ALKBH5通过抑制上皮-间质转化降低人滋养细胞的迁移和侵袭能力

ALKBH5 suppresses migration and invasion of human trophoblast cells by inhibiting epithelial-mesenchymal transition

目的探讨去甲基酶ALKBH5对滋养细胞HTR-8/SVneo迁移、侵袭功能及对上皮-间质转化(EMT)过程的影响。方法将ALKBH5高表达、抑制及其阴性对照NC质粒转染人滋养细胞系HTR-8/SVneo细胞,采用实时荧光定量PCR(qRT-PCR)及Westen boltting(WB)实验检测各组细胞中ALKBH5 mRNA及ALKBH5蛋白的表达水平。并通过Transwell实验检测转染后滋养细胞迁移、侵袭功能变化,同时通过WB实验检测各组细胞中上皮-间质转化(EMT)相关蛋白:Vimentin、Fibronectin、E-cadherin、N-cadherin、MMP9及MMP2的表达水平。结果ALKBH5组与未转染组(Control)和无意义序列组(NC)相比,ALKBH5 mRNA及蛋白高表达效果显著(P<0.05);shALKBH5组与未转染组(Control)和无意义序列组(NC)相比,ALKBH5 mRNA及蛋白抑制效果显著(P<0.05)。ALKBH5高表达后HTR-8/Svneo细胞迁移、侵袭功能降低(P<0.05),EMT相关蛋白中上皮标记物E-cadherin蛋白表达上调,间质标记物Fibronectin、N-cadherin、MMP2蛋白表达下调,差异有统计学意义(P<0.05)。ALKBH5表达抑制后HTR-8/Svneo细胞迁移、侵袭功能增强(P<0.05),EMT相关蛋白中上皮标记物E-cadherin蛋白表达下调,间质标记物Fibronectin、N-cadherin、MMP2蛋白表达上调,差异有统计学意义(P<0.05)。结论ALKBH5通过抑制EMT过程降低滋养细胞迁移、侵袭能力,从而参与子痫前期的发病机制。

Objective To investigate the effects of ALKBH5 on migration,invasion and epithelial-mesenchymal transition(EMT)of human trophoblast cells.Methods The expression plasmid of ALKBH5 or a negative control plasmid(ALKBH5-NC)was transfected in human trophoblast HTR-8/SVneo cells,and the expressions of ALKBH5 mRNA and protein were detected by qRT-PCR and Western blotting.Transwell assay was used to assess the changes in migration and invasion abilities of the trophoblast cells after the transfection.Western blotting was performed to detect the expressions of EMT-related proteins in the cells including vimentin,fibronectin,E-cadherin,N-cadherin,MMP9 and MMP2.Results ALKBH5 mRNA and protein expressions were significantly higher in ALKBH5 group than in the control group(P<0.05).Over-expression of ALKBH5 significantly attenuated migration and invasion abilities of HTR-8/Svneo cells(P<0.05).Compared with the control cells,the cells overexpressing ALKBH5 showed an up-regulated expression of E-cadherin and down-regulated expressions of vimentin,fibronectin,N-cadherin,MMP9 and MMP2(P<0.05).Conclusion ALKBH5 is involved in the pathogenesis of preeclampsia by inhibiting EMT of trophoblast cells and hence reducing their migration and invasion abilities.