雌激素通过ERα介导lncRNA 1200076调节卵巢ERα（+）细胞生物学行为

We have observed that estrogen(E2)deregulated lncRNA 1200076 expression in ERα（+）ovarian cells. Through bioinformatic analysis, we found that lncRNA 1200076 might be regulated by ERα and regulate its predicted cis target ING4,which might contrubute to cell proliferation, apoptosis, motility and migration. Based on such finding, we are trying to do further research in the current study which include three parts: (1) verifying that lncRNA-TC0101441 is regulated by ERα through a serial of assays including using competitive antagonist of E2 and ERα-shRNA, dual-luciferase reporter gene and ChIP assay; (2) comfirming that ING4 is the cis target of lncRNA-TC0101441 and elucidating the regulation mechanism of lncRNA-TC0101441 on ING4 through a serial of assays including over-expression/interference technology, RNA pulldown and mass spectral analysis, Delete assay, RIP and ChIP assay; (3) investigating roles of lncRNA1200076 on ERα（+）ovarian cell proliferation, apoptosis, motility and migration both in vitro and in vivo. Through all these experiments, we believe that the results might help provide the perspective of lncRNA to get a novel insight into the regulation mechanism of E2-ERα pathway,which may be helpful to better understand the pathogenesis of estrogen dependent ovarian diseases.

我们发现：雌激素（E2）降调节卵巢ERα(+)细胞长链非编码RNA(lncRNA 1200076)。生物信息学预测：E2可介导ERα降调lnRNA1200076，调控临近靶基因ING4，介导卵巢ERα（+）细胞生物学行为。本课题拟研究以下三方面：①通过ER抑制剂、ERα干扰、荧光素酶报告基因、ChIP实验，证明lncRNA 1200076受E2/ERα调控；②通过过表达/干扰技术、RNA Pulldown与质谱分析、启动子区域截取研究、RIP、ChIP实验，证明ING4是lncRNA 1200076临近靶基因，探索其中调控机制；③过表达/干扰lncRNA 1200076，通过体内外实验，研究lncRNA 1200076对卵巢ERα（+）细胞增殖、凋亡、运动及迁移能力影响。本课题将从lncRNA角度揭示E2/ERα途径调控卵巢ERα(+)细胞的新机制，以利于研究雌激素依赖性卵巢疾患发病机制。

前期芯片发现雌激素（E2）显著上调卵巢ERα阳性(+)细胞长链非编码RNA- TC0101441（命名为ElncRNA1）的表达。但E2上调ElncRNA1的机制以及ElncRNA1在卵巢ERα(+)细胞中的功能尚未涉及。基于此，本项目展开的一系列实验研究，结果如下：①生物信息学分析发现ERE序列位于ElncRNA1转录起始位点上游59 bp。通过ERα抑制剂、ERα干扰、ChIP及双荧光素酶报告基因实验证实E2诱导ElncRNA1的上调是经典ERα-ERE途径依赖的。②借助ERα(+)SKOV3、CAOV3及HO8910细胞模型，展开体外细胞功能实验，发现在细胞增殖、凋亡、周期、迁移及侵袭表型中，干扰和过表达ElncRNA1对ERα(+)卵巢细胞迁移侵袭能力的影响最为显著，提示ElncRNA1在ERα(+)卵巢细胞迁移侵袭中扮演着重要的角色。③活体成像及大体解剖结果均表明干扰ElncRNA1显著抑制裸鼠腹腔种植瘤的播散转移，提示在体内，ElncRNA1能促进ERα(+)卵巢细胞的转移。④借助转移基因PCR芯片及qRT-PCR、Western验证发现受ElncRNA调节的基因多为 EMT相关基因，初步表明ElncRNA1可能通过调控EMT赋予了ERα(+)卵巢细胞转移的表型。这些结果将从lncRNA角度揭示E2/ERα途径调控卵巢ERα(+)细胞的新机制，以利于研究雌激素依赖性卵巢疾患发病机制。