De-SUMOylation Protease SENP3 Is Involved In Testosterone Induced Autophagy In Granulosa Cells Of Women With PCOS

Objective:1.To explore the effect of testosterone on autophagy of ovarian granulosa cells in PCOS patients;2.To study whether SENP3 is involved in autophagy induced by testosterone and its effect on cell function;3.To study the changes in autophagy and cell function under the simultaneous action of chloroquine,sh-SENP3 and testosterone.Methods:1.Using western and immunofluorescence staining detected the expression and localization of autophagy protein and SENP3 of granulosa cells in PCOS and control group;2.Treating KGN cells with testosterone at different concentrations and time,western was used to detect protein LC3BII/I,SQSTM1/p62,CYP19A1 and SENP3 expression.CCK-8 method was used to disclose the cell viability;3.After SENP3 knockdown and overexpression,KGN cells were treated with 10μM testosterone for 0 h,6 h,12 h,and 24 h,respectively.The protein LC3BII/I,SQSTM1/p62,CYP19A1 and SENP3 expression was used to detected by western;4.Under the conditions of SENP3 knockdown,chloroquine and testosterone,western was used to detect the expression of protein LC3BII/I,SQSTM1/p62,CYP19A1 and SENP3.Results:1.Western results showed that the enhanced autophagy(increased LC3BII/I)and decreased SENP3 expression of granulosa cells in PCOS,and immunofluorescence staining showed that SENP3 is mainly located in the nucleus,and highly accumulated in the nucleolus;2.Testosterone up-regulated autophagy and down-regulated SENP3 and aromatase CYP19A1 in KGN cells in a dose-and time-dependent manner,but cell viability had no significant change;3.SENP3 knockdown enhanced the LC3B-II levels and decreased aromatase expression in KGN cells.This enhanced autophagy response to testosterone treatment and discounted estrogen signing were obviously amplificated in the combined context of sh-SENP3 and chloroquine treatment;4.The overexpression of SENP3 weakened the effect of testosterone on KGN cell autophagy and aromatase.Conclusion:1.Granulosa cells from women with PCOS showed enhanced autophagic activation and decreased SENP3 expression;2.Hyperandrogenism led to enhanced autophagy and decreased estrogen signaling in PCOS by downregulating SENP3 in granulosa cells;3.We presumed that SENP3 was a negative regulator of autophagy in the granulosa cells.SENP3 and de SUMOylation might be involved in the pathological mechanism of PCOS and become a potential therapeutic target for PCOS.