LRRC47基因拷贝数缺失致自然流产的作用机制研究

LRRC47, which locates in the copy number deletion region identified by chromosomal microarray, was found to be significantly down-regulated in villus tissue of patients with spontaneous abortion. Our previous study found that silencing LRRC47 could significantly reduce the invasiveness of human trophoblast cells. Bioinformatic analysis and experiments suggested that the effect of LRRC47 may be related to the JAK/STAT3 signaling pathway which has been confirmed to be associated with trophoblast invasion and spontaneous abortion. Therefore, we hypothesize that LRRC47 could affect trophoblast invasion through JAK/STAT3 signaling pathway. In this study, we will investigate the relationship between LRRC47 and trophoblast invasion as well as spontaneous abortion. Using overexpression and knockdown strategies, we intend to explicit the function and mechanism of LRRC47 in trophoblast cells invasion dysregulation. Furthermore, we will use pull-down and rescue experiment strategies to analyze the downstream regulation mechanism of LRRC47 in modulating JAK/STAT3 signaling pathway. Moreover, we will evaluate the effect of LRRC47 on trophoblast invasion and spontaneous abortion by transfecting pregnant mice. The study will provide new clues and potential intervention targets for the prevention and treatment of spontaneous abortion.

LRRC47是我们通过染色体基因芯片在自然流产患者遗传诊断中获得的、位于拷贝数缺失区域、差异低表达于自然流产绒毛组织的基因。前期发现，LRRC47沉默表达显著降低滋养细胞侵袭；生物信息学分析及实验均提示LRRC47的作用可能与JAK/STAT3通路有关，已证实JAK/STAT3通路与滋养细胞侵袭、自然流产密切相关；因此，我们提出LRRC47通过调控JAK/STAT3通路影响滋养细胞侵袭、参与自然流产发生的假说。本研究拟临床评估LRRC47表达与滋养细胞侵袭、自然流产的相关性；采用过表达、沉默策略，论证LRRC47对滋养细胞侵袭的影响；以JAK/STAT3通路为切入点，通过Pull-down、挽救实验论证LRRC47通过调控JAK/STAT3通路影响滋养细胞侵袭、参与自然流产发生的机制；借助动物模型，评估LRRC47对滋养细胞侵袭及自然流产的作用。研究将为自然流产防治提供新线索及干预靶标。

自然流产是妊娠期最常见的并发症，但其发病机制，尤其是拷贝数变异在其中的作用，目前尚不明确。因此，本课题对前期工作中通过染色体基因芯片筛选所得的位于拷贝数缺失区域的LRRC47基因进行后续功能性研究。研究发现，LRRC47在自然流产患者绒毛中的mRNA和蛋白表达水平均低于对照组。通过沉默表达滋养细胞HTR-8/SVneo中的LRRC47水平，发现LRRC47的沉默表达可抑制HTR-8/SVneo细胞的侵袭能力。通过对LRRC47沉默表达后HTR-8/Svneo细胞进行转录组测序，我们发现了LRRC47沉默表达所影响的下游基因以及相关信号通路。这些结果初步揭示了LRRC47沉默表达在自然流产发生过程中所起的作用及相应的机制。为了扩展对自然流产遗传学病因的认识，我们对自然流产患者进行了全转录组测序及生物信息学分析，初步揭示了自然流产新的遗传学病因。此外，我们自主建立了一种新型的、耗时短、价格便宜的分子检测技术，为自然流产遗传学病因检测提供了新方法。