胎盘WNT5A表达异常通过影响滋养细胞侵袭参与子痫前期发生

Abnormal expression of placental WNT5A participates in preeclampsia by affecting trophoblast invasion

目的:探讨WNT5A在子痫前期(PE)患者胎盘组织中的表达及其对滋养层细胞侵袭的影响和潜在作用机制。方法:收集27例PE孕妇及38例正常孕妇的胎盘组织,实时荧光定量聚合酶链式反应(RT-qPCR)检测WNT5A表达;Transwell侵袭实验检测人重组WNT5A蛋白(200ng/mL)处理对人永生化绒毛外滋养细胞系(HTR8/SVneo)侵袭的影响;Western blot检测WNT5A对人类原代EVT细胞胞浆内活化的β-catenin蛋白水平作用。基于空白对照组和重组BMP2蛋白(25ng/mL)处理组的HTR8/SVneo转录组测序数据,明确BMP2对WNT5A的调控作用,并在HTR8/SVneo和原代人类EVT细胞中,通过RT-qPCR和Western blot分别在mRNA和蛋白水平上验证BMP2对WNT5A和活化β-catenin的调控作用;结合BMP2受体抑制剂DMH-1预处理HTR8/SVneo细胞,验证BMP2调控WNT5A表达所需受体及信号通路。结果:与正常孕妇相比,PE患者胎盘组织中WNT5A水平显著降低。胎盘WNT5A mRNA水平可较好地用于PE诊断,曲线下面积(AUC)为0.844(95%CI为0.74~0.95,P<0.001)。外源性人重组WNT5A蛋白添加可明显提高HTR8/SVneo细胞侵袭力,并抑制EVT细胞内活化β-catenin表达(P<0.05)。人重组BMP2蛋白处理后可降低HTR8/SVneo和EVT细胞中WNT5A表达,提高细胞内活化β-catenin表达水平(P<0.05)。BMP2蛋白处理后可提高HTR8/SVneo细胞磷酸化的SMAD1/5/9蛋白表达,BMP2受体拮抗剂DMH-1预处理可阻断这一上调作用。结论:PE患者胎盘组织中WNT5A表达下调,其表达受到BMP2激活的SMAD1/5/9信号通路调控,并通过影响滋养细胞侵袭,参与PE发生。

Objective:To explore the expression of WNT5A in placentas of patients with pre-eclamptic(PE)and its effect on trophoblast invasion as well as potential underlying mechanism.Methods:A total of 27 PE women and 38 normal pregnant women were included to acquire placental tissues.RT-qPCR was conducted to detect the expression of WNT5A.Transwell invasion assay was used to evaluate the effect of recombinant human WNT5A protein(200ng/mL)on human immortalized trophoblast cell line(HTR8/SVneo).Western blot was utilized to examine the activeβ-catenin protein level in human primary EVT cells following WNT5A treatment.RNA-seq data based on blank control group and BMP2 recombinant protein(25ng/mL)treated group were used to analyze the regulation of BMP2 on WNT5A.The impact of BMP2 on WNT5A and activeβ-catenin was further confirmed by RT-qPCR and Western blot in HTR8/SVneo and EVT cells.In addition,DMH-1 inhibitor was used to pre-treat HTR8/SVneo cells to verify the receptor and signaling pathways required for BMP2-regulated WNT5A expression.Results:Compared to the control group,WNT5A mRNA levels in placenta of PE group were significantly reduced.Additionally,the placental WNT5A mRNA level proved to be a reliable diagnostic marker for PE with an area under the curve(AUC)of 0.844(95%CI=0.74~0.95,P<0.001).Exogenous WNT5A protein supplementation significantly increased HTR8/SVneo cell invasion and suppressed activeβ-catenin expression in EVT cells(P<0.05).Furthermore,treatment with human recombinant BMP2 protein decreased WNT5A expression in HTR8/SVneo and EVT cells,while increasing activeβ-catenin expression(P<0.05).BMP2 treatment also increased the phosphorylation of SMAD1/5/9 proteins in HTR8/SVneo cells,an effect blocked by pre-treatment with the BMP2 receptor antagonist DMH-1.Conclusion:The deficiency of WNT5A expression in placental tissues of PE patients could be regulated by BMP2-phsophorylated SMAD1/5/9 signaling pathway and participates PE pathogenesis by regulating trophoblast invasion.