Mechanisms Of SGK3 Involved In The Dysfunctional Folliculogenesis And Estradiol Biosynthesis In PCOS

Aim Polycystic ovary syndrome(PCOS)is the most common cause of anovulatory infertility in reproductive aged women,but the etiology remains poorly understood.The dysfunction in proliferation,apoptosis and steroidogenesis of the ovarian granulosa cells is believed to be associated with the disturbance of follicle development.Serum-glucocorticoid-inducible kinase 3(SGK3)is a Ser/Thr kinase,which belongs to the “AGC” kinase family and acts as downstream of PI3 K.SGK3plays an important role in Akt-independent signaling in human cancer by regulating ion channels,glucose homeostasis,cell proliferation,survival,and migration.Our previous study has found that the expression of SGK3 was significantly up-regulated in granulose cells of PCOS patients compared to that of non-PCOS group.In this study,we aim to investigate the function of SGK3 in the granulosa cell and its possible regulatory mechanism.MethodsThe follicular fluid and serum of PCOS patients and non-PCOS group were collected on the oocytes retrieval day,and their granulosa cells were obtained for RNA and protein extraction.The abundance of SGK3 m RNA in uncultured granulosa cells were measured by q-RT PCR,and protein of SGK3 were measured by western blot.T In vitro,to detected the effect of dihydrotestosterone(DHT)on the expression of SGK3,cultured granulosa cells and KGN cell line were treated with various DHT concentrations with and without androgen receptor(AR)blockage.Furthermore,after knocking down of SGK3 with small interfering RNA,the proliferation of cultured granulosa cells and KGN cell line were detected by CCK8 assay,Ed U assay,and the immunofluorescence staining of Ki-67.The effect of knocking down of SGK3 on cell cycle progression is analyzed by flow cytometry;meanwhile the expression of CDK protein family members is detected by western blot.The estradiol concentrations in the culture medium were assayed by ELISA,and the expression of aromatase were detected by q-RT PCR and western blot.Results(1)The abundance of SGK3 m RNA and protein level were significantly increased in granulosa cells of PCOS patients,and there was a positive correlation between SGK3 m RNA abundance and the level of serum basal testosterone.(2)Androgen promoted the expression of SGK3 in a dose-dependent manner in KGN cell line and cultured granulosa cells.(3)Androgen stimulates SGK3 gene transcription via androgen receptor in KGN cell line and cultured granulosa cells.(4)Knockdown of SGK3 expression promoted the proliferation of KGN cell line and cultured granulosa cells.(5)Knockdown of SGK3 expression promoted the G1 to S transition in KGN cell line and cultured granulosa cells.(6)Knockdown of SGK3 expression promoted aromatase expression and estradiol biosynthesis in KGN cell line and cultured granulosa cells.ConclusionsPCOS women had increased expression of SGK3 in granulosa cells,and the expression of SGK3 is associated with higher circulating androgen level.Androgen promoted the expression of SGK3 in KGN cell line and cultured granulosa cells,and the effect could be blocked by knock down of AR.The knockdown of SGK3 in KGN and cultured granulosa cells promoted cell proliferation by promoting the G1 to S transition.In addition,knockdown of SGK3 promote aromatase expression and estradiol production.Therefore,our findings shows that increased SGK3 in granulosa cells of PCOS patients is due to the increased androgen level,which may contribute to the aberrant folliculogenesis and abnormal aromatase activity,and in turn leads to hyperandrogensim drastically.