子宫内膜异位症过程中LncRNA HAND2-AS1对HAND2的转录调控机制

孕激素耐受与内膜异位症及不育症密切相关。前期研究发现HAND2是孕激素在子宫内膜中的主要效应分子，但其调控机制及在异位内膜症中的作用机理不明。本课题组发现，lncRNA HAND2-AS1与其毗邻的HAND2在内膜细胞中协同表达，该lncRNA有可能通过改变与其共享的双向启动子甲基化参与HAND2的调控。我们拟采用人为干扰HAND2-AS1的表达、双向启动子报告质粒的构建及RNA/DNA免疫共沉淀等方法从不同角度论证HAND2-AS1调控HAND2转录的分子机制；采用体内外细胞培养手段探讨该调控机制异常对内膜基质细胞生长、分化、粘附及侵袭能力的影响；通过对临床标本中双向启动子甲基化分析探讨该调控的紊乱与内膜异位症及相关不孕症的关联。以证明科学假说: HAND2-AS1通过双向启动子的去甲基化维持内膜中HAND2的表达，该调控上的紊乱所引起的孕激素的耐受与内膜异位症及不孕症。

Progesterone resistance is closely associated with pathogenesis of endometriosis and the related infertility. Early studies have shown that HAND2 is a critical molecule for endometrial response to progesterone stimulation. The molecular mechanism underlying HAND2 regulation and the impact of this regulation in the process of endometriosis remains largely unknown. Our recent studies have shown that lncRNA HAND2-AS1 and its neighboring target HAND2 are coordinately expressed in endometrial cells. HAND2-AS1 may control HAND2 transcription via affecting methylation status of the bidirectional promoter. In this application, we propose to perform HAND2-AS1 interference, construction of reporter plasmid with the bidirectional promoter, and RNA/DNA-ChIP in endometrial stromal cells to elucidate the molecular mechanism by which HAND2-AS1 regulates HAND2 transcription; To employ in vitro and in vivo culture techniques to investigate how HAND2 dysregulation affects endometrial stromal cell proliferation, differentiation, adhesion and invasion. We also plan to explore whether HAND2 dysregulation is associated with development of endometriosis and the related infertility by analyzing methylation change in the bidirectional promoter in the eutopic endometrium and ectopic lesions of patients. The outcome of these studies is expected to confirm our hypothesis: HAND2-AS1 maintains HAND2 expression and progesterone sensitivity in endometrial cells via demethylation of the bidirectional promoter. Dysregulation of HAND2 expression leads to progesterone resistance that will eventually contribute to pathogenesis of endometriosis, as well as the disease-related infertility.