow cytometry. Interaction between hsa_circ_0002021, microRNA-125a-5p (miR-125a-5p), and cyclin-dependent kinase 6 (CDK6) was investigated using dual-luciferase assays and RNA immunoprecipitation (RIP). A POF mouse model was induced with CTX, treated with HUMSCs-Exo, and analyzed histologically and via immunofluorescence staining. Gene expression was quantified using RT-qPCR and Western blot. hsa_circ_0002021 was under expressed in both in vivo and in vitro POF models and was effectively delivered by HUMSCs-Exo to KGN cells, showing a capability to reduce GC senescence. Overexpression of hsa_circ_0002021 in HUMSCs-Exo significantly enhanced these anti-senescence effects. This circRNA acts as a competitive adsorbent of miR-125a-5p, regulating CDK6 expression, which is crucial in modulating cell cycle and apoptosis. Enhanced expression of hsa_circ_0002021 in HUMSCs-Exo ameliorated GC senescence in vitro and improved ovarian function in POF models by modulating oxidative stress and cellular senescence markers. This study confirms that hsa_circ_0002021, when delivered through HUMSCs-Exo, can significantly mitigate GC senescence and restore ovarian function in POF models. These findings provide new insights into the molecular mechanisms of POF and highlight the therapeutic potential of circRNA-enriched exosomes in treating ovarian aging and dysfunction.

Yang G ，Zhang B ，Xu M ，Wu M ，Lin J ，Luo Z ，Chen Y ，Hu Q ，Huang G ，Hu H ... -  《-》

HucMSCs-exosomes containing miR-21 promoted estrogen production in ovarian granulosa cells via LATS1-mediated phosphorylation of LOXL2 and YAP.

Premature ovarian failure (POF) is one of the common disorders found in women leading to 1% female infertility. Clinical features of POF are hypoestrogenism or estrogen deficiency. With the development of regenerative medicine, human mesenchymal stem cells (hMSCs) therapy brings new prospects for POF. This research aims to reveal the therapeutic effects and potential mechanisms of human umbilical cord mesenchymal stem cells (hucMSCs)-derived exosomes on POF. The mRNA and protein expressions in hucMSCs and ovarian granulosa cells (KGN and SVOG cells) were assessed using qRT-PCR and western blot. ELISA assay was performed to evaluate estradiol (E2) secretion in granulosa cells. The binding relationship between miR-21 and LATS1 was verified by dual-luciferase reporter assay and RNA binding protein immunoprecipitation assay (RIP) assay. Additionally, Immunoprecipitation assay was carried out to confirm Lysyl oxidase like 2 (LOXL2) was phosphorylated by large tumor suppressor 1 (LATS1). Finally, the binding relationships between Yes-associated protein (YAP), StAR and LOXL2 were verified by dual-luciferase reporter assay and/or chromatin immunoprecipitation assay (ChIP) assay. Here our results displayed that miR-21 was overexpressed in hucMSCs and hucMSCs-derived exosomes, compared with that ovarian granulosa cells. hucMSC-exo with overexpressing miR-21 could markedly promote the secretion of estrogen in ovarian granulosa cells. LATS1 overexpression in ovarian granulosa cells reduced the secretion of estrogen. We subsequently confirmed that LATS1 was the target of miR-21. In addition, LATS1 could regulate StAR expression by phosphorylating LOXL2 and YAP. miR-21 carried by hucMSCs-derived exosomes could downregulate LATS1, thereby reducing phosphorylated LOXL2 and YAP, and ultimately promoting estrogen secretion in ovarian granulosa cells.

Cai JH ，Sun YT ，Bao S 《-》