LncRNA-PVT1作为ceRNA通过Wnt2信号通路调控滋养细胞侵袭力的机制研究

Unexplained recurrent spontaneous abortion is a common complication of human pregnancy. The inadequate invasion of trophoblast cells in maternal-fetal interface is the key to the occurrence of abortion. Our previous study found that PVT1 was involved in the occurrence of URSA by regulating the invasiveness of trophoblastic cells by regulating Wnt2 signals, but the specific mechanism was unknown. It is reported that PVT1 can act as ceRNA binding miR-199a-5p to regulate the invasiveness of tumor cells, while miRNA-199a-5p can affect the migration of tumor cells by regulating Wnt2 signals. Therefore, we speculate that in the course of pregnancy, PVT1 can regulate Wnt2 signaling act as ceRNA competitively binding miR-199a-5p and affect the invasiveness of trophoblastic cells, leading to the occurrence of miscarriage. The aim of this project is to elucidate the regulatory role and molecular mechanism of PVT1-miR-199a-5p-Wnt2 loop in trophoblastic cell invasiveness at cellular level through gene transfection and RIP experiments. Constructing mouse models of recurrent abortion to verify the effect of loop related molecules on the pregnancy process, and further proving the effect of the loop on the pregnancy outcome at the tissue level. This research is to explore the molecular events of URSA and to provide new targets for the prevention and treatment of URSA.

不明原因复发性流产（URSA）是人类妊娠常见合并症。母胎界面滋养细胞侵袭不足是流产发生的关键。我们前期研究发现PVT1通过调控Wnt2信号影响滋养细胞侵袭力参与URSA的发生，但具体机制不明。文献报道，PVT1可作为ceRNA竞争性结合miR-199a-5p调控肿瘤细胞的侵袭力；而miRNA-199a-5p可通过调控Wnt2信号影响肿瘤细胞迁移。因此，我们推测在妊娠过程中，PVT1以ceRNA方式竞争性结合miR-199a-5p来调控Wnt2信号，影响滋养细胞侵袭力，导致流产的发生。本项目拟在细胞水平通过基因转染、RIP等实验深入阐明PVT1-miR-199a-5p-Wnt2环路对滋养细胞侵袭力的调控作用及分子机制；构建复发性流产小鼠模型，验证环路相关分子对妊娠过程的影响；在组织水平进一步证明环路对妊娠结局的影响。本研究深入探索URSA发生的分子事件，为URSA的防治提供新的干预靶点。

复发性流产（Recurrent spontaneous abortion，RSA）是指与同一性伴侣连续发生2次及以上20周内的自然流产，发生率逐年上升，严重损害育龄妇女的身心健康。在妊娠早期，适度的滋养细胞侵袭和迁移对胚胎着床和胎盘发育至关重要，滋养细胞侵袭力不足是流产发生的关键。PVT1（Plasmacytoma variant translocation 1）是一种新发现的长链非编码RNA（Long non-coding RNA，lncRNA），可促进多种肿瘤细胞的增殖和迁移，而在母-胎界面滋养层细胞中PVT1的生物学功能仍知之甚少。本研究通过RNA测序发现，RSA组和对照组绒毛组织中PVT1表达水平存在差异，进一步收集临床组织发现，PVT1在RSA绒毛组织中显著下调，且PVT1的下调可显著抑制滋养细胞的侵袭、迁移、增殖和黏附。联合生物信息学分析和ChIP实验证实YY1可直接靶向PVT1启动子进而调节PVT1表达。深入探究后发现下调的PVT1可通过抑制Wnt2的表达，影响滋养细胞的侵袭能力，从而参与流产的发生。此外，进一步发现PVT1可以调节自噬小体的形成及自噬相关蛋白mTOR、ULK1、LC3、Beclin-1和p62的表达进而影响滋养细胞的生物学行为。本项目在细胞及临床样本两个层面系统阐述lncRNA-PVT1在调控滋养细胞侵袭力和维持妊娠稳态中的重要作用，为揭示RSA的发生机制提供理论依据，同时为临床上治疗流产提供新靶点与新思路。