锌指蛋白KLF12转录抑制SPHK1调控卵巢颗粒细胞凋亡与卵泡发育的机制研究

卵巢颗粒细胞凋亡启动卵泡闭锁的调控机制不清。我们最新研究发现转录因子KLF12显著增加颗粒细胞凋亡和caspase 3活化，并抑制颗粒细胞中抗凋亡分子1-磷酸鞘氨醇（S1P）的生成。腺病毒介导的KLF12过表达明显抑制细胞中AKT和FOXO1的磷酸化修饰，但增加FOXO1的乙酰化修饰。生物信息学分析发现鞘氨醇激酶1（SPHK1）为KLF12可能的下游调控靶分子。推测颗粒细胞中KLF12通过转录抑制SPHK1表达，减少细胞中S1P生成，进而调节AKT磷酸化和FOXO1的磷酸化及乙酰化修饰，加速FOXO1蛋白的核输入，促进颗粒细胞凋亡，加速各级卵泡的闭锁，是导致卵巢功能衰退的一个新机制。本研究将重点探讨KLF12抑制细胞中S1P生成调控颗粒细胞凋亡的分子机制；并利用Klf12卵巢颗粒细胞特异敲入小鼠模型整体实验研究Klf12在小鼠卵泡自主发育中的作用，以阐明颗粒细胞凋亡导致卵巢功能衰退的机制。

The precise mechanisms of granulosa cell apoptosis leading to accelerated follicular atresia in mammalian ovaries are unknown. Our studies demonstrated that the Krüppel-like factor 12 (KLF12) significantly induces granulosa cell apoptosis and caspase 3 activation. Moreover, the adenovirus-mediated overexpression of KLF12 markedly increases intracellular levels of pro-survival sphingosine-1-phosphate (S1P) in cultured human luteinized granulosa cells (hLGC) and KGN cells. Western blot analysis reveals that the level of AKT and FOXO1 phosphorylation decreased and FOXO1 acetylation increased in KLF12-overexpressing and H2O2-treated KGN cells. Based on the human SPHK1 promoter sequence deposited in the transcriptional regulatory element database, we found that SPHK1 is a potential KLF12 downstream target gene. So, our hypothesis on the possible mechanism for follicular atresia is that KLF12 induces granulosa cell apoptosis by repression of the expression of its target gene SPHK1, which decreases S1P production and the post-translational modification of FOXO1 and nuclear accumulation. We will further address the mechanism of granulosa cell apoptosis and follicular atresia through KLF12-mediated SPHK1/S1P pathway in vitro and in Klf12 knock-in mice model. Our study might be elucidating of the molecular control of ovarian dysfunction by transcription factor KLF12.