转录因子KLF12转录抑制LIF参与子宫内膜容受性调控的机制研究

Uterine receptivity plays a key role in the establishment of successful pregnancies, and its impairment may limit ART success and contribute to the subfertility in some gynecological diseases. Our studies demonstrated that Krüppel-like factors 12 (KLF12) expression is markedly up-regulated in the endometrium of RIF (repeated implantation failure) women and decreased after decidualization stimulated by 8-Br-cAMP plus medroxyprogesterone acetate (MPA). Moreover, adenovirus-mediated overexpression of KLF12 significantly down-regulated the endometrial receptivity marker LIF mRNA expression in Ishikawa cells and markedly decreased the embryo implantation rates in mouse uterus. Based on the LIF promoter sequence deposited in the transcriptional regulatory element database,,we found that LIF maybe the target gene of KLF12. Therefore, our hypothesis is that in RIF patients, dysregulated expression of KLF12 impairs endometrial receptivity and embryo implantation through down-regulating LIF expression and STAT3 phosphorylation. Here, the main focus of this study was on the role of KLF12 impaired uterine receptivity by regulating the expression of LIF in vitro and in Klf12 knock-in mice model. Our study might be necessary for evaluation of KLF12 and its target genes may be a novel therapeutic target to overcome repeated implantation failure.

子宫内膜容受性缺陷是导致胚胎种植失败的主要因素，也是制约IVF-ET妊娠成功率的瓶颈，但具体调控机制不清。我们临床调查发现RIF患者子宫内膜组织中KLF12蛋白表达异常升高，而在内膜间质细胞体外蜕膜化过程中表达明显减少；过表达KLF12明显抑制Ishikawa细胞中LIF mRNA的表达和下游分子STAT3的磷酸化并显著降低小鼠胚泡的着床率。作为子宫内膜容受性的标志分子，LIF启动子区含有KLF12保守的结合序列CAGTGGG。因此，我们推测RIF患者内膜组织中异常高表达的KLF12转录抑制容受性标志分子LIF的表达，并通过影响STAT3磷酸化，抑制胚胎黏附，最终导致胚胎种植失败。本研究将重点探讨KLF12参与容受性分子LIF调控的机制；并通过Klf12基因敲入小鼠模型在体研究Klf12对子宫内膜容受性的影响，期望阐明IVF-ET周期中反复种植失败患者胚胎不能正常着床的分子机制。

辅助生殖技术的不断发展，单个周期移植的妊娠率和着床率不断提高，但仍有10%-15%的不孕症患者经历多次移植仍未获妊娠，其中三分之二是子宫内膜容受性缺陷，但反复种植失败的具体机制仍不甚清楚。我们研究发现反复种植失败患者子宫内膜组织中KLF12的表达异常增高，同时着床标志分子LIF的表达降低，两者表达呈负相关；细胞学实验证实KLF12高表达可以显著抑制LIF的mRNA及和蛋白的表达水平，尤其明显抑制E2联合MPA诱导的LIF表达，进而抑制LIF下游通路STAT3的Tyr705位点磷酸化，影响下游通路的激活，但不影响STAT3总蛋白本身的表达。通过生物信息学分析LIF启动子包含2个KLF12特异结合位点，ChIP/PCR及ABCD实验证实KLF12均可直接结合于启动子区的两个CAGTGGG序列；高表达KLF12抑制LIF启动子的转录活性，影响LIF mRNA和蛋白的表达，进而显著抑制BeWo细胞球黏附率，补充人同源重组LIF蛋白（hLIF）可以逆转KLF12对BeWo细胞球粘附的抑制作用；同样的，利用小鼠囊胚-上皮细胞黏附模型亦发现KLF12高表达明显影响小鼠囊胚黏附的稳定性。.构建Klf12子宫特异性敲入的小鼠模型中，小鼠的繁殖能力受影响，虽然在一窝产仔数上并无明显差异，子宫特异性敲入的鼠只生育2-4代。且子宫特异性高表达Klf12后，小鼠子宫组织中Lif蛋白的表达明显降低，Stat3的磷酸化受抑制，而Stat3总蛋白无明显变化，与体外实验结果一致。综上，反复种植失败患者子宫内膜组织中KLF12的表达异常增加，通过直接抑制LIF转录及表达，影响胚胎黏附，最终导致胚胎种植失败，这可能是反复种植失败患者胚胎不着床的一个重要新机制。