外泌体携带miR-214对子宫内膜异位症纤维化的作用及其机制研究

Endometriosis (EMs) is a common gynecologic disease affecting women’s health, while EMs fibrosis is the main cause of female pelvic pain and infertility. Our previous experiments have shown that the connective tissue growth factor (CTGF/CCN2) was associated with fibrosis in EMs in vitro and might be directly regulated by miR-214. In animal experiments, the expression levels of sustained response gene CCN2 and its downstream fibrosis associated proteins were remarkly suppressed after animal models received exosome carried with miR-214 mimics. The level of miR-214 in circulating exosome was significantly lower than that in total serum. Thus, we hypothesize that exosomal miR-214 could inhibit the fibrosis in EMs through directly regulating CCN2, and miR-214 could be mainly packaged and delivered by exosome to be involved in celluar membrane transportation. In order to verify this hypothesis, we intend to test the binding site between miR-214 and CCN2 by luciferase reporter assay, and to explore the possibility that miR-214 could inhibit the fibrosis formation in human EMs fibrosis cells model and the ectopic endometrium tissue of EMs animal model based on the condition that the CCN2 was silenced. Lastly, we plan to compare the level of miR-214 in total serum to its level in exosome. Based on these experiments, we aim to investigate the function and the underlying mechanism of miR-214 targeting CCN2 in the development of fibrosis in EMs, and to investigate the feasibility that circulating exosome carried with miR-214 could be served as a non-invasive biomarker for early detection of EMs. The results may indicate an innovative method of the molecular therapy on EMs.

子宫内膜异位症（EMs）是危害女性健康的常见疾病，EMs纤维化是引发盆腔痛和不孕的重要因素。本课题前期体外实验发现，结缔组织生长因子（CCN2）与EMs纤维化相关，并很可能受miR214调控。动物实验载有miR214拟似物的外泌体可降低纤维化持续应答基因CCN2及下游纤维化蛋白表达。临床血清miR214低于血清外泌体内miR214。由此提出假说：miR214通过直接调控CCN2作用EMs纤维化，miR214可由外泌体介导跨细胞膜传递而发挥作用。为验证假说，本项目拟通过荧光素酶报告质粒验证miR214与CCN2的结合位点；细胞和动物实验抑制CCN2基因后，调整miR214水平检测纤维化蛋白变化；同时对比EMs与非EMs血清外泌体miR214水平差异。以阐明miR214调控CCN2在EMs纤维化产生中的作用及机制，探索外泌体携带miR214作为纤维化无创标记物的可能，提出EMs分子治疗新思路。

子宫异位内膜组织纤维化是导致患者不孕或盆腔疼痛的主要原因，为了探究纤维化发生的机制，本项目根据前期结果提出：miR214通过直接调控CCN2作用EMs纤维化，miR214可由外泌体介导跨细胞膜传递而发挥作用。为验证该假说，我们首先进行组织水平检测，发现与EMs在位内膜组织及非子宫内膜异位症病人内膜组织相比，纤维化相关蛋白Collagen1及α-SMA表达升高，同时EMs异位组织中miR-214显著下调，CCN2显著上调。进一步对原代分离的间质细胞，分别转染miR-214（miR-214-3p或miR-214-5p分别实验，最后明确miR-214-3p是主要发挥作用的分子）拟似物和抑制剂，在此基础上使用qRT-PCR检测miR-214-3p及CCN2表达水平，证实miR-214-3p通过靶向作用于CCN2抑制EMs纤维化发生。同时通过细胞培养上清液外泌体检测试剂盒SBI ExoQuick-TC成功提出外泌体，使用Wb的方法检测其标志性分子CD63及CD9的表达。为了明确外泌体在miR-214-3p细胞间转运的核心作用，我们首先使用PKH67绿色荧光标记外泌体，红色荧光标记的miR-214-3p分子追踪miR-214-3p转运过程。进一步通过细胞共培养实验，通过外泌体分泌抑制剂GW4869阻断外泌体功能，反向验证实验结果。最终得出结论外泌体携带miR-214-3p在EMs间质细胞间发挥转运作用，调节下游基因CCN2，从而调节子宫内膜异位症纤维化过程。同时，我们成功构建了动物模型，这些实验结果在动物实验中得到进一步验证。本项目最后证实目EMs病人血清中外泌体miR-214-3p水平低于非子宫内膜异位症病人。进而得到外泌体miR-214-3p通过靶向抑制CCN2进而抑制EMs纤维化发生，miR-214-3p可作为EMs的生物标志物并具有潜在的治疗价值。