Peng Bo, Zhu Hua, Leung Peter C K
Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Vancouver V5Z 4H4, Canada.
J Clin Endocrinol Metab. 2015 Jan;100(1):E19-29. doi: 10.1210/jc.2014-1897.
GnRH and its receptor, GnRHR, were shown to promote trophoblastic cell invasion. Detection of elevated early development-related transcription factor TWIST and adhesion molecule N-cadherin in the invasive trophoblastic cells could suggest that GnRH promotes trophoblastic cell invasion through TWIST-regulated N-cadherin pathway.
This study sought to investigate the regulatory effect of GnRH on TWIST and N-cadherin expression as well as invasiveness in human trophoblastic cells.
The expression of GnRHR, TWIST, and N-cadherin was first examined in human first-trimester chorionic villi by immunohistochemistry. Expression levels of GnRHR, TWIST, and N-cadherin were tested in primary extravillous trophoblastic (EVT) cells and an immortalized EVT cell line HTR-8/SVneo cells with incubation of GnRH and its antagonist, Antide. Small interfering RNA strategy was used to study the roles of TWIST and N-cadherin in basal- and GnRH-regulated trophoblast invasion. Matrigel-mediated transwell invasion assays were employed to assess cell invasion capacity.
GnRHR, TWIST, and N-cadherin were detected at the invasive site of first-trimester human placenta. GnRH treatment significantly increased TWIST and N-cadherin expression in primary EVT as well as HTR-8/SVneo cells. Pretreatment with the GnRH receptor antagonist Antide attenuated the effects of GnRH on TWIST and N-cadherin expression. Invasive capacity of primary EVT and HTR-8/SVneo cell was reduced following siRNA-mediated knockdown of either TWIST or N-cadherin. Furthermore, by knocking down endogenous TWIST, the expression level of N-cadherin was reduced as well as GnRH-induced HTR-8/SVneo cell invasion. Treatment with GnRH induces AKT phosphorylation and Phosphoinositide3-kinase inhibitor LY294002 attenuates the effects of GnRH on TWIST and N-cadherin expression and trophoblastic cell invasion.
Our results suggest that GnRH acts via its receptor to induce AKT phosphorylation, which contributes to elevated TWIST expression. Increased levels of TWIST subsequently induce N-cadherin expression, which promotes human trophoblastic cell invasion in vitro.
促性腺激素释放激素（GnRH）及其受体（GnRHR）可促进滋养层细胞侵袭。侵袭性滋养层细胞中早期发育相关转录因子TWIST和黏附分子N-钙黏蛋白水平升高，提示GnRH可能通过TWIST调控的N-钙黏蛋白途径促进滋养层细胞侵袭。
本研究旨在探讨GnRH对人滋养层细胞中TWIST和N-钙黏蛋白表达及侵袭能力的调控作用。
首先采用免疫组化法检测人孕早期绒毛膜中GnRHR、TWIST和N-钙黏蛋白的表达。用GnRH及其拮抗剂Antide处理原代绒毛外滋养层（EVT）细胞和永生化EVT细胞系HTR-8/SVneo细胞，检测GnRHR、TWIST和N-钙黏蛋白的表达水平。采用小干扰RNA策略研究TWIST和N-钙黏蛋白在基础状态及GnRH调控的滋养层细胞侵袭中的作用。采用基质胶介导的Transwell侵袭实验评估细胞侵袭能力。
在人孕早期胎盘的侵袭部位检测到GnRHR、TWIST和N-钙黏蛋白。GnRH处理显著增加原代EVT细胞及HTR-8/SVneo细胞中TWIST和N-钙黏蛋白的表达。用GnRH受体拮抗剂Antide预处理可减弱GnRH对TWIST和N-钙黏蛋白表达的影响。siRNA介导的TWIST或N-钙黏蛋白敲低后，原代EVT细胞及HTR-8/SVneo细胞的侵袭能力降低。此外，敲低内源性TWIST后，N-钙黏蛋白的表达水平降低，同时GnRH诱导的HTR-8/SVneo细胞侵袭也受到抑制。GnRH处理可诱导AKT磷酸化，磷脂酰肌醇-3激酶抑制剂LY294002可减弱GnRH对TWIST和N-钙黏蛋白表达及滋养层细胞侵袭的影响。
我们的结果提示，GnRH通过其受体诱导AKT磷酸化，这有助于TWIST表达升高。随后TWIST水平升高诱导N-钙黏蛋白表达，从而促进体外人滋养层细胞侵袭。
