Mechanism Of TGF- Beta Regulation Of Boar Sertoli Cells Proliferation

As a regulatory factor of cell proliferation, transforming growth factor-β(TGF-β) plays an important role in mediating Sertoli cell proliferation. However, the detailed mechanism involved and especially the role of miR-24 role in this process are still unclear. The objective of the study is to identify whether TGF-β regulate Sertoli cell proliferation through Smad dependence and non- Smad dependence pathway, decide whether activation of Smad pathway by TGF-β could mediate the expression of P15, P21, c-Myc and Skp2 expression, and explore whether TGF-β regulate the expression of miR-24 to mediate the activity of Smad to promote Sertoli cell proliferation.Firstly, 17β-estradiol stimulated cultured boar immature Sertoli cells in different time to detect the concentration of TGF-β in medium by enzyme linked immunosorbent assay(ELISA). Secondly, different concentrations of TGF-P stimulated cultured Sertoli cells in different time to analysis cell viability and cell cycle through CCK-8 kit and flow cytometry. Thirdly, TGF-β stimulated cultured Sertoli cells in different time to detect the activity of Smad3, ERK1/2 and PI3K by Western blotting. Sertoli cells were pretreated with or without 10-DEBC, LY2109761 and U0126 for 2hr, before stimulation with TGF-β to explore the role of Smad dependence and non- Smad dependence pathway on cell cycle and cell viability. Fourthly, Sertoli cells were pretreated with or without LY2109761 for 2hr, before stimulation with TGF-β to explore the role of Smad dependence pathway on the expression of P15, P21 protein and c-Myc and Skp2 by Western blotting and fluorescence quantitative PCR. Finally, TGF-β stimulated cultured Sertoli cells in different time to detect the expression of miR-24 by fluorescence quantitative PCR. Sertoli cells were treated with or without miR-24 minic and inhibitor with TGF-βto explore the role of miR-24 in the process by Western blotting and fluorescence quantitative PCR.The results are as fellow:(1)17β-estradiol (1×10-9 mol/L) promotes the production of TGF-βin time dependent manner.(2) TGF-β(0-300 pg/mL) regulates cell viability and cell cycle process in time and concentration dependent manner with the TGF-P(180 pg/mL) peaking at 24 h.(3)TGF-β inhibits the phosphorylation of Smad3,but increases the phosphorylation of ERK1/2 and. PI3K in time dependent manner. U0126 (5.0 umol/L),10-DEBC (2.0 umol/L) and LY2109761 inhibits TGF-β induced cell viability and cell cycle, and shows addiative effect.(4)Pretreatment of LY2109761 promotes the expression of P15 and p21, but inhibits the expression of c-myc mRNA and Skp2 mRNA expression.(5)TGF-β enhances the expression of miR-24 in time-dependence manner. However, miR-24 minic inhibits the activity of Smad3, promotes the expression of c-Myc and Skp2 mRNA, and increase cell viability, while miR-24 minic had opposite effect.In conclusion, estrogen can stimulate the production of TGF-β. TGF-β enhances the expression of miR-24,which inhibits the activity of Smad. The reduced activity of Smad reduced the expression of P15 and P21,increases c-Myc and Skp2 mRNA,which promotes Sertoli cell proliferation.On the other hand, TGF-β also regulates Sertoli cell proliferation via activation of ERK1/2 and PI3K signaling pathways.