Melatonin Inhibits Granulosa Cells Apoptosis Of Polycystic Ovary Syndrome By SP1/EGR1-mediated REV-ERBs Upregulation

ObjectiveThe apoptosis of granulosa cells in patients with polycystic ovary syndrome(PCOS)is closely related to follicular developmental disorders,but further studies are needed to elucidate the specific mechanism.This study aimed to investigate the molecular mechanism by which melatonin upregulates REVERBs through SP1/EGR1 mediation to inhibit apoptosis in PCOS granulosa cells.The study utilized both in vitro and in vivo experiments,using the KGN cell line and a rat PCOS model as research objects.MethodsqRT-PCR and Western blot were performed to detect the m RNA and protein levels of REV-ERBα and REV-ERBβ in melatonin-treated KGN cells.TUNEL was used to detect the apoptosis of KGN cells;Ki-67 was used to detect the proliferation of KGN cells;Dual-luciferase reporter assay was used to assess the fluorescence activity of the promoter of REV-ERBα and REV-ERBβ in melatonintreated KGN cells.In order to identify the effect of melatonin on the core promoter regions that regulate transcription,we constructed truncated and mutated versions of the REV-ERBα and REV-ERBβ promoters respectively.Western blot was used to detect the protein expression of SP1 and EGR1 in melatonin-treated KGN cells.RNA interference(RNAi)was used to investigate the effect of transcription factors SP1 and EGR1 on the expression of REV-ERBα and REVERBβ.Effect of SP1 and EGR1 on Bax and Bcl-2 expression were measured by Western blot.To further determine the function of melatonin,PCOS model rats were used to confirm the effect of melatonin on REV-ERBα and REV-ERBβ expression and on the follicle development.ResultsTreatment with melatonin significantly increases the expression of both REV-ERBα and REV-ERBβ in KGN cells.The increase in expression of these genes shows a concentration-dependent and time-dependent effect on growth.After treating KGN cells with melatonin,the apoptosis of KGN cells was significantly decreased and cell proliferation was significantly enhanced compared with the control group.The dual-luciferase reporter assay was used to confirm that melatonin could significantly increase the promoter activity of REV-ERBα and REV-ERBβ.By constructing truncated and mutated fluorescent reporter genes of the REV-ERBα and REV-ERBβ promoters,it was found that melatonin acts at SP1/EGR1 binding sites in REV-ERBα promoter(–261 bp upstream of the transcription start site)and REV-ERBβ promoter(-1981 bp upstream of the transcription start site).when we inhibit SP1 and EGR1 expression by RNA interference,we find that interfering with the expression of SP1 and EGR1 can affect the expression of REV-ERBα and REV-ERBβ proteins in KGN cells,as well as cell apoptosis and proliferation.In the animal experiment,melatonin can reverse the expression of REV-ERBα protein and REV-ERBβ protein in ovarian tissue,and improve abnormal ovarian follicular development.ConclusionsMelatonin can up-regulate the expression of REV-ERBα and REV-ERBβ in granulosa cells,inhibit cell apoptosis and promote cell proliferation.Melatonin can positively regulate the expression of REV-ERBα and REV-ERBβ m RNA by modulating the binding of transcription factors SP1/EGR1 to their promoters.This can inhibit cell apoptosis.Melatonin can up-regulate the expression of REVERBα and REV-ERBβ in PCOS model rats,and effectively inhibit abnormal ovarian follicular development.