Effects of MiR-375-BMPR2 as a Key Factor Downstream of BMP15/GDF9 on the Smad1/5/8 and Smad2/3 Signaling Pathways
Effects of MiR-375-BMPR2 as a Key Factor Downstream of BMP15/GDF9 on the Smad1/5/8 and Smad2/3 Signaling Pathways
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MiR-375-BMPR2 作为 BMP15/GDF9 下游关键因子对 Smad1/5/8 和 Smad2/3 信号通路的影响
DOI：
10.1159/000488424
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发表时间：
2018-01-01
影响因子：
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通讯作者：
Zhang, Jiabao
中科院分区：
文献类型：
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作者：
Liu, Chang;Yuan, Bao;Zhang, Jiabao
背景/目标：骨形态发生蛋白15（BMP 15）和生长分化因子9（GDF 9）是由卵母细胞分泌的，是卵泡生长发育和卵巢功能的重要调节因子。这两种因子通过调节Smad信号通路，可以调节卵丘细胞的增殖和凋亡。研究表明，BMP 15和GDF 9可以影响miR-375的水平，而miR-375的靶基因是BMP 15和GDF 9的II型受体BMPR 2。然而，BMP 15/GDF 9-miR-375-BMPR 2通路是否或如何通过调节Smad信号通路影响牛卵丘细胞的增殖和凋亡仍不清楚。方法：本研究首先从卵丘-卵母细胞复合体（COCs）中获得卵丘细胞。在体外培养过程中加入适当浓度的BMP 15和GDF 9。采用CCK-8法和流式细胞仪检测BMP 15/GDF 9对牛卵丘细胞增殖和凋亡的影响。随后，合成miR-375模拟物、miR-375抑制剂和BMPR 2 siRNA并用于转染实验。Western Blot分析转染前后BMP 15/GDF 9 I型受体ALK 4、ALK 5和ALK 6的表达水平，BMP 15/GDF 9下游关键信号通路蛋白Smad 2/3和Smad 1/5/8的磷酸化水平，卵丘细胞增殖关键基因PTX 3、HAS 2和PTGS 2的表达水平，卵丘细胞增殖相关基因的表达水平，卵丘细胞增殖相关基因的表达水平。和Bcl 2/Bax，它们是参与细胞凋亡的基因。结果如下：添加100 ng/mL BMP 15或200 ng/mL GDF 9或50 ng/mL BMP 15和100 ng/mL GDF 9联合添加可有效抑制牛卵丘细胞凋亡并促进细胞增殖。BMP 15/GDF 9负调控miR-375表达，正调控BMPR 2表达。高水平的miR-375和BMPR 2的抑制导致ALK 4的表达增加和PTX 3、HAS 2和PTGS 2的表达减少，而miR-375的抑制导致相反的结果。BMP 15和GDF 9显著激活p-Smad 2/3和p-Smad 1/5/8的水平，而miR-375通过负性调节BMPR 2抑制p-Smad 2/3和p-Smad 1/5/8的水平，并导致细胞凋亡。结论：BMP 15和GDF 9具有协同作用，可通过miR-375影响I型受体ALK 4和II型受体BMPR 2的表达水平，激活Smad信号通路，进而影响卵丘细胞的增殖、铺展和凋亡。
Background/Aims: Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are secreted by oocytes, are important regulators of follicular growth and development and ovarian function. These two factors can regulate the proliferation and apoptosis of cumulus cells via modulation of the Smad signaling pathway. Studies have shown that BMP15 and GDF9 can affect the level of miR-375, whereas the target gene of miR-375 is BMPR2, the type II receptor of BMP15 and GDF9. However, whether or how the BMP15/ GDF9-miR-375-BMPR2 pathway affects the proliferation and apoptosis of bovine cumulus cells through regulation of the Smad signaling pathway remains unclear. Methods: In this study, cumulus cells were first obtained from cumulus-oocyte complexes (COCs). Appropriate concentrations of BMP15 and GDF9 were added during the in vitro culture process. Cell Counting Kit-8 (CCK-8) analyses and flow cytometry were used to determine the effects of BMP15/GDF9 on bovine cumulus cells proliferation and apoptosis. Subsequently, miR-375 mimics, miR-375 inhibitor and BMPR2 siRNA were synthesized and used for transfection experiments. Western Blot analysis was used to detect changes before and after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4, ALK5 and ALK6; the phosphorylation levels of Smad2/3 and Smad1/5/8, which are key signaling pathway proteins downstream of BMP15/GDF9; the expression levels of PTX3, HAS2 and PTGS2, which are key genes involved in cumulus cells proliferation; and Bcl2/Bax, which are genes involved in apoptosis. Results: The addition of 100 ng/mL BMP15 or 200 ng/mL GDF9 or the combined addition of 50 ng/mL BMP15 and 100 ng/mL GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation. BMP15/GDF9 negatively regulated miR-375 expression and positively regulated BMPR2 expression. High levels of miR-375 and inhibition of BMPR2 resulted in increased expression of ALK4 and decreased expression of PTX3, HAS2 and PTGS2, whereas miR-375 inhibition resulted in the opposite results. BMP15 and GDF9 significantly activated the levels of p-Smad2/3 and p-Smad1/5/8, whereas miR-375 inhibited the levels of p-Smad2/3 and p-Smad1/5/8 by negatively regulating BMPR2 and also led to apoptosis. Conclusion: BMP15 and GDF9 have synergistic effects and can act through miR-375 to affect the expression levels of type I receptor ALK4 and type II receptor BMPR2 and the activation of Smad signaling pathway, which subsequently affected the proliferation, spread and apoptosis of cumulus cells.