The Role Of FHL2 In Follicular Growth And Ovulatory Obstacles Of Polycystic Ovary Syndrome

Aims The follicular growth and ovulatory dysfunction mechanisms underlying polycystic ovary syndrome(PCOS)are not completely understood.There is no effective therapy for PCOS so far.The aim of this study was to explore the role of FHL2 in follicular growth and ovulatory obstacles of polycystic ovary syndrome.Methods 1.We measured the expression of FHL2 and other related-genes in human granulosa cells(h GCs)from patients with and without PCOS;2.Human primary granulosa cells were cultured to investigate the effects of h CG on the expression of FHL2 and other ovulation-related genes;3.We used human granulosa-like KGN cells for in vitro experiments to investigate the molecular mechanisms underlying FHL2-related pathways in follicular growth and ovulatory obstacles of PCOS;4.We analysed the correlation between the basal testosterone and the abundance of FHL2 in patients with PCOS(37 samples)using Spearman rank correlation analysis and used DHT to determine whether androgen stimulation can enhance the expression of FHL2 in vitro;5.We established the FHL2-overexpressing rat ovaries via intrabursal injection of lentivirus vectors to further explore the role of FHL2 in regulating ovulation in vivo;6.KGN cells were cultured for proliferation assays and cell cycle analysis after transfection with FHL2 si RNA,and the proliferation-related genes were examined after transfection;7.KGN cells were cultured to investigate the effect of IL-1β on proliferation assays,cell cycle analysis and the expression of proliferation-related genes;8.KGN cells were cultured to investigate the molecular mechanisms underlying FHL2- related pathways in cell proliferation and cell cycle;8.We measured the abundance of IL-1β and CCND1 in human granulosa cells(h GCs)from patients with and without PCOS;Using Spearman rank correlation analysis,we analysed the correlation between the abundance of FHL2 and the abundance of IL-1β in patients with PCOS(37 samples).Results Part 1 1.Compared with non-PCOS patients,increased FHL2 and androgen receptor(AR)m RNA and decreased C/EBPβ m RNA were observed in h GCs from patients with PCOS.2.Treatment with h CG negatively regulated the m RNA and protein abundance of FHL2 and AR in h GCs,and increased the expression levels of phosphorylated ERK1/2,C/EBPβ,COX2 and HAS2.3.Treatment with Forskolin mimicked LH surge on KGN cells presented similar expression patterns of ovulation-related genes as h CG-induced expression patterns in h GCs;FHL2 inhibited the expression of ovulation-related genes,including phosphorylated ERK1/2,C/EBPβ,COX2 and HAS2,and it was partially by interacting with AR to act as its co-regulator to inhibit C/EBPβ expression and by binding to ERK1/2 to inhibit its phosphorylation.4.FHL2 abundance in h GCs was positively correlated with the basal serum testosterone concentration of patients with PCOS,and dihydrotestosterone(DHT)-induced FHL2 upregulation was mediated by AR signalling in KGN cells.5.Lentiviral-mediated functional FHL2 overexpression in rat ovaries for 1 week contributed to an impaired superovulatory response,displaying decreased numbers of retrieved oocytes and a lower MII oocyte rate;3-week FHL2 overexpression rat models without superovulation led to acyclicity and polycystic ovary morphology.Part 2 1.Knockdown of FHL2 inhibited cell proliferation and cell cycle progression;The expression levels of PCNA and Cyclin D1 were decreased by FHL2 knockdown.2.Treatment with IL-1β increased cell proliferation,cell cycle progression and the expression levels of NF-κB p-p65/p65-total,PCNA and Cyclin D1.3.Knockdown of NF-κB p65 inhibited cell proliferation and cell cycle progression,also decreased the expression of PCNA and Cyclin D1;NF-κB p65 increased the expression of Cyclin D1 by binding to the promoter of CCND1.4.FHL2 promoted the expression of Cyclin D1 by binding to p65 to increase its phosphorylation in order to act as its co-regulator.5.Treatment with IL-1β in the presence or absence of FHL2 si RNA demonstrated that IL-1β increased cell proliferation and cell cycle,which was partially mediated by FHL2.6.Compared with non-PCOS patients,increased IL-1β and CCND1 m RNA were observed in h GCs from patients with PCOS;FHL2 abundance in h GCs was positively correlated with the abundance of IL-1β of patients with PCOS.Conclusions We have demonstrated in this study that upregulated FHL2 may be a contribution to acyclicity,polycystic ovary morphology and anovulation in PCOS patients,and the possible underlying mechanisms that FHL2 inhibited ovulation by targeting AR and ERK signalling pathways.Up-regulated FHL2 in h GCs of PCOS may promote cell proliferation and cell cycle by binding to p65 to increase its phosphorylation in order to act as its co-regulator to promote the expression of CCND1.IL-1β increased cell proliferation and cell cycle,which may be partially mediated by FHL2.Therefore,we assumed that FHL2-induced granulosa cell proliferation and cell cycle progression may relate to follicular growth obstacles.