Hyperandrogen induces apoptosis of human ovarian granulosa cells and up-regulation of PDCD4

Abstract

Objective To investigate the role of PDCD4 in apoptosis of human granulosa cells and to investigate whether hyperandrogen can promote the up-regulation of PDCD4 and participate in the apoptosis of human ovarian granulosa cells. Methods The PDCD4 was overexpressed by transfection of PDCD4 expression construct in human ovarian granulosa cell line KGN cells and the transfection efficiency was detected by fluorescence quantitative PCR. The effect of overexpression of PDCD4 on apoptosis of KGN cells was detected by flow cytometry and Annexin V staining. The KGN cells were treated with testosterone of different concentration gradients (0, 10-7 mol/L, 10-6 mol/L, 10-5 mol/L). The expression of PDCD4 was detected by real-time quantitative PCR and Western blotting after 24 h and 48 h of testosterone treatment. The apoptotic effect of testosterone on granulosa cells was detected by flow cytometry after 48 h. PDCD4 small interfering RNA was used to knock down the expression of PDCD4 in KGN cells treated with high concentration of testosterone (10-5 mol/L). Flow cytometry was used to detect the effect of PDCD4 knockdown on apoptosis induced by high androgen. Results After overexpression of PDCD4 in KGN cells, results of Annexin V-PE staining and flow cytometry showed a significant increase in apoptosis of KGN cells (P=0.014). KGN cells were treated with different concentrations of testosterone. Compared with NC group, the expression level of PDCD4 mRNA in 10-5 mol/L group and 10-6 mol/L group was up-regulated (24 h: P=0.035, P=0.038; 48 h: P=0.029, P=0.009); the expression level of PDCD4 protein was also up-regulated. After treatment of KGN cells with high concentration of testosterone (10-6 mol/L and 10-5 mol/L) for 48 h, apoptosis of granulosa cells increased significantly (P=0.036 in 10-6 mol/L group compared with NC group; P=0.028 in 10-5 mol/L group compared with NC group). After knockdown of PDCD4 expression in KGN cells treated with 10-5 mol/L testosterone, the apoptosis rate was significantly lower than that in the non knockdown group (P=0.038). Conclusion PDCD4 can promote the apoptosis of human ovarian granulosa cells. High concentration of testosterone can induce the apoptosis and up-regulate the expression of PDCD4 in granulosa cells. The mechanism of apoptosis of granulosa cells induced by high concentration of testosterone may be related to apoptotic pathway mediated by PDCD4. Key words: Androgen; Ovarian granulosa cells; Apoptosis; PDCD4; Polycystic ovary syndrome