LncRNA NR-038395作为ceRNA调控miR200s在子宫内膜异位症上皮-间质转化机制中的作用研究

The exact pathogenic mechanism of endometriosis remains unclear and there is no effective therapy in clinic. Epithelial-mesenchymal transition (EMT) might be a key mechanism underlying the induction of invasion and metastasis of tumors. Due to the invasive behavior and cytoskeletal rearrangement of endometrial epithelial cells during ectopic implantation, some studies have demonstrated that EMT might be involved in the development of endometriosis. However, the mechanism of this uterine disorder is poorly understood. Recent studies have found that the decreased expression of miR200s lead to EMT in ectopic endometriosis lesions, promote endometrial invasion and metastasis. LncRNA NR_038395 as a ceRNA could inhibit the expression of miR200s. We hypothesized that lncRNA NR_038395 may be involved in the regulation of miR200s in EMT of endometriosis. Biochemistry, immunology and molecular biology methods will be used to study whether lncRNA NR_038395 could upregulate the expression of target genes ZEB1 and ZEB2 by inhibiting the expression of miR200s in endometriosis and induce the occurrence of EMT and promotes the ectopic endometrial invasion and metastasis by inhibiting E-cadherin expression. This research is to elucidate the molecular mechanism of lncRNA NR_038395 regulatiing miR200s in EMT of endometriosis.

子宫内膜异位症（内异症）的发病机制一直是妇科基础与临床的热点。上皮间质转化（EMT）在许多生命过程和肿瘤侵袭转移中起关键作用。近年来研究表明EMT参与内异症的发生和发展，机制不明。研究发现miR200s在内异症异位病灶中表达下降，通过调控EMT参与异位子宫内膜的侵袭和转移。而lncRNA NR_038395可作为ceRNA可抑制miR200s表达。提示：lncRNA NR_038395可能通过调控miR200s表达参与内异症EMT的发生。为此，本研究采用生物化学、免疫学及分子生物学等方法，研究lncRNA NR_038395在内异症中是否通过抑制miR200s的表达，上调靶基因ZEB1和ZEB2，抑制E-cadherin的表达、导致EMT发生，促进异位子宫内膜的侵袭和转移，并阐明lncRNA NR_038395调控miR200s参与内异症EMT发生的分子机制。

研究目的. 1. 明确HIF-1α、lncRNAUBOX5-AS1及EMT与内异症的关系。. 2. 研究在低氧条件下，HIF-1α调控lncRNAUBOX5-AS1在子宫内膜上皮细胞上皮间质转化中的作用。.研究方法. 1. 利用RT-PCR检测正常内膜组织、内异症患者的在位内膜组织和异位病灶组织中lncRNAUBOX5-AS1的表达情况。. 2. 在低氧（含1%O2）条件下培养EECs和Ishikawa细胞系，利用RT-PCR检测不同时间点lncRNAUBOX5-AS1的表达情况；并利用核浆分离技术检测lncRNAUBOX5-AS1的细胞定位。. 3. 常氧条件下过表达HIF-1α和靶向沉默HIF-1α，检测lncRNAUBOX5-AS1和EMT相关分子标志物的表达；低氧条件下靶向沉默HIF-1α，检测上述指标。. 4. 常氧条件下过表达lncRNAUBOX5-AS1和靶向沉默lncRNAUBOX5-AS1后，检测Ishikawa细胞中EMT相关分子标志物的表达，低氧条件下靶向沉默lncRNAUBOX5-AS1，检测上述指标。. 5. 用Transwell模型检测在以上处理后细胞的迁移和侵袭能力。.研究结果. 1. 与正常内膜组织相比，卵巢子宫内膜异位症患者异位病灶的lncRNA UBOX5-AS1表达明显增加。在位内膜中lncRNA UBOX5-AS1的表达与正常内膜相比无明显差异。. 2. 低氧可诱导EECs和Ishikawa细胞中lncRNA UBOX5-AS1和HIF-1α的表达上调，促进EMT的形成。. 3. LncRNA UBOX5-AS1主要表达在细胞核内，在低氧环境中，细胞浆内lncRNA UBOX5-AS1所占比例有所增加。. 4. 常氧条件下过表达HIF-1α可上调lncRNA UBOX5-AS1，促进内异症细胞的EMT，低氧条件下敲低HIF-1α可降低lncRNA UBOX5-AS1的表达，阻止EMT的发生发展。. 5. 常氧条件下过表达lncRNA UBOX5-AS1促进EMT，敲低后再行低氧处理可阻止EMT进程。.结论. 低氧介导HIF-1α上调lncRNAUBOX5-AS1表达，促进EMT的发生，增强异位内膜侵袭转移，参与内异症的形成和发展。