CSRP2BP Regulated The Function Of Trophoblasts And Vascular Smooth Muscle Cells In Preeclampsia

IntroductionPreeclampsia(PE)is a gestation-specific syndrome,which is also one of the leading causes of maternal and perinatal morbidity and mortality.Inadequate invasions of trophoblast cells cause shallow invasion and impairs placental vascular formation,which leads to further ischemic and hypoxic placenta,both of them processed the occurrence and development of PE.There is still lack of effective prediction methods for PE,so elucidation of the molecular mechanism of PE,and defined new diagnostic markers of PE,are significant for the early diagnosis and treatment of PE.The roles of Epigenetic factors in PE are the currently research hotspots.The Cysteine-Rich Protein 2-Binding Protein(CSRP2BP)is a newly defined histone acetyltransferase,which is one of the core members of ATAC chromosome remodeling complex,which has an important biological function in the development of stem cells and mouse embryos.However,the functional studies are still very limited in human cells.Our preliminary analysis found that CSRP2BP is down-expressed in the placenta of PE,suggesting that it may be involved in the pathological process of PE.Therefore,we further explored the role of CSRP2BP in human trophoblast cells and vascular smooth muscle cells as well as its potential mechanisms in the development of PE.Objective1.Explore the expression of CSRP2BP in human placenta tissues.2.Analyze the relationship between CSRP2BP expression and the clinical features in PE patients.3.Study the role of CSRP2BP in trophoblast cells proliferation and apotosis.4.Study the role of CSRP2BP in trophoblast cells migration and invasion.5.Explore the molecular mechanisms of CSRP2BP in trophoblast cells.6.Study the role and molecular mechanisms of CSRP2BP in human vascular smooth muscle cells.Methods1.IHC and Western Blotting were used to investigate the expression and localization of CSRP2BP in human first-trimester placenta villi and terminal placenta,and further to compare the expression of CSRP2BP as well as the acetylation of H3 in PE patients and normal pregnancy women placenta tissues;2.Using SPSS20.0 to analyze the relationship between the expression level of CSRP2BP and clinical indicators in PE patients.3.Using human extravillous trophoblast cell line HTR-8 cell as a cell model to study the effect of CSRP2BP in the biological function of trophoblast cells:to detect the localization of CSRP2BP in HTR-8 cells by immunofluorescence,qRT-PCR and Western Blotting;to silence the expression of endogenous CSRP2BP in human trophoblast HTR-8 cells by shRNA and siRNA or to increase CSRP2BP expression by lentivirus vector;to detect the effect of CSRP2BP on the proliferation and cell cycle of HTR-8 cells by plate colony formation assay,EDU assay and flow cytometry analysis;to observe the effect of CSRP2BP on the migration and invasion of HTR-8 cells by wound healing experiments and invasion assay;to observe the effect of CSRP2BP on apoptosis of HTR-8 cells by flow cytometry analysis.4.Using gene expression microarray to analyze the changes of gene expression profiles after silencing endogenous CSRP2BP in HTR-8 cells,and screen possible target genes of CSRP2BP.5.Using immunohistochemistry and immunofluorescence to detect the expression and localization of CSRP2BP in human placenta tissues and primary human vascular smooth muscle cells,and the expression of CSRP2BP in placenta vascular smooth muscle of PE group and normal group was compared.6.After overexpression or silencing of CSRP2BP in 10T1/2 cells or human vascular smooth muscle cells,the expression of SMA,SM-MHC,Calponin and SM22a which affect the differentiation and function of smooth muscle cells were detected by immunofluorescence and qRT-PCR.7.Isolating the primary human primary vascular smooth muscle cells and silencing their endogenous CSRP2BP by siRNAs,and exploring the effect of CSRP2BP on the migration of human smooth muscle cells was analyzed by wound healing experiments.8.Constructing mutants with different functional domains of CSRP2BP and studying the mechanism of CSRP2BP in the regulation of human smooth muscle related genes expression.Results1.The expression of CSRP2BP was enriched in chorionic villous cells and vascular smooth muscle cells.2.We found that CSRP2BP expression and the acetylation of H3 was markedly down-regulated in PE patients' placenta tissue samples,in comparison to that in normal pregnancy patients' placenta tissue samples.3.Immunohistochemistry showed a significant difference in CSRP2BP expression in PE patients(n=85)and normal pregnant placenta tissues(n=58).4.Colony formation assays showed a drastic reduction of cell proliferation in HTR-8 cells in which CSRP2BP expression was knocked down.5.EdU experiments showed that the silencing endogenous CSRP2BP in HTR-8 cells reduced the incorporation of EdU.Cell cycle experiments showed that silencing endogenous CSRP2BP in HTR-8 cells were arrested in G0/G1 phase and HTR-8 cells with overexpression of CSRP2BP were arrested in G2/M phase.6.Wound healing experiments showed that down-expression of CSRP2BP in HTR-8 cells reduced the migration.7.Transwell assays showed that down-expression of CSRP2BP in HTR-8 cells reduced the invasion and overexpression of CSRP2BP in HTR-8 cells improved the invasion.8.Apotosis assays showed that there is no difference in apotosis rate between HTR-8 cells with CSRP2BP down-expression and control cells.9.FOXA1 expression was inhibited or increased after silencing or overexpression CSRP2BP in HTR-8 cells.10.CSRP2BP was differently expressed in human placenta smooth muscle cells,as well as the number of the blood vessels were different between the placental tissues of the PE group and the normal pregnancy group.11.After silencing CSRP2BP in human vascular smooth muscle cells,the expression of smooth muscle-related genes SMA,SM22a,caiponin,and SM-MHC was decreased,while after overexpressed CSRP2BP in 10T1/2 cells,the expression levels of these related genes were increased.12.After silencing the expression of endogenous CSRP2BP in human vascular smooth muscle cells,the migration function was weakened.13.There was an interaction among CSRP2BP,SRF and Myocardin,the activity of CSRP2BP requires the participation of its HAT domain and its interaction domain with SRF and Myocardin.14.The expression of CSRP2BP was significant different between PE group and normal control group,and was correlated with systolic blood pressure,diastolic blood pressure and number of births in patients.Conclusions1.CSRP2BP was down regulation in PE placenta tissues;the expression of CSRP2BP was associated with some clinical features of PE patients.2.CSRP2BP promoted the proliferation,invasion and migration of HTR-8 cells,but had no influence in apoptosis.3.CSRP2BP affected the function of human vascular smooth muscle cells.4.CSRP2BP regulated the expression of human smooth muscle genes through the interaction with SRF.5.CSRP2BP might be a novel and useful prognostic marker of PE.