靶向调控子宫内膜异位症性不孕在位内膜囊性纤维化跨膜调节因子(CFTR)的microRNA筛选与鉴定研究

Endometriosis associated infertility is the common gynecologic disorder in women of reproductive age. Endometriosis seriously affects female fertility. However, the pathogenesis is unclear. It is considered that the peritoneal environment, ovarian function and endometrial receptivity could be potentially involved in. Based on our previous microRNA microarray, combined with bioinformatics and genetics, we speculate that in eutopic endometrium of endometriosis down-regulated microRNAs may regulate cystic fibrosis transmembrane conductance regulator (CFTR), which leads to repaired endometrial receptivity, resulting in embryo implantation failure and infertility. In this study we plan to verify the microRNA microarray result and select the goal microRNA, detect the expression and localization of CFTR in eutopic endometrium of women with endometriosis. Then transfection experiment and luciferase reporter assay will be used to determine the regulation of miRNA on CFTR expression and its mechanism. The endometriosis animal model of nude mice is used to investigate expression of microRNAs and its downstream gene CFTR in vivo. The purpose of the study is to explore the pathogenesis of endometriosis associated infertility, which will provide new experimental basis for clinical practice.

子宫内膜异位症性不孕是育龄期妇女的常见病，严重影响女性生育力。迄今，其导致不孕的机制尚不明确，可能涉及盆腔内环境、卵巢功能和在位内膜容受性等。我们根据前期microRNA芯片结果，利用生物信息学软件预测到内异症在位内膜中低表达的microRNAs可能靶向调控囊性纤维化跨膜调节因子(CFTR)的表达，从而引起在位内膜容受性异常，影响胚胎着床导致不孕。本研究首先验证芯片结果，进一步筛选microRNA，并检测CFTR在内异症在位内膜中的表达情况，通过转染、双荧光素酶实验确定microRNA对CFTR的调控作用和靶标位点，并在内异症裸鼠模型验证相关microRNA的影响及干预效果。通过研究，探索内异症性不孕的发病机制，为临床诊治提供理论依据。

子宫内膜异位症在位内膜容受性异常被认为是胚胎着床失败和早期自然流产的重要原因之一。既往研究表明子宫内膜中囊性纤维化跨膜调节因子（CFTR）在胚胎着床窗口发挥关键作用，并提示CFTR异常与早期自然流产相关。根据microRNA芯片和生物信息学软件预测结果，内异症在位内膜低表达的miR-505-3p可能靶向调控CFTR表达。本研究首先检测内异症合并早期流产患者和非内异症早期妊娠正常妇女在位内膜中miR-505-3p和CFTR的表达情况，并通过双荧光素酶实验和转染Ishikawa细胞株体外实验确定miR-505-3p对CFTR的靶标位点和体外调控作用，最后在小鼠模型中验证miR-505-3p对CFTR的体内调控作用及对妊娠结局的影响。结果显示，与对照组相比，RT-qPCR、免疫组化显示内异症早期流产患者在位内膜中miR-505-3p显著下调，同时CFTRmRNA和蛋白水平显著上调。双荧光素酶报告实验证实miR-505-3p对CFTR的靶向调控位点。在ISK细胞株和内异症裸鼠模型中，过表达miR-505-3p显著抑制CFTR，而抑制miR-505-3p后CFTR表达明显升高。在早期自然流产小鼠模型中，转染miR-505-3p后流产率降低，而CFTR过表达则使流产率升高。miR-505-3p表达抑制后流产率有升高趋势，同时CFTR表达抑制流产率则显著下降。有趣的是，我们还发现了miR-505-3p对小鼠植入胚胎数量的影响。这些新发现提示内膜miR-505-3p不但是胚胎植入的保护性因素，而且miR-505-3p靶向CFTR可降低小鼠的流产率。本研究提示内异症在位内膜miR-505-3p异常是内异症性不孕子宫内膜容受性受损导致胚胎着床失败和早期妊娠丢失的一个关键性原因，从而为临床上开发特异性的诊疗策略提供了潜在线索和理论依据。