PDLIM1 Reduces The Invasiveness Of Extravillous Trophoblasts By Regulating The Ubiquitin-proteasome Degradation Pathway Of ACTN4 In Preeclampsia

Objective: The objectives of this study were to investigate the expression of PDLIM1 in placental trophoblast cells in preeclampsia,to investigate the effect of abnormal PDLIM1 expression on trophoblast cell function,to elucidate the relationship between PDLIM1 and ACTN4 protein,and to reveal the role of PDLIM1 on the post-translational modification of ACTN4 protein and the specific mechanism,in order to provide a new research direction for the elucidation of the pathogenesis of preeclampsia and to provide potential targets for the prediction and treatment of preeclampsia.Methods: The protein binding between PDLIM1 and ACTN4 in trophoblast cells was detected by immunoprecipitation combined with liquid chromatography mass spectrometry,and the structural domain of PDLIM1 interacting with ACTN4 was detected by immunoprecipitation,GST-pulldown and immunofluorescence.The localization and expression of PDLIM1 in normal and PE placental tissues were detected by immunofluorescence,immunohistochemistry and Western blot;PDLIM1and/or ACTN4 were knocked down or overexpressed in trophoblast cells by sh RNA lentivirus infection or plasmid transfection,and the invasion and migration ability of each group of cells were detected by Western blot,wound healing assay,matrigel invasion assay and Transwell migration assay.The proliferation and apoptosis levels of each group of cells were detected by Ed U assay,CCK-8 assay and flow apoptosis.The protein and m RNA expression levels of PDLIM1 and ACTN4 in trophoblast cells were detected by Western blot and RT-q PCR,respectively,and their correlation was revealed.We further elucidated the effect of PDLIM1 on the ubiquitination level of ACTN4 by immunoprecipitation.Finally,immunoprecipitation combined with mass spectrometry was used to screen the preferential ubiquitination sites of ACTN4 in trophoblast cells.Results: Immunoprecipitation,GST-pulldown,and immunofluorescence showed that there was direct binding between PDLIM1 and ACTN4 in trophoblast cells,and immunoprecipitation results after plasmid cotransfection indicated that the Middle domain of PDLIM1 interacted with ACTN4,and the RD domain of ACTN4 played an important role in the mutual binding with PDLIM1.Immunofluorescence,immunohistochemistry and Western blotting showed that PDLIM1 was mainly expressed in placental CTB cells and i EVT cells,and its expression was significantly higher in PE placental trophoblast cells compared with the normal group.Western blot,wound healing assay,matrigel invasion assay and Transwell migration assay showed that the absence of PDLIM1 significantly enhanced the invasive migration ability of trophoblast cells while Ed U assay,CCK-8 assay and flow apoptosis showed that knockdown of PDLIM1 expression in trophoblast cells did not affect the level of cell proliferation and apoptosis.PDLIM1 prolonged the half-life of ACTN4 protein,decreased the ubiquitination level of ACTN4,and increased the expression of ACTN4 protein.Further study revealed that K140,K166,K217 and K622 of ACTN4 were its preferential ubiquitination sites.Conclusion: PDLIM1 is directly bound to ACTN4 in trophoblast cells.PDLIM1 is mainly expressed in placental CTB cells and i EVT cells,and its expression is significantly higher in PE placental trophoblast cells compared to normal group.The PDLIM1/ACTN4 axis is involved in regulating trophoblast invasion and migration,while PDLIM1 is not involved in regulating trophoblast proliferation.PDLIM1 can increase the ubiquitination degradation of ACTN4 protein and decrease the stability of ACTN4 protein,and K140,K166,K217 and K622 of ACTN4 are its preferential ubiquitination sites.