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

Abstract

Background 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). Methods 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. Results 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. Conclusions 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.

Bone marrow mesenchymal stem cells in premature ovarian failure: Mechanisms and prospects

“…Cai et al also found that hUMSCs-derived miR-21 could inhibit the expression of LATS1, so as to reduce phosphorylated LOXL2 and YAP, and ultimately promote E2 secretion in ovarian GCs (196). Moreover, hUMSCs-derived miR-126-3p promote proliferation while inhibit the apoptosis of GCs through PIK3R2/PI3K/AKT/mTOR pathway (197). Similarly, Yang et al demonstrated that hUMSCs exosome transplantation could ameliorate ovarian function through promoting angiogenesis by activating PI3K/AKT signaling pathway (198).…”

“…also found that hUMSCs-derived miR-21 could inhibit the expression of LATS1, so as to reduce phosphorylated LOXL2 and YAP, and ultimately promote E2 secretion in ovarian GCs ( 196 ). Moreover, hUMSCs-derived miR-126-3p promote proliferation while inhibit the apoptosis of GCs through PIK3R2/PI3K/AKT/mTOR pathway ( 197 ). Similarly, Yang et al.…”

Stem cell-derived extracellular vesicles: A novel and potential remedy for primary ovarian insufficiency

“…There are round or oval membranous vesicles, which can be aggregated and distributed, and their membrane structure is clearly defined ( Vohra et al, 2020 ). hUCMSCs-EVs express the EVs-specific four-transmembrane protein markers CD9, CD63, CD81 and tumor susceptibility gene 101 protein (TSG101), heat shock protein70 (HSP70), and the multivesicular biosynthesis-related protein ALIX ( Sun et al, 2017 ; Yang et al, 2019 ; Zhang et al, 2020 ; Deng et al, 2021 ; Cai et al, 2022 ; Gao et al, 2022 ; Qu et al, 2022 ). In 2017, hUCMSCs- EVs were first described for application to POI by Sun et al (2017) .…”

“…Not surprisingly, stem cell-based therapies hold tremendous potential for treating POI in both preclinical and clinical trials ( Ding et al, 2018 ; Yin et al, 2020 ; Fu et al, 2021 ; Mashayekhi et al, 2021 ; Wang et al, 2022a ). Recent research suggests stem cells may provide therapeutic effects by paracrine means, specifically using extracellular vesicles (EVs) that include exosomes ( Zhang et al, 2019a ; Liu et al, 2021 ; Qu et al, 2022 ). The diameters of EVs range from approximately 30 nm–3,000 nm, and their biomolecular composition determines their functions, as well as their source and conditions ( Riazifar et al, 2017 ).…”

Messenger roles of extracellular vesicles during fertilization of gametes, development and implantation: Recent advances

“…EVs play a critical role in each process. For example, EVs can recruit stem cells ( Liang et al, 2020 ; Wang D et al, 2022 ) and transmit stem cell information ( Qu et al, 2022 ). Cells in the lesion site will release EVs in addition to recruitment factors to recruit surrounding tissues or peripheral stem cells ( Chen et al, 2022 ).…”