长链非编码RNA SPRY4-IT1调控EMT过程参与子痫前期发病机制研究

Preeclampsia (PE) is characterized by a series of maternal syndromes that includes hypertension, proteinuria, etc. Uterine spiral arteries remodeling dysfunction is an important pathology in PE. Recently, the relationship between epithelial - mesenchymal transition (EMT) process and uterine spiral arteries remodeling dysfunction has became a new point in PE studies. Previous studies have shown that LncRNA SPRY4-IT1 could reduce trophoblast migration, which might be associated with EMT regulation. Wnt pathway is an important mechanism mediated EMT. The WNT2 gene expression was found to be 7 times lower than the normal controls in PE placenta, and its transcription levels could be reduced by SPRY4-IT1. Studies have reviewed that the function of LncRNA is mainly by recruiting proteins, thus as methyl transferase EZH2. Further experiments found that EZH2 could inhibit trophoblast WNT2 gene expression. Accordingly, we are proposing an assumption: SPRY4-IT1 recruits EZH2 and guides to the WNT2 gene, thus plays a guiding role in epigenetic regulation of WNT2 gene expression. Finnally, this mechanism results in EMT and uterine spiral arteries remodeling dysfunction, mediating in PE. This notion will be validated in human pancenta,trophoblast cells and mice.We brought SPRY4-IT1 into the mechanism study of PE,and provided a new angle to reveal the mechanism of regulation of spiral arteries remodeling dysfunction. So it is hopeful to provide a theoretical basis for PE prevention in the future.

子痫前期（PE）系以高血压、蛋白尿为主要表现的妊娠特发疾病。子宫螺旋动脉重铸障碍是PE重要病理改变，滋养细胞上皮-间质转化（EMT）受阻参与螺旋动脉重铸障碍机制成为研究新热点。前期研究发现LncRNA SPRY4-IT1能降低滋养细胞迁移能力，可能与EMT调控相关。Wnt通路是介导EMT的重要机制，实验发现WNT2基因在PE胎盘中近7倍下调，而SPRY4-IT1可抑制其转录。LncRNA主要通过募集甲基转移酶EZH2等发挥转录调控作用。进一步实验发现EZH2也可抑制滋养细胞WNT2基因转录。据此，课题组提出假设：SPRY4-IT1募集EZH2并发挥引导作用，表观遗传学水平调控WNT2基因表达，导致滋养细胞EMT过程受阻及螺旋动脉重铸障碍，介导PE发生。本研究拟从临床标本、细胞及整体水平，系统地探索SPRY4-IT1作用机制，从新的角度揭示螺旋动脉重铸障碍机制，为PE预测及防治提供理论依据。

子痫前期是妊娠20周后出现以高血压、蛋白尿为主要表现一种妊娠特发疾病，是导致孕产妇及围产儿发病和死亡的主要原因之一。PE病因学说众说纷纭，子宫螺旋动脉重铸障碍是影响其早期发病的因素之一。妊娠早期，为了保证胚胎发育所需要的营养和氧气的供应，滋养细胞获得迁移和侵袭能力，可侵入蜕膜、子宫肌层来保证子宫螺旋动脉正常的重铸使母胎之间保持高流低阻的血液供应。滋养细胞这种获得迁移和侵袭能力的现象是上皮间质转化所需具备的最重要的条件。课题组前期研究发现，SPRY4-IT1在子痫前期胎盘组织中高表达，且SPRY4-IT1可以抑制滋养细胞迁移和侵袭，其表明SPRY4-IT1可能参与EMT调控。由于Wnt通路是介导EMT过程的重要路径，且前期有文章报道SPRY4-IT1可以通过绑定特定的蛋白调控基因表达。课题组提出假设：SPRY4-IT1可能通过结合某种特定的蛋白从而影响下游把基因的表达，使滋养细胞EMT过程受阻及螺旋动脉重铸障碍，介导PE的发生。本研究通过：（1）扩大样本量通过定量PCR检测50对正常孕妇和PE孕妇的胎盘组织中SPRY4-IT1表达，结果显示子痫前期组SPRY4-IT1表达量明显高于正常组。（2）用已检测过的转染效率高的干扰序列和质粒分别同时转染HTR-8/SVneo细胞。通过transwell实验，划痕试验，检测其对细胞迁移、侵袭的影响。结果发现，在敲低SPRY4-IT1后，迁移能力增强。在过表达SPRY4-IT1后结果相反。与此同时，检测了细胞的表型变化。（3）在转染HTR-8/SVneo后，分别提取细胞蛋白并采用Western blotting和定量PCR检测EMT相关 指标的表达水平，结果发现干扰SPRY4-IT1后，E-cadherin, β-catenin表达下调，vimentin表达水平上调。过表达后结果与之相反。此外，免疫荧光实验检测结果趋势与之相同。（4）核质分离实验检测SPRY4-IT1在HTR-8/SVneo细胞中的分布情况，结果显示其大部分在细胞质中。（5）RIP实验检测结果显示SPRY4-IT1与HuR结合，β-catenin和HuR结合，干扰了HuR后定量PCR检测β-catenin（6）定量PCR检测Wnt家族成员的表达量，结果显示wnt3和wnt5b有差异表达。本研究结果从新的角度揭示螺旋动脉重铸障碍机制,为PE预测及防治提供理论。