let-7i在子痫前期胎盘滋养细胞自噬中的作用机制研究

Pre-eclampsia (PE) is a gestational disease that has high morbidity.The expression of let-7i was significantly down-regulated in the PE placenta. Bioinformatics analysis and experiments prove Atg4B is directly targeted by let-7i; the expression of Atg4B in placenta of PE patients was up-regulated; Silence of let-7i in trephocytes could up-regulate the expression Atg4B the autophagy activity was increased. In present study, we would like to: analyze the correlation between the expression of let-7i/Atg4B and the autophagy index with large samples; observe the relationship among the over-expression or silence of let-7i, the expression of Atg4B, and the autophagy of trephocytes, the trophoblast function infulenced by autophagy; clarify the molecular mechanism that let-7i regulates the expression of Atg4B and modulates the autophagy of trephocytes by technics including gene over-expression, gene silencing; evalute the function of let-7i in the process of trephocytes autophagy reverse, protecting PE occurrence in vivo. The study that let-7i involves in the autophagy of trephocytes and relates to the incidence of PE has never been reported and PE caused by autophagy is new theory; Our study will contribute to the clarification of the pathogenesis of PE and provide a new idea for the early prevention and treatment of PE.

子痫前期是以胎盘滋养细胞功能异常为重要特征、高发病率的妊娠期疾病。let-7i是我们采用芯片技术筛选出的、显著低表达于子痫前期患者胎盘组织中的miRNA。软件分析和实验证实自噬相关Atg4B基因为其作用靶标，而滋养细胞过度自噬诱发子痫前期的研究已见报道；沉默let-7i表达可显著上调Atg4B表达，促进滋养细胞的自噬，提示let-7i可通过调控Atg4B表达诱发滋养细胞自噬，导致子痫前期发生。本研究拟通过：①评估let-7i、Atg4B表达、滋养细胞自噬与子痫前期发生的相关性；②以Atg4B为机制线索，采用过表达、沉默和挽救实验策略，论证let-7i诱发滋养细胞自噬参与子痫前期的作用与机制；③在体评估let-7i诱发子痫前期发病的作用。目前未见let-7i通过自噬途径参与子痫前期的报道，本研究有望为子痫前期疾病的防治提供新靶标。

自噬，即II型程序性细胞死亡，现已被认为与子痫前期（PE）(一种高发病率的妊娠疾病)的病理生理有关。本研究旨在探讨let-7i在滋养细胞自噬中的功能及作用机制。我们收集重度子痫前期（SPE）患者或正常孕妇的胎盘组织，结果发现SPE患者胎盘组织中let-7i表达明显降低。在SPE组织中，我们观察到自噬空泡，微管相关蛋白轻链3（LC3）II/I比值升高。我们以滋养细胞为研究对象，合成let-7i inhibitor、let-7i mimics转染HTR-8/SVneo滋养细胞系(HTR-8)、人胎盘绒毛膜癌细胞系JEG-3细胞，结果显示let-7i mimic抑制HTR-8和JEG-3的自噬活性，而let-7i inhibitor则增强其自噬活性。除此之外，我们发现自噬相关Atg4B基因作为let-7i的潜在靶标，在SPE中表达明显升高。荧光素酶报告基因实验结果显示let-7i负调控Atg4B的表达。在滋养细胞转染let-7i mimic或let-7i inhibitor后， Atg4B在滋养细胞的mRNA和蛋白表达均发生的显著变化。进一步研究，我们发现，Atg4B的过表达可以部分逆转let-7imimic减少的LC3II/I表达，而Atg4B沉默可以部分抵消let-7i inhibitor引起的LC3II/I表达上调。以上结果表明，let-7i可能通过调节Atg4B的表达来调节自噬活性，导致了了PE的发生。