缺氧/复氧经DNA甲基转移酶途径减弱人绒毛外滋养细胞的迁移和侵袭

Hypoxia/reoxygenation attenuates migration and invasion of human extracellular villus trophoblast cells via DNA methyltransferase pathway

目的探讨缺氧/复氧(hypoxia/reoxygenation,H/R)对人绒毛外滋养细胞(extravillous trophoblast,EVT)侵袭能力的调控机制。方法体外培养EVT永生化细胞系HTR-8/SVneo、早孕胎盘绒毛分离的原代EVT细胞和绒毛外植体,1%O2和20%O2浓度转换条件下模拟EVT细胞侵入子宫时的H/R环境,Western blot、免疫荧光技术检测细胞侵袭等关键因子的表达,Transwell小室法检测细胞迁移和侵袭。结果经H/R诱导后,HTR-8/SVneo细胞迁移和侵袭能力减弱,体外培养的绒毛外植体的外延能力和分离的原代EVT细胞迁移、侵袭能力均减弱;H/R条件下,HTR-8/SVneo细胞中基质金属蛋白酶MMP-2和MMP-9,整合素integrin-β1和integrin-α5蛋白水平降低(P<0.05),DNA甲基转移酶(DNA methyltransferase,DNMTs)的蛋白表达亦下调(P<0.01);H/R条件下,原代EVT细胞中上皮-间充质转化(epithelial-mesenchymal transition,EMT)的正调控因子Slug、Snails等被抑制(P<0.05),上皮细胞标志因子E-cadherin表达上升(P<0.01),MMP-2和-9、integrin-β1和-α5蛋白水平下降(P<0.05),细胞侵袭能力下降并伴随DNMT1、DNMT3A的表达下降(P<0.05)。HTR-8/SVneo细胞中过表达DNMT1、DNMT3A和DNMT3B可促进间质型标志因子N-cadherin、MMP-2和-9的蛋白表达上调(P<0.05)。结论H/R通过DNMTs调控MMP-2/9等EMT相关因子的表达从而减弱EVT细胞迁移和侵袭能力。

Objective To explore the regulative mechanism of hypoxia/reoxygen(H/R)for invasion ability of extravillous trophoblast(EVT).Methods Immortalized human EVT cell line HTR-8/SVneo in early pregnancy,primary EVT cells isolated from the villi of placenta in early pregnancy,and villi explants were cultured respectively.A simulation model of H/R environment in EVT cells invading the uterus was established under the conversion conditions of 1%O_2 for 24 h to 20%O_2 for another 24 h.Western blotting and immunofluorescence assay were used to detect the expression of key factors such as cell invasion,and Transwell chamber test was employed to observe cell migration and invasion.Results After H/R induction,the migration and invasion abilities of HTR-8/SVneo cells were weakened,and the epitaxial ability of cultured villi explants and the migration and invasion abilities of isolated primary EVT cells were also weakened.Under H/R conditions,the expression levels of MMP-2 and MMP-9,integrin-β1 and integrin-α5 were decreased in HTR-8/SVneo cells(P0.05),and the protein expression of DNA methyltransferase(DNMTs)was also down-regulated(P0.01).In the primary EVT cells under H/R condition,the expression levels of epithelial-mesenchymal transition(EMT)positive regulatory factors,Slug and Snails,were inhibited(P0.05),that of epithelial cell marker E-cadherin was increased(P0.01),protein levels of MMP-2 and MMP-9,integrin-β1 and integrin-α5 were decreased(P0.05),and cell invasion ability was reduced along with decreased DNMT1 and DNMT3A expression(P0.05).However,overexpression of DNMT1,DNMT3A and DNMT3B in HTR-8/SVneo cells promoted the up-regulation of interstitial markers N-cadherin,and MMP-2 and MMP-9(P0.05).Conclusion H/R regulates the expression of EMT-related factors such as MMP-2/-9 through DNMTs,and thereby reduces the migration and invasion abilities of EVT cells.