The Research On The Molecular Mechanism Of MicroRNA-28-5p On Rat PCOS Ovary Granulosa Cells

ObjectivesPolycystic ovary syndrome(PCOS)is a common endocrine disorder affecting women of reproductive age,with elucidated etiology.MicroRNAs(miRNAs)are noncoding single-stranded RNA,regulating gene expression via translational repression.Methylation is one of the major regulators that control miRNA expression.Studies show that miRNAs are involved in PCOS pathology.In this study,we tested the expression of miRNA-28-5p and its possible target gene PROKl in ovary granulosa cells and the influences on cell proliferation and apoptosis of miRNA-28-5p methylation and miRNA-28-5p/PROK1 signaling axis as well as the signaling pathway,aiming to provide theoretical basis for the application of miRNA-28-5p in the early diagnosis and treatment of PCOS.MethodsWe built PCOS rat models.QRT-PCR and MSP were used to test miRNA-28-5p expression and methylation status in its promoter region respectively of normal and PCOS ovary granulosa cells.CCK-8,EDU,flow cytometry and Western blot were adopted to see the influences on granulosa proliferation and apoptosis of 5-Aza-CdR treatment.PROK1 was screened as the possible target gene of miRNA-28-5p,whose expression in normal and PCOS ovary granulosa cells were shown by qRT-PCR and Western blot.Dual-luciferase reporter gene was employed to confirm the binding between miRNA-28-5p and PROK1.CCK-8,EDU,flow cytometry and Western blot were adopted to study the the influences on granulosa proliferation and apoptosis as well as the signaling pathway of PROK1 overexpression.ResultsMiRNA-28-5p expression was low with methylated CpG in promoter region of PCOS ovary granulosa cells while was high with unmethylated of normal ovaries.Its methylation can be inhibited by 5-Aza-CdR with the expression elevated,leading to the suppression of cell viability and proliferation and promotion of cell cycle arrest and apoptosis of granulosa cells.PROK1 was the possible target gene of miRNA-28-5p,high in PCOS ovary granulosa cells and low in normal ones.Dual-luciferase reporter gene indicated that miRNA-28-5p inhibited PROK1 expreesion by biding to PROK13'-UTR.PROK1 overexpression increased cell viability and proliferation and decreased cycle arrest and apoptosis of granulosa cells.PI3K/AKT/mTOR was one of the signaling pathways of miRNA-28-5p/PROKl signaling axis.ConclusionsMiRNA-28-5p influence granulosa cell proliferation and apoptosis via regulating PROK1 expression through PI3K/AKT/mTOR signaling pathway,which may be involved in the development of PCOS.