Study On Molecular Mechanisms Of BMP2 Regulating Ovulation-related Genes Expression In Human Granulosa Cells And Its Correlation With PCOS

Normal follicular development is a complex dynamic physiological process,in addition to strict regulation of pituitary-derived gonadotrophins,locally produced growth factors and cytokines also exert their effects in a paracrine/autocrine fashion.As an important member of the TGF-β superfamily,BMP2 is significantly expressed in ovarian granulosa cells and corpus luteum.Previous studies showed that BMP2 promotes the expression of aromatase and FSH receptors in granulosa cells to increase estrogen production,and on the other hand,reduces the production of progesterone through inhibiting the expression of StAR and LH receptors,thereby exerting its regulatory effects on follicular development.However,studies of BMP2 on female reproduction still need further investigation.So,the current study focused on the following three parts to explore the regulation of BMP2 on ovulation-related genes in human granulosa cells and its correlation with PCOS,in order to provide molecular evidence for the prevention and treatment of ovulation abnormalities and PCOS in clinical applications:1.To investigate the effect of BMP2 on the expression of SERPINE2 and the underlying mechanisms.2.To investigate the effect of BMP2 on the regulation of GREM2 expression and explore its inhibitory effect on GDF8 induced signaling pathway and biological function.3.To investigate the effect of BMP2 on the regulation of IL-6 expression and its correlation with PCOS.PartⅠ Regulation of BMP2 on the expression of plasminogen activator inhibitor SERPINE2 in human granulosa cells and its mechanismObjective:To elaborate the molecular mechanism of BMP2 regulating the expression of SERPINE2 and verify its function in human granulosa cellsMethods:Different concentration of BMP2 were prepared base on gradient dilution,then treated immortalized human granular cells(SVOG)and primary cultured human granular cells(hGL)for 12 hours and 24 hours,respectively.RT-PCR and Western Blot was used to detect the dose-dependent effect of BMP2 on the regulation of SERPINE2 mRNA and protein expression.SVOG cells were treated with BMP2(50 ng/mL)for 1,3,6,12,and 24 hours to explore the time dependent effect.TGF-βsuperfamily specific receptor inhibitors and small interfering RNA were applied to specifically knock down the endogenous expression of related molecules in the signaling pathways to explore the molecular mechanisms.The levels of uPA and tPA in the cell culture medium after siSERPINE2 were detected by ELISA.Results:In a dose-and time-dependent manner,BMP2 up-regulates the expression of SERPINE2.TGF-β family-specific type I receptor inhibitors dorsomorphin and DMH-1 or siALK3 can completely block the promotion of BMP2 on the expression of SERPINE2.BMP2 can activate the canonical SMAD1/5/8 signaling pathway and the non-canonical SMAD2/3 and P38 MAPK signaling pathways.The non-canonical SMAD2/3 and P38 MAPK signaling pathways are involved in the up-regulation of SERPINE2 by BMP2.BMP2 inhibits the production of uPA and tPA by up-regulating the expression of SERPINE2.Conclusion:BMP2 up-regulates the expression of SERPINE2 through non-canonical SMAD2/3 and P38 MAPK signaling pathways,thereby inhibiting the production of uPA and tPA.Part II Regulation of BMP2 on the expression of GREM2 and its inhibitory effect on GDF8 induced signaling pathwayObjective:To investigate the effect of BMP2 on the regulation of GREM2 expression and explore its inhibitory effect on GDF8 induced signaling pathwayand biological function.Methods:Different concentration of BMP2 were prepared base on gradient dilution,then treated immortalized human granular cells(SVOG)and primary cultured human granular cells(hGL)for 12 hours and 24 hours,respectively.RT-PCR and ELISA was used to detect the dose-dependent effect of BMP2 on the regulation of GREM2 mRNA and protein expression.SVOG cells were treated with 50 ng/mL BMP2 for 1,3,6,12,and 24 hours to explore the time dependent effect.TGF-β family specific receptor inhibitors and small interfering RNA were applied to specifically knock down the endogenous expression of related molecules in the signaling pathways to explore the underlying molecular mechanisms.BMP2 and GREM2 treated SVOG and hGL cells simultaneously for 60 minutes,the protein expression of P-SMAD 2,P-SMAD 3 and P-SMAD 1/5/8 were detected by Western Blot to verify the inhibitory effect of GREM2 on BMP2 induced signaling pathways.BMP2 pretreated SVOG and hGL cells for 24 hours,GDF8 treated SVOG cells for another 60 minutes,the protein expression of P-SMAD 2 and P-SMAD 3 were detected by Western Blot to explore the inhibitory effect of BMP2 on GDF8 induced signal pathways and biological function.Results:In a dose-and time-dependent manner,BMP2 significantly up-regulated the expression of GREM2.The TGF-β family-specific type I receptor inhibitors DMH-1 and dorsomorphin or siALK2 or siALK3 totally blocked the increase of GREM2 expression by BMP2.The classic SMAD1/5/8 signaling pathway was involved in the up-regulation of GREM2 induced by BMP2.GREM2 significantly inhibited the phosphorylation of SMAD1/5/8 and SMAD2/3 induced by BMP2.GREM2 also significantly inhibited the phosphorylation of SMAD2 and SMAD3 induced by GDF8.BMP2 inhibited GDF8-induced phosphorylation of SMAD2/3 and PAI-1 expression by up-regulating the production of GREM2.Conclusion:BMP2 inhibits signaling pathways and biological function induced by GDF8 through up-regulating the expression of GREM2.Part III Regulatory mechanism of BMP2 on the expression of interleukin 6(IL-6)in human granulosa cells and its correlation with PCOSObjective:To illustrate the molecular mechanism of BMP2 regulating the expression of IL-6 and elaborate its correlation with the development of PCOS in human granulosa cellsMethods:Different concentration of BMP2 were prepared base on gradient dilution,then treated immortalized human granular cells(SVOG)and primary cultured human granular cells(hGL)for 1 hours and 2 hours,respectively.RT-PCR and Western Blot was used to detect the dose-dependent effect of BMP2 on the regulation of IL-6 mRNA and protein expression.TGF-β superfamily specific receptor inhibitors and small interfering RNA were applied to specifically knock down the endogenous expression of related molecules in the signaling pathways to explore the molecular mechanisms.The follicular fluid of PCOS and non-PCOS patients were harvested,the expression of IL-6 mRNA and protein expression were determined after extracting the granulosa cells.The correlation of BMP2 and IL-6 levels in PCOS and non-PCOS patients were detected using ELISA.Results:BMP2 up-regulates the mRNA and protein expression of IL-6 expression significantly.TGF-β family-specific type Ⅰ receptor inhibitors dorsomorphin and DMH-1 or siALK3 totally block the up-regulating effect of BMP2 on IL-6 expression.Inhibition of SMAD or p-NFkB signaling pathway both partially block the regulation of BMP2 on IL-6 expression.Using siRNA and inhibitors to block the SMAD and p-NFkB signaling pathway simultaneously totally inhibited the promotion of BMP2 on IL-6 expression.BMP2 and IL-6 expression in the granulosa cells of PCOS patients are higher than those in non-PCOS patients,moreover,there is a significant positive correlation between them.Conclusion:BMP2 up-regulates the expression of IL-6 through SMAD and p-NFkB signaling pathways.BMP2 is involved in the development of PCOS by promoting IL-6 expression.