miR-155诱导AT1R/B2R异源二聚体致子痫前期发病的通路机制研究

Immuno-inflammatory response plays an important role in the pathogenesis and organ damage of preeclampsia.It has been proven that increased B2-receptor can lead to the form of AT1-B2-receptor heterodimer and the increased AT1-B2-receptor heterodimer mediates an increased angiotensin II sensitivity in preeclampsia. At the same time miRNA-155, induced by LPS, can promote the development of inflammation through inhibiting SHIP-1, then induce the transcription of B2R.Our preliminary study found that an ultra-low-dose LPS injection protocal in early pregnant rats could induce an animal model of preeclampsia with miRNA-155 upregulation in placenta. When miRNA-155 was competitively inhibited by the "sponge" of anti-miRNA-155,the symptom of preeclampsia disappeared. So the hypothesis of our research is that miRNA-155, induced by LPS, can induce the form of AT1-B2-receptor heterodimer,which activate angiotensin-II-mediated signaling persistently and lead to preeclampsia. This study will use pathologic, immunologic, and molecular biologic technologies, using in vivo and in vitro models, in order to explore one main pathogenesy of preeclampsia, that miRNA-155 activates RAS system through mediating immuno-inflammatory system, which can provide new insight into therapeutics target point of preeclampsia.

子痫前期(PE)是我国孕产妇死亡第二原因，但发病机制不清。PE胎盘形成及母体PE样病理变化与发病机制研究关联密切。免疫炎症过度激活是PE胎盘形成重要机制，我们发现PE患者及免疫炎症过度激活的大鼠PE模型中微小RNA-155(miR-155)高表达，当竞争抑制miR-155后大鼠PE样症状消失。已有研究证明miR-155靶向作用于SHIP-1可上调缓激肽受体(B2R)表达；另外B2R高表达促异源二聚体B2R/AT1R形成，致血管紧张素II敏感性增加是母体PE样病理变化重要基础。由此提示miR-155是关联PE胎盘形成及PE样病理变化的关键因子。本研究围绕PE临床标本、动物模型及体外实验，通过免疫共沉淀、Western Blotting、qRT-PCR等研究miR-155介导SHIP-1/异源二聚体B2R/AT1R通路，阐明其为PE重要发病分子机制，为药物治疗提供新靶点，为临床防治提供新支撑。

（1）我们在前期验证miR-155与相关基因存在密切关联的基础上，设计miR-155高表达和低表达HTR-8细胞模型，发现miR-155高表达后，B2R表达显著减少，反之亦然。然而miR-155高表达和低表达后SHIP-1、TNF-a及AT1R无明显变化。同时我们还利用双荧光素酶报告基因实验进一步证实miR-155对B2R 3’UTR的负向调节作用。.（2）本课题组通过IP(免疫共沉淀)实验验证B2R/AT1R异源二聚体的存在，然而miR-155高表达后，B2R/AT1R表达不稳定。同期德国学者在cell杂志上发表了PE患者的二聚体表达异常及药物干预的研究与拟定研究计划高度重合，根据我们研究实际情况和文献检索结果，我们将研究重心调整至B2R及相关分子对胎盘的影响上。.（3）通过对临床样本的研究，发现B2R主要在合体滋养细胞和绒毛外滋养细胞中表达，且早发型重度子痫前期（sPE）患者胎盘组织中B2R的表达明显低于孕龄相当的非感染性早产（nPTB）患者。.（4）通过体外细胞实验，证实高表达B2R提高滋养细胞存活率、抑制其凋亡，且可促进G0/G1期细胞向S期过渡，促进了细胞的增殖活性。B2R分别通过CCND-1和VEGF-A促进滋养细胞增殖和迁移、侵袭及成环；通过PI3K/AKT信号通路促进PLGF表达和分泌，并促进HUVECs的血管形成功能。.（5）我们利用syncytin启动子介导的miR-155高表达质粒构建了胎盘特异性高表达 miR-155的小鼠模型。无论父源性或母源性胎盘高表达miR-155均导致孕鼠出现高血压、肾脏损伤、胎儿生长受限等PE症状。胎盘转录组芯片分析提示B2R显著降低，目前正基于该模型从动物实验角度进一步验证前期细胞水平相关通路的影响及对通路中分子靶位点干预后PE症状的改变。.本研究阐明了miR-155调控缓激肽受体2低表达作为子痫前期胎盘形成障碍的重要作用机制，为子痫前期防治提供新支撑。