miR-155-5p/CEBPB对子宫腺肌病内膜上皮细胞功能的调节机制

Adenomyosis is a common gynecological disease characterized by the presence of endometrial stroma and glands located deep within the myometrium. The aetiology and pathogenic mechanisms of adenomyosis are poorly understood, which further lead to the lack of effective diagnosis and treatment. Recently，we identified the aberrant expression of miR-155-5p/CEBPB in eutopic endometrium of patients with adenomyosis. We hypothesized that miR-155-5p/CEBPB may involve in the pathogenesis of adenomyosis through regulating the function of endometrial epithelial cells. In the present project, we will explore the molecular mechanism in three levels including the clinical samples, cell lines, and animal models. We will analyze the regulating role of miR-155-5p on CEBPB expression; investigate the involvement of miR-155-5p/CEBPB pathway in the pathological function of endometrial epithelial cells; explore the downstream genes and signal pathways regulated by C/EBPβ; reveal the role of miR-155-5p/CEBPB pathway in vivo by using uterus specific miR-155-5p knockdown and miR-155-5p/CEBPB blocked mouse models. Our project may improve our understanding of the pathogenesis of adenomyosis and provide theory basis for further clinical treatment and new drug development.

子宫腺肌病（AM）是妇科常见严重疾病，其发病机制尚不完全清楚，缺乏有效诊治手段。依据本课题组前期研究发现miR-155-5p/CEBPB在AM中存在异常表达，提出miR-155-5p/CEBPB通路调控子宫内膜上皮细胞增殖、侵袭等生物学功能，参与AM发生发展的科学假说。本项目将在临床样本、细胞系和动物模型三个层面，探索miR-155-5p/CEBPB通路在AM发生中的作用机制。研究miR-155-5p对CEBPB异常表达上调的调节作用；探讨miR-155-5p/CEBPB通路对人内膜上皮细胞功能的影响；从转录调控水平探寻C/EBPβ下游关键靶基因和信号通路；建立子宫局部miR-155-5p敲低和特异性阻断miR-155-5p/CEBPB相互作用小鼠模型，在体研究miR-155-5p/CEBPB的作用机制。研究结果将为探索AM发病机理提供理论基础，为AM临床治疗和新药研发提供技术依据。

子宫腺肌病（AM）患者的在位内膜在疾病的发病中有至关重要的作用，然而在位内膜中与AM发病机理有关的特定基因仍有待阐明。通过从AM患者的在位子宫内膜中鉴定差异表达基因（DEGs）和分子网络，在转录水平为疾病的机制研究提供新的途径。通过Ingenuity Pathway Analysis（IPA）分析DEGs的功能、经典通路和下游生物过程，通过MassArray系统进行CEBPB基因的DNA甲基化定量分析。生物信息学分析预测，AM中白介素6（IL-6）信号和ERK/MAPK信号被激活。DEGs主要参与调控下游细胞增殖和侵袭。子宫腺肌病CEBPB的表达增加和DNA低甲基化状态。采用qPCR检测miR-155-5p和CEBPB在AM的在位内膜、异位病灶和对照组子宫内膜中的表达水平，采用免疫组化检测CCAAT增强子结合蛋白β（CCAAT enhancer binding protein beta, C/EBPβ）的定位和表达水平。miR-155-5p的表达在AM在位内膜和异位病灶中显著低于对照组内膜，而CEBPB在AM在位内膜和异位病灶中的表达显著高于对照组内膜。通过CCK8和克隆形成实验进行检测子宫内膜上皮细胞Ishikawa的增殖能力，通过Transwell实验检测细胞迁移和侵袭能力，miR-155-5p过表达显著抑制Ishikawa增殖、迁移和侵袭能力，而抑制miR-155-5p表达则效果相反。采用双荧光素酶报告基因实验验证miR-155-5p与CEBPB的靶向调控作用。双荧光素酶报告实验也证实miR-155-5p靶向CEBPB的3’UTR区调控其转录活性。回复实验表明miR-155-5p通过CEBPB调节Ishikawa细胞的增殖、迁移和侵袭抑制作用。研究揭示了AM在位内膜的关键途径和网络，首次提出CEBPB与AM之间的相关性。miR-155-5p可能通过靶向CEBPB影响子宫内膜上皮细胞的增殖、迁移和侵袭的能力，从而参与AM的发病机制。