miR-378a-3p participates in the decline of ovarian reserve function by targeting ZFP36L2 to regulate granulosa cell mitochondrial function and apoptosis

Diminished ovarian reserve (DOR) is associated with impaired female fertility; however, its molecular mechanisms remain incompletely understood. Our previous studies found that individuals with DOR have lower levels of microRNA-378a-3p (miR-378a-3p) expression in their granulosa cells (GCs), yet its role in DOR has not been investigated in depth.

To explore the effects of miR-378a-3p on apoptosis and mitochondrial function in the pathogenesis of DOR.

Identifying the target gene of miR-378a-3p using bioinformatics prediction and dual-luciferase reporter gene analysis, and its expression was quantitatively assessed using qRT-PCR and Western blotting. By transfection of KGN cells to alter the level of miR-378a-3p expression. The role of miR-378a-3p in mitochondrial function and GC apoptosis was investigated via flow cytometry. The levels of miR-378a-3p and its target genes in GCs from patients with DOR and normal ovarian reserve (NOR) were examined, and their expression levels and correlation with assisted reproductive technology (ART) outcomes were explored.

miR-378a-3p directly bound to the 3'UTR of ZFP36L2, confirming ZFP36L2 as its direct target. Compared to the negative control group, inhibition of miR-378a-3p resulted in a marked decrease in mitochondrial membrane potential (MMP) (P < 0.05), along with a significant elevation in reactive oxygen species (ROS) levels (P < 0.05) and a reduction in mitochondrial DNA (mtDNA) content (P < 0.05). Furthermore, transfection with the miR-378a-3p inhibitor significantly reduced the proportion of viable cells and increased the percentages of both early and late apoptotic cells (P < 0.05). miR-378a-3p expression in the DOR group was significantly lower than in the NOR group (P < 0.01), while ZFP36L2 mRNA and protein were significantly upregulated (P < 0.01). ZFP36L2 mRNA in GCs was strongly negatively correlated with miR-378a-3p (r = -0.874, P < 0.01). Furthermore, the expression level of miR-378a-3p showed significant positive correlations with basal ovarian reserve markers AMH (r = 0.736, P < 0.001) and AFC (r = 0.774, P < 0.001), and was also positively correlated with better clinical assisted reproductive outcome parameters, particularly evidenced by a higher quantity of retrieved oocytes (r = 0.680, P < 0.001) and embryos (r = 0.575, P < 0.001).

miR-378a-3p promotes mitochondrial dysfunction and apoptosis in GC by targeting ZFP36L2, and may be involved in the pathogenesis of DOR. This study provides new clues for the molecular mechanism of DOR, but has limitations such as the need for additional rescue/interaction experiments and animal studies.