Umbilical cord mesenchymal stem cells-derived extracellular vesicles improve excessive autophagy of granulosa cells through METTL3.

Abstract

Objective: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder impacting women's fertility. We assessed the effect of umbilical cord mesenchymal stem cell-derived extracellular vesicles (UC-MSC-EVs) on PTEN-induced kinase 1 (PINK1)/Parkin-mediated excessive autophagy of ovarian granulosa cells (GCs) through methyltransferase-like 3 (METTL3). Methods: Human ovarian GC line KGN was cultured and treated with dehydroepiandrosterone (DHEA) and UC-MSC-EVs. Cell apoptosis and viability, autophagy-related protein levels, adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) level, and microtubule-associated protein 1 light chain 3 beta (LC3B) and translocase of outer mitochondrial membrane 20 (TOMM20) co-localization were assessed by flow cytometry, CCK-8, western blot, kit, and immunofluorescence. PINK1 N6-methyladenosine (m6A) modification, METTL3 levels, and PINK1 mRNA stability were determined by Me-RIP, RT-qPCR and western blot. The PCOS mouse model was established and treated with UC-MSC-EVs. Serum hormone and ovarian tissue autophagy-related protein levels were determined by ELISA. Results: DHEA decreased KGN cell viability and p62 level, increased PINK1, Parkin, LC3BII/I and Beclin-1 protein levels, ATP content, MMP level, TOMM20⁺LC3B⁺ cell number and apoptosis, which were partly abrogated by UC-MSC-EVs treatment. PINK1 had m6A modification sites. METTL3 was a PINK1 m6A-modified Writer protein. After DHEA treatment, KGN cells showed elevated METTL3 and PINK1 m6A modification levels and mRNA stability, while UC-MSC-EV treatment caused the opposite results. METTL3 overexpression partly averted UC-MSC-EVs-improved PINK1/Parkin-mediated mitophagy. UC-MSC-EVs curbed PINK1/Parkin-mediated excessive autophagy through METTL3 and improved ovarian function in PCOS mice. Conclusions: UC-MSC-EVs suppressed PINK1/Parkin-mediated mitophagy of ovarian GCs through METTL3, thereby improving PCOS.