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KLF12转录抑制FOXO1表达导致子宫内膜蜕膜化障碍研究
【作者】 张慧；
【导师】 孙海翔；
【作者基本信息】 南京医科大学 ， 妇产科学， 2015， 硕士
【摘要】 Krüppel样转录因子家族成员KLF12抑制蜕膜化过程中标志基因泌乳素(Prolactin,PRL)和胰岛素样生长因子结合蛋白(Insulin-like growth factor binding protein 1,IGFBP1)的表达及人子宫内膜间质细胞(human endometrial stromal cells,HESCs)细胞骨架的重构,负向调控HESCs的蜕膜化过程。但具体调控机制及生物学意义不清。本文旨在探讨KLF12调控子宫内膜间质细胞蜕膜化的分子机制及其与临床反复种植失败疾病的相关性。自然妊娠实验结果显示KLF12高表达小鼠(n=11)胚胎种植过程受抑制,胚胎种植率较对照组(n=11)下降38.6%(p=0.0051)。人工蜕膜化动物实验进一步提示KLF12抑制子宫内膜的蜕膜化过程,导致假孕胚胎数减少,诱导蜕膜化侧子宫角重量减轻,胎盘形成障碍,并且小鼠蜕膜化标志分子dPRP表达明显下降(降低约200倍)。此外,启动蜕膜化过程的重要分子FOXO1的mRNA水平下降。实时定量PCR和Western Blot实验结果显示,过表达KLF12可抑制FOXO1mRNA和蛋白的表达,8-Br-cAMP联合醋酸甲羟孕酮(medroxyprogesterone,MPA)体外诱导蜕膜化后KLF12对FOXO1的抑制作用更显著。染色质免疫共沉淀/PCR、链亲和素生物素偶联DNA沉淀反应及荧光素酶报告基因实验证实KLF12可直接结合于FOXO1基因转录起始位点上游-2624/-2617bp区的CAGTGGG序列,直接转录抑制子宫内膜间质细胞中FOXO1的表达。临床子宫内膜样本调查研究显示,反复种植失败(repeated implantation failure,RIF)病人子宫内膜组织中KLF12表达异常增高,而其靶分子FOXO1的表达则较正常对照组明显降低。相关性分析结果显示RIF病人内膜组织中FOXO1与KLF12蛋白水平呈中度负相关关系。综上,我们推测RIF病人内膜组织中异常高表达的KLF12通过直接转录抑制蜕膜化关键分子FOXO1的表达负向调控子宫内膜蜕膜化,抑制胚胎种植,这可能是RIF病人胚胎种植失败的潜在分子机制,为临床原因不明的RIF病人提供诊断和治疗依据。
【Abstract】 KLF12,a member of Krüppel-like transcriptional factor family,repressed decidual marker genes PRL and IGFBP1 expression.When KLF12 overexpression,human endometrial stromal cells(HESCs)displayed a poorly formed,diffuse network of actin cytoskeleton.KLF12 negatively regulated HESCs decidualization,however,little is known about the effector mechanism involved.Based on the previous description,this study aimed to explore the mechanism of KLF12 during the decidualization of endometrial stromal cells and provide a new line of evidence correlating the repeated implantation failure(RIF).Using natural pregnant mouse,we reported that KLF12 inhibited the mouse embryo implantation process.The rate of embryo implantation decreased 38.6% which was observed in the uteri with KLF12 overexpression(n=11,p=0.0051).We further demonstrated that KLF12 interfered with the pseudopregnant implantation sites and uterine weight ratio of the stimulus to control,as well as placental formation via artifical decidualization mouse model.Using real-time PCR,we showed that the expression of mouse decidual marker gene dPRP was significantly decreased(about200 times);FOXO1 mRNA level decreased as well.Using isolated uterine stromal cells,we showed adenovirus-mediated overexpression of KLF12 in HESCs markedly attenuated the level of FOXO1 mRNA and protein.Moreover,the decidualized stimulus of 8-Br-cAMP plus MPA enlarged the downregulation.By chromatin immunoprecipitation(ChIP)PCR,Avdin-biotin conjugate DNA precipitation and luciferase reporter gene assay,we observed KLF12 bound to the CAGTGGG sequence in the promotor of FOXO1,which was located in the-2624/-2617 bp from the transcription start site,and transcriptionally repressed FOXO1.Clinical endometrial sample studies showed that KLF12 were abnormally overexpressed in the endometrium of women with repeated implantation failure(RIF).By contrast,mRNA and protein level of FOXO1 were less than that of control group.Concomitantly,the protein level of FOXO1 was moderately negatively correlated with that of KLF12 in patients with RIF(r=-0.4272395,p=0.04202).In conclusion,all the results above indicated that,by transcriptional regulation FOXO1 expression,KLF12 repressed the decidualization process in vivo and vitro.We further explained the potential molecular mechanism of RIF from the point of impaired decidualization,which would served as a diagnose and treatment evidence for the unexplained RIF.