Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biol
Open Access
ISSN: 2375-4508
ISSN: 2375-4508
Hanieh Motahari Rad*, Fatemeh Akbarian, Zohreh Hashemian, Seyed Javad Mowla, Mojtaba Rezazadeh Valojerdi
Purpose: Polycystic Ovarian Syndrome (PCOS) is a metabolic syndrome that is regulated by several genes and miRs. In a previous study, we showed that miR-647 was significantly downregulated in PCOS patients compared with normal controls. Furthermore, miR-647 exhibited a negative correlation with CYP11A1 in both follicular fluid and granulosa cells. The purpose of this study was to gain a preliminary understanding of the role of miR-647 in granulosa cells and its impact on the prognosis of PCOS patients.
Methods: A miR-647 Precursor (premiR) and a Sponge (miR-sp) were designed for the overexpression and knockdown of miR-647. Wild-type and mutant 3'-UTRs of CYP11A1 were also prepared as specific binding sites for miR-647. On the basis of bioinformatics studies, the low level of miR-647 in HEK-293T cells makes it suitable for testing the effectiveness of the premiR construct. A dual luciferase reporter assay was conducted to assess the function of miR-sp. The effect of miR-sp was investigated in GCN-01 (granulosa cells) cells by determining the expression of related genes via qRT?PCR. ELISA was used to quantify steroid hormones in the conditioned media of miR-sp-GCN-01.
Results: The significant reduction in miR-647, along with the significant increase in AR and CYP family genes in miR-sp-GCN-01, revealed a molecular pattern that closely resembled androgenic PCOS. The results of the luciferase reporter assay confirmed the specificity of miR-sp. Moreover, the levels of the hormones estradiol and progesterone significantly decreased in miR-sp-GCN-01.
Conclusion: Our data demonstrate that PCOS conditions can be simulated at the cellular level by downregulating miR-647, making it a potential in vitro model for PCOS.
Published Date: 2025-08-19; Received Date: 2024-07-31