miR‐221‐3p regulates apoptosis of ovarian granulosa cells via targeting FOXO1 in older women with diminished ovarian reserve (DOR)

Abstract In our earlier study, we showed that the expression of microRNA‐221‐3p (miR‐221‐3p) was significantly lower in women of advanced age with diminished ovarian reserve (DOR) compared with young women with normal ovarian reserve (NOR). Therefore, in this study, we aimed to explore how miR‐221‐3p regulates apoptosis of granulosa cells and the pathogenesis of DOR. Bioinformatics prediction and dual‐luciferase reporter assay were conducted to identify the target gene of miR‐221‐3p. miR‐221‐3p expression was manipulated by transfecting KGN cells with miR‐221‐3p mimics, inhibitor, and negative control. Following transfection, apoptosis of granulosa cells was determined by flow cytometry, and the expression of the target gene was measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis (WB). In addition, the expression of the target gene in granulosa cells of DOR patients and NOR patients was measured. miR‐221‐3p were found to directly bind the 3ʹ untranslated region of Forkhead box O1 ( FOXO1 ). Transfection with miR‐221‐3p mimics significantly decreased the apoptosis rate of KGN cells compared with transfection with miR‐221‐3p inhibitors. The expression level of miR‐221‐3p was negatively correlated with the messenger RNA and protein levels of the FOXO1 gene. Besides, FOXO1 expression was upregulated in DOR patients. In conclusion, these results provide evidence that downregulation of miR‐221‐3p expression promotes apoptosis of granulosa cells by upregulating FOXO1 expression, thus serving an important role in DOR pathogenesis.