lncEMRLasp1/miR-203a-3p/Lasp1信号通路在子宫内膜异位症(EMs)间质细胞增殖和迁移中的作用

Based on previous and our recent results, we hypothesize that the expression of lncEMRLasp1 might be upregulted and act as “molecular sponge” to suppress the expression of Lasp1-associated miR-203a-3p, lead to an increased expression of Lasp1 in endometriosis (EMs); Lasp1 would facilitate cell proliferation and migration of eutopic endometrial stromal cells (EESCs) and thus promote the progress of EMs via acting as an actin binding cytoskeletal protein. In this project, we plan to collect the ectopic and paired eutopic endometriums from pathologically confirmed patients with endometriosis, and also the eutopic endometriums from control samples, then we will determine the gene expression levels of lncEMRLasp1 and miR-203a-3p, and also the mRNA and protein expression of Lasp1; construct corresponding transgenic cells of lncEMRLasp1/miR-203a-3p/Lasp1, respectively; then we will explore the potential interactions and molecular mechanisms among the lncEMRLasp1/miR-203a-3p/Lasp1 with RIP, RNA Pull-Down and Dual-Luciferase Reporter Assay System. Finally, we will assess the cellular biological effects of these transgenic cells in vivo, with the aims of elucidating the potential roles of lncEMRLasp1/miR-203a-3p/Lasp1 signaling pathway in the process of cell proliferation and migration in EESCs of EMs, and providing novel insights for further exploring the molecularly biological mechanism for the initiation and progression of EMs.

在总结前人与自身工作基础上，我们假设：EMs时lncEMRLasp1上调，发挥分子海绵作用，抑制miR-203a-3p，上调Lasp1表达；Lasp1发挥其细胞迁移支架蛋白之作用，参与EMs异位间质细胞的增殖和迁移。拟选择临床病理确诊EMs患者在位、异位子宫内膜及对照样品子宫内膜，检测lncEMRLasp1、miR-203a-3p及Lasp1 mRNA与蛋白；构建lncEMR Lasp1/miR-203a-3p/Lasp1相应转基因细胞，应用RIP、RNA Pull-Down、双荧光素酶报告基因技术等探讨lncEMRLasp1/miR-203a-3p/Lasp1间相互作用及其机制；检测母本/转基因细胞生物学特征并在裸鼠体内验证。以求阐明lncEMRLasp1/miR-203a-3p/Lasp1信号通路在EMs间质细胞增殖和迁移中的作用，为深入探讨EMs发生、发展之分子生物学机制提供新思路。

背景:我们前期的双向电泳实验显示LASP1在子宫内膜异位症患者中的表达明显高于对照子宫内膜。然而，LASP1在子宫内膜异位症/子宫腺肌症中调控的分子机制尚不清楚。方法:采用qPCR方法分析LASP1和miR218-5p在子宫内膜异位症(endometriosis, Ems)细胞与对照细胞之间的表达水平。采用荧光原位杂交技术检测miR-218-5p在异位子宫内膜与正常子宫内膜中的表达水平。转染miR-218-5p mimic或inhibitor后，进行transwell实验，观察miR-218-5p对子宫内膜基质细胞(endometrial stromal cell, ESCs)迁移的影响。EdU法测定细胞增殖率。双荧光素酶报告基因检测用于验证hsa-miR-218-5p与LASP1 3’utr的结合。采用Western blot和免疫荧光分析方法检测子宫内膜组织中LASP1和EMT标志物的蛋白表达模式。结果:miR-218-5p主要分泌于血管，表达于子宫内膜周围的肌肉层，在正常的ESCs中，miR-218-5p通过结合LASP1的3’utr区，抑制LASP1的表达水平。miR-218-5p的过表达阻碍上皮-间充质转化(EMT)，阻止ESCs的迁移和Ems中Vimentin的表达。结论:我们的研究结果显示，子宫内膜微环境中的miR-218-5p通过抑制LASP1抑制异位子宫内膜间质细胞的迁移。