Abstract

Background. Diminished ovarian reserve (DOR) is a common cause of female infertility. Our previous high-throughput sequencing study demonstrated a specific expression profile of follicular fluid-derived exosomal microRNAs (miRNAs) in DOR patients, suggesting that exosomal miRNAs may play a critical role in the pathogenesis and progression of DOR. Using a miRNA PCR array, we identified that miR-483-3p is markedly upregulated in exosomes derived from the follicular fluid of DOR patients. Bioinformatic and experimental analyses revealed that METTL3 is a potential target gene of miR-483-3p. This suggests that excess accumulation of miR-483-3p may have cytotoxic effects and also exert an inhibitory effect on METTL3-mediated N6-methyladenosine (m6A). Therefore, this study aimed to investigate the effects of miR-483-3p on METTL3 expression, m6A modification levels, cell proliferation, and mitochondrial function in ovarian granulosa cells (GCs).

Methods. The targeting interaction between miR-483-3p and METTL3 was confirmed using a dual-luciferase reporter gene assay. RT-qPCR, cellular immunofluorescence, Western blot, and Dot blot were used to evaluate the effects of miR-483-3p mimics, cyclophosphamide (CTX), and STM2457 on METTL3 and m6A expression levels in KGN cells. Additionally, cell counting kit-8 (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) assays, along with mitochondrial membrane potential (MMP) measurements, were used to examine the impact of these treatments on KGN cell proliferation and mitochondrial function.

Results. miR-483-3p directly targets the 3 ′ untranslated region (3 ′ UTR) of METTL3, and its mimics significantly suppress METTL3 gene expression. In stark contrast, CTX robustly promoted METTL3 gene expression, concomitant with a substantial elevation in global m6A levels. STM2457 treatment also showed a similar trend. The inhibitory effect of miR-483-3p overexpression on METTL3 protein expression and m6A levels was modest, suggesting the presence of complex post-transcriptional regulatory mechanisms that may buffer protein and m6A changes despite gene expression suppression. Regarding cellular phenotypes, miR-483-3p overexpression, CTX, and STM2457 all exerted significant inhibitory effects on KGN cells. All three treatments consistently suppressed the proliferative activity of KGN cells, reduced the EdU-positive cell ratio, and decreased MMP levels.

Conclusion. This study demonstrated that aberrantly high expression of miR-483-3p significantly suppresses METTL3 mRNA expression but only moderately reduces METTL3 protein and m6A levels, indicating complex post-transcriptional regulatory mechanisms. Furthermore, overexpression of miR-483-3p markedly inhibited the proliferation of GCs and impaired their mitochondrial function. These findings suggested that overaccumulation of miR-483-3p may contribute to the pathogenesis of DOR.