滋养层细胞自噬异常导致原因不明习惯性流产的分子机制研究

Unexplained recurrent miscarriage (RM) is a common complication of human pregnancy. Recently, study found that the incidence of the disease is associated with impaired trophoblast proliferation and invasion. Our previous studies also found that autophagy-related genes, LC3B and Beclin1 expression is increased significantly in villi tissue of RM patients compared with healthy controls, and further demonstrated these proteins could regulate the invasion ability of trophoblasts in vitro. On the basis, this project aims to study the molecular mechanisms of LC3B and Beclin1 regulation of trophoblast invasion. First, we detected the signaling pathway involved in invasion ability of trophoblast by knockdown or overexpression of LC3B and Beclin1, and screened gene expression of the trophoblast by using mRNA microarray. Using molecular biology methods and animal experiments, we confirmed on the up- and down-stream molecules of LC3B and Beclin1 regulated trophoblast invasion. Finally, these results will clarify the molecular mechanisms of LC3B and Beclin1 regulation trophoblast invasion. Our research will help discover and understand the biological function of LC3B and Beclin1, expanding our understanding of the development of unexplained recurrent spontaneous abortion disease, and provide a new theoretical basis for treatment of unexplained recurrent miscarriages disease.

不明原因的习惯性流产是人类妊娠中常见的合并症。研究发现该类疾病的发生常常与滋养层细胞侵袭力不足有关。我们前期的mRNA芯片结果也发现自噬相关基因LC3B和Beclin1基因在原因不明自发性流产患者的滋养层组织表达显著增加。初步的实验结果表明该分子具有调控滋养层细胞的侵袭能力。在此基础上，本课题拟深入研究LC3B和Beclin1调控滋养层细胞侵袭的分子机制。本项目检测LC3B和Beclin1对参与滋养层细胞侵润的重要通路的影响，并通过芯片筛查LC3B和Beclin1敲低和过表达对滋养层细胞中基因表达的影响。运用分子生物学和动物实验方法确认LC3B和Beclin1调控滋养层细胞侵袭的上下游分子，最终阐明LC3B和Beclin1调控滋养层细胞侵袭的分子机制。我们的研究将有阐明LC3B和Beclin1的功能，拓展我们对妊娠相关疾病发生发展的认识，为妊娠相关疾病的预防及治疗提供理论依据。

在项目的支持下，我们的研究发现在复发性流产患者的绒毛组织中FXR1基因的表达显著降低，进一步通过细胞实验我们发现FXR1调控cox-2蛋白的表达，通过分子机制的实验我们发现FXR1可以结合到cox-2 3’-UTR区，从而调控cox-2 mRNA的稳定性。这些结果已经发表在生殖医学权威杂志 “Reproduction, Fertility and Development”上面发表。此外，我们发现在复发性流产病人的绒毛组织中TTP蛋白表达显著增加，过表达TTP显著的降低滋养层细胞的侵袭和增值能力。在绒毛外植体模型中敲低TTP可以显著的增加STMN1蛋白的表达。这些结果已经发表在PLoS one杂志上面发表。更进一步，我们使用实时定量PCR芯片实验，我们发现在复发性流产病人的绒毛组织中ALKBH5表达显著增加，敲低ALKBH5显著的增加滋养层细胞的侵袭和增值能力。在绒毛外植体模型中敲低ALKBH5可以显著的降低绒毛细胞生长和侵袭能力。RNA测序实验发现改变ALKBH5蛋白表达可以显著影响编码RNA表达，特别是CYR61表达发生显著改变。我们进一步探索了ALKBH5和CYR61表达改变和复发性流产之间的关联。这些结果已经发表在生物治疗权威杂志Theranostics上面投稿并修回。