AMPK经NET1A调控RhoA-ROCKⅡ通路介导的滋养细胞侵袭相关机制研究

Compromised trophoblast invasion into uterus wall is the physiopathological basis for the development of preeclampsia（PE）, however, the regulatory mechanism of trophoblast invasion remains to be addressed. RhoA-ROCKⅡsignaling pathway is the key pathway regulating stress fiber contraction, which is key process of cell invasion. Recent study demonstrated that most of the AMP-activated protein kinase（AMPK） substrates are involved in regulation of cell mobility, adhesion and migration， indicating it might be also involved in trophoblast invasion. In addition， a downstream target of AMPK，NET1A， has been found regulate cell migration and invasion by activating RhoGTPase, implying AMPK may regulate RhoA-ROCKⅡsignaling pathway and cell invasion through NET1A. Our preliminary data showed that AMPK is hyperactivated in PE associated placentas. Moreover，AMPK negatively regulate trophoblast invasion in vitro. These results strongly suggest that abnormal AMPK activation could impair trophoblast invasion and ultimately lead to PE development. The proposed study will focus on the role of AMPK on trophoblast invasion， and investigate the regulatory mechanism of trophoblast invasion by AMPK and NETIA through RhoA-ROCKⅡ signaling pathway.

滋养细胞侵袭能力下降作为PE发生的病理生理学基础之一，其调控机制仍有待进一步阐明。最新研究发现绝大多数AMPK磷酸化底物与细胞运动、粘附和迁移相关，提示AMPK可能参与调控细胞侵袭。其中一种AMPK磷酸化靶标蛋白NET1A同时也是Rho的活性活性调节因子，而RhoA-ROCKⅡ通路作为介导应力纤维收缩的关键通路，已知参与了对滋养细胞侵袭的调控。提示AMPK有可能经NET1A调控RhoA-ROCKⅡ通路介导的细胞侵袭。本项目组前期工作发现，PE胎盘组织中存在AMPK异常活化。此外，我们还证实了AMPK对于滋养细胞侵袭具有负调控作用，表明AMPK异常激活可能是导致滋养细胞侵袭力减弱并最终导致PE发生的直接原因之一。本研究拟以AMPK为研究对象，以滋养细胞侵袭为切入点，深入探讨AMPK经NET1A调控RhoA-ROCKⅡ通路介导的滋养细胞侵袭的具体分子机制。

滋养细胞侵袭能力下降作为PE发生的病理生理学基础之一，其调控机制仍有待进一步阐明。最新研究发现AMPK有可能调控NET1A磷酸化，影响其核质转运，从而对RhoA-ROCKⅡ通路介导的细胞侵袭起调控作用。本项目组前期工作发现，PE胎盘组织中存在AMPK异常活化。此外，我们还证实了AMPK对于滋养细胞侵袭具有负调控作用，表明AMPK异常激活可能是导致滋养细胞侵袭力减弱并最终导致PE发生的直接原因之一。本项目提出“滋养细胞AMPK异常激活，导致NET1A46S磷酸化水平升高，NET1A核质转运失衡，从而抑制 RhoA-ROCKⅡ通路活性，导致滋养细胞侵袭力下降，诱发 PE 临床症状”。本项目中，我们通过提取原代滋养细胞，证实了PE患者滋养细胞AMPK的异常激活，NET1A的核质转运失衡以及RhoA-ROCKⅡ通路的抑制。然后在细胞层面，AMPK的激活可以促进NET1A46S的磷酸化，并且促进其向核内转运，导致下游RhoA的激活受到抑制，RhoA-ROCKⅡ介导的应力纤维形成抑制，细胞运动侵袭受到抑制；当NET1A46S突变后，AMPK的改变不再对下游RhoA-ROCKⅡ通路，应力纤维或滋养细胞运动产生影响，证实了AMPK通过改变NET1A46S磷酸化来调控RhoA-ROCKⅡ通路；而ROCK II 敲降后，滋养细胞侵袭受到抑制，且AMPK对滋养细胞侵袭的调节作用消失，证实了AMPK是通过RhoA-ROCKⅡ通路来影响滋养细胞的侵袭。并在RUPP子痫前期小鼠模型和胎盘特异性AMPK敲除小鼠中进行了验证，在体证实了NET1A/RhoA/ROCK II通路介导的AMPK过度激活对PE发病的影响。在实验进程中，我们发现NET1A调控RhoA-ROCKⅡ通路参与了AMPK对滋养细胞侵袭的调控。本项目为子痫前期的发病机制提供新的认识，证实了AMPK可以作为PE的新的治疗靶点。本项目的完成将为PE的防治提供新的靶点。