Overaccumulation of miR-483-3p exerts acute toxic effects on ovarian granulosa cells by impairing cell proliferation, mitochondrial function, and METTL3-mediated m6A modification

Background Diminished ovarian reserve (DOR) is a common cause of female infertility. Prior high-throughput sequencing showed specific follicular fluid exosomal miRNA profiles in DOR patients, implicating exosomal miRNAs in DOR pathogenesis. Using miRNA PCR arrays, we found miR-483-3p was significantly upregulated in DOR follicular fluid exosomes. Bioinformatics and experiments indicated METTL3 as a potential miR-483-3p target, suggesting excess miR-483-3p exerts cytotoxicity and suppresses METTL3-mediated N6-methyladenosine (m6A) modification. This study thus investigated miR-483-3p effects on METTL3 expression, m6A 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. qRT-PCR, immunofluorescence, Western blot, and Dot blot were used to evaluate the effects of miR-483-3p mimics, cyclophosphamide (CTX), and STM2457 on METTL3 protein expression and m6A modification levels in KGN cells. Additionally, Cell Counting Kit-8 (CCK-8) 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 targeted the 3′ untranslated region (3′UTR) of METTL3, and its mimics significantly suppressed METTL3 gene expression. In contrast, CTX robustly promoted METTL3 gene expression, accompanied by a substantial elevation in global m6A levels. STM2457 treatment also showed a similar trend. But miR-483-3p overexpression modestly inhibited METTL3 protein and m6A levels. Regarding cellular phenotypes, miR-483-3p overexpression, CTX, and STM2457 all exerted significant inhibitory effects on KGN cells. All three treatments consistently suppressed the cell viability of KGN cells, reduced the EdU-positive cell ratio, and decreased MMP levels. Conclusion This study provides preliminary evidence suggesting a specific regulatory interaction between miR-483-3p and METTL3. We demonstrate that aberrantly high expression of miR-483-3p significantly suppresses METTL3 mRNA levels but only moderately reduces METTL3 protein and m6A levels, suggesting the involvement of complex post-transcriptional regulatory mechanisms. Furthermore, overaccumulation of miR-483-3p markedly inhibits the proliferation of ovarian GCs and impairs their mitochondrial function. Further investigation into the role of the candidate miR-483-3p-METTL3-m6A axis in DOR will be conducted using in vivo models alongside an expanded cohort of clinical samples.