miR-6388 regulates granulosa cell function in sheep by targeting GDF9 and modulating the TGF-β signaling pathway

Litter size is an economically important trait in sheep and is closely associated with ovarian follicular development and granulosa cell (GC) function. This study investigated the association between GDF9 polymorphisms and litter size, and examined the post-transcriptional regulation of GDF9 by miR-6388 in ovine GCs. Variants were initially identified by Sanger sequencing in 20 ewes, and subsequently genotyped in 377 three-year-old ewes, including 231 Sonid (SN) sheep and 146 Ujimqin (UM) sheep for association analysis. Candidate miRNAs targeting litter size-associated variants in the GDF9 3'UTR were predicted, and the miR-6388-GDF9 interaction was evaluated using dual-luciferase reporter assays. RT-qPCR, Western blotting, EdU incorporation, and flow cytometry were used to assess endogenous GDF9 expression and GC function. Twelve single-nucleotide polymorphisms were identified, including the putatively novel variant g.42114076C > G. The linkage disequilibrium block comprising g.42116936C > T, g.42113821T > A, and g.42113962G > A polymorphisms of GDF9 was significantly associated with litter size in both SN and UM sheep, whereas the c.477G > A was associated with litter size only in UM sheep. Reporter assays showed that the GDF9 3'UTR region carrying the G allele of g.42113962G > A was more responsive to miR-6388-mediated repression than the region carrying the A allele. miR-6388 overexpression reduced GDF9 mRNA and GDF9 protein levels, inhibited GC proliferation, altered cell-cycle distribution, and promoted apoptosis, whereas miR-6388 knockdown increased GDF9 expression and GC proliferation and reduced apoptosis. These cellular changes were accompanied by altered expression of cell-cycle and apoptosis-related genes and TGF-β signaling-related components.