miR‑206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis

Abstract

An increasing number of studies have reported that microRNAs (miRNAs) have an important role in polycystic ovary syndrome (PCOS). Downregulation of miR-206 in patients with PCOS has been found, however, its specific role remains unclear. The present study aimed to investigate the roles of miR-206 in (PCOS) and to determine the underlying molecular mechanisms. Reverse transcription-quantitative PCR (RT-qPCR) was performed to analyze the expression levels of miR-206 in normal ovarian surface epithelial IOSE80 cells and human ovarian granulosa cell-like KGN cells. TargetScan was used to predict the target gene of miR-206, which was subsequently verified using a dual-luciferase reporter gene assay. The mRNA expression levels of cyclin D2 (CCND2) and the transfection efficiencies of the miR-206 mimic and CCDN2 overexpression plasmid were determined using RT-qPCR analysis. The protein expression levels of CCND2, cleaved-caspase-3 and pro-caspase-3 were analyzed using western blotting, and an MTT assay and flow cytometric analysis were used to evaluate the cell viability and levels of apoptosis, respectively, in the cells following transfection. Finally, the activity of caspase-3 was analyzed using a caspase-3 activity assay kit. The results of the present study revealed that the expression levels of miR-206 were downregulated in KGN cells compared with IOSE80 cells. CCND2 was predicted and verified to be a direct target gene of miR-206, and the mRNA and protein expression levels of CCND2 were discovered to be upregulated in KGN cells compared with IOSE80 cells. The miR-206 mimic and CCND2 overexpression plasmid significantly upregulated the expression levels of miR-206 and CCND2, respectively, in KGN cells. The miR-206 mimic also downregulated the expression levels of CCND2 in KGN cells, while this effect was reversed following the transfection with the CCND2 overexpression plasmid. Compared with the mimic control group, the miR-206 mimic significantly decreased the cell viability, induced the levels of apoptosis, increased the activity of caspase-3, upregulated cleaved-caspase-3 protein expression levels and downregulated pro-caspase-3 protein expression levels in KGN cells following transfection; these effects were reversed following the overexpression of CCND2. In conclusion, the findings of the present study suggested that miR-206 may serve an important role in PCOS through modulating ovarian granulosa cell viability and apoptosis.