MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells

BACKGROUND: We elucidated the role of specific MicroRNAs (miRNAs) in the development of polycystic ovary syndrome (PCOS) and explained the changes in the proliferation of granulosa cells. Excised ovarian cortex specimens were collected for miRNA profiling analysis (n = 20 PCOS females and 5 non-PCOS females). Insulin-treated ovarian granulosa cells isolated from mice were used for mechanical studies. RESULTS: High miR-140 expression was observed in PCOS samples and insulin-treated granulosa cells compared to that in non-PCOS and unstimulated cells, respectively. However, the Ras-related protein Rap-2a precursor (RAP2A) was downregulated in in PCOS. MTT assay and EdU staining showed that an miR-140 inhibitor attenuated viability in insulin-treated granulosa cells; cell viability increased with miR-140 overexpression. Reduced expression of miR-140 and the expression of the miR-140 mimic resulted in marked cell apoptosis, as evidenced by the results of PI flow cytometry and Annexin V-FITC; miR-140 overexpression results in downregulated RAP2A expression, and the miR-140 mimic directly bound to the RAP2A 3′-UTR, causing increase in RAP2A levels in insulin-treated granulosa cells; RNA-mediated silencing of RAP2A in insulin-treated granulosa cells restored cell proliferation and apoptosis to normal levels. Phosphorylated AKT was found to be negatively regulated through cross-talk between miR-140 and RAP2A. CONCLUSIONS: In conclusion, PCOS ovarian cortex specimens and insulin-treated granulosa cells showed elevated expression of miR-140, which could lead to increased proliferation and reduced apoptosis of cells by targeting RAP2A. This study may pave the way for future research on the properties of granulosa cells in PCOS.