miR-3074-5p对人滋养细胞生理活性的调控作用机制及其与复发性流产的关联

Recurrent miscarriage (RM) is an adverse pregnancy outcome characterized by the dysfunction of extravillous trophoblast cells (EVT). Our previous research found that, the expression of miR-3074-5p was remarkably decreased at the maternal-fetal interface in mice, but significantly increased in the placental villus of RM, and the over-expression of miR-3074-5p in HTR8/SVneo cells promoted cell apoptosis but inhibited cell invasion, being accompanied by the significantly elevated expressions of p27. Thus, we hypothesized that, the up-regulated expression of miR-3074-5p might lead to the early pregnancy loss by disturbing the appropriate cellular activities of EVT.. To authenticate this hypothesis, in this study we plan to investigate the in vitro effects of miR-3074-5p on the expression levels of molecules involved in p27-pathway in HTR8/SVneo cells, as well as the effects of inhibitors of p27-pathway on the cellular physiological effects of miR-3074-5p; meanwhile, the in vivo effects of miR-3074-5p on the embryo implantation, and the effects of steroid hormone on the expression of miR-3074-5p at the maternal-fetal interface in mice will be also observed；Furthermore, the venous blood samples will be collected from RM patients and normal early pregnant women to evaluate the potential role of circulating miR-3074-5p and other miRNAs as novel biomarkers for RM.

复发性流产（RM）是临床难治性不育症，其发病与绒毛外滋养细胞（EVT）生理活性的异常密切相关。我们发现miR-3074-5p在正常妊娠小鼠母胎界面呈低表达，在RM患者绒毛中高表达；并证实它能诱导人EVT来源的HTR8/SVneo细胞凋亡并抑制其侵袭，且伴随p27蛋白表达上调。由此推测miR-3074-5p很可能通过干扰EVT的生理活性而导致妊娠失败。为验证此推测，本项目将检测miR-3074-5p对HTR8/SVneo细胞中p27信号通路相关蛋白表达水平的影响；观察miR-3074-5p的细胞生理学效应是否受p27信号通路抑制剂的影响；验证miR-3074-5p对小鼠早期妊娠的影响，及雌孕激素与miR-3074-5p表达的关系；通过筛查早孕妇女外周血中miR-3074-5p等的含量，分析其与RM的相关性，以期能阐明miR-3074-5p对EVT的调控作用机制，并发现新的RM生物标志分子。

母胎界面血管重铸是妊娠建立与维持的关键环节，绒毛外滋养细胞（EVT）功能异常可致血管重铸障碍而继发复发性流产（RM）和子痫前期（PE）。前期发现miR-3074-5p在RM患者绒毛中显著高表达，并推测其可能通过FOXD1/P27途径调控EVT细胞的功能。本研究发现：1、miR-3074-5p能抑制EVT细胞侵袭而促进其凋亡，其表达变化可导致多量与细胞分化相关基因的差异表达；2、p27和ERβ是miR-3074-5p的靶基因，但FOXD1并不是，鉴于miR-3074-5p过表达EVT细胞后，P27表达显著上调，而降调P27并不改变miR-3074-5p所诱导的细胞功能变化，提示miR-3074-5p不是直接通过靶向P27来调控EVT细胞的生理活性；3、miR-3074-5p在孕早期各类滋养细胞中均有少量表达，在胚胎植入前小鼠子宫组织中表达丰度亦极低，于囊胚植入后开始明显增加；4、用miR-3074-5p激动剂处理或上调其在囊胚中的表达，均可导致小鼠胚胎停育，说明miR-3074-5p异常高表达可能会引发早期妊娠丢失；5、人外周血中未检出miR-3074-5p，但miR-146a-5p等miRNAs在RM患者外周血中的含量存在显著异常；6、RM患者绒毛组织中ERβ的表达显著上调，而蜕膜组织中ERβ和NDRG1的表达均呈下调；7、PE患者胎盘中miR-3074-5p显著低表达，P27显著高表达，其下游CCND1则显著低表达，且PE患者外周血中miR-146a-5p的含量显著下降。上述结果提示，孕早期EVT中miR-3074-5p的高表达可能通过抑制NDRG1的表达而干扰血管重铸，进而导致自然流产；孕中晚期胎盘滋养细胞中miR-3074-5p的低表达可能通过促进P27的表达而影响胎盘功能，进而导致PE发生；外周血中的miR-146a-5p等miRNAs是RM和/或PE潜在的预测指标。