The Mechanisms Of TET2 Regulates Invasion And Migration Of Trophoblast Cells And Pathogenesis Of Preeclampsia

Preeclampsia is a major pregnancy related disease in the world,leading maternal adverse pregnancy outcomes.It seriously threatens maternal and children’s health.At present,clinical medical research has not yet solved this problem.In recent years,the incidence of preeclampsia has been increased year by year.Effective preventing and treating preeclampsia,reducing the risk of pregnant women during pregnancy,and increasing the birth rate and survival rate of pregnant women and fetuses have become the top priority for contemporary obstetrics and gynecology research.Adverse uterine conditions caused by changes in epigenetic modifications may be related to pathogenesis of preeclampsia,including dysregulation of DNA methylation and demethylation.Studies have shown that the degree of methylation in placental tissue changes significantly in patients with preeclampsia.TET2 protein can oxidize 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hmC)to regulate the methylation level of DNA,and TET2 is involved in the occurrence of various diseases,such as leukemia.In this paper,preeclampsia placental samples,trophoblast cell culture model and methylation analysis were used to conduct indepth studies on the TET2 in the regulation of MMP9 promoter methylation and it’s relevance to preeclampsia.This article explores the following aspects:In placental tissue samples,the distribution of TET2 in placenta tissue was detected by immunohistochemical staining.Quantitative PCR was used to detect the transcriptional changes of TET2 and MMP9 genes.Western blot was used to detect the changes of TET2 and MMP9 protein levels.Pyrosequencing was used to detect the methylation level of MMP9 promoter.At the cellular level in vitro,expression of the TET2(sh-TET2)gene was knocked down by lentivirus in HTR8 cells.The CCK-8 assay was used to detect the proliferation of sh-TET2 cells.Transwell assay was used to detect the invasion and migration of sh-TET2 cells.The changes of cytokines and related proteins in sh-TET2 cells were detected by anti-angiogenic factor protein microarray.The expression of MMP9 in sh-TET2 cells was detected by quantitative PCR and Western blotting.The methylation status of MMP9 promoter in sh-TET2 cells was detected by BSP sequencing.Enzyme-linked immunosorbent assay for the secretion of MMP9 in sh-TET2 cells.Dual-luciferase reporter assay was used to detect the effect of DNA methylation on transcriptional activity of MMP9 promoter.Functional recovery of sh-TET2 cells by vitamin C was determined.The results of this study are as follows:In cases of clinical samples of preeclampsia,the results show:The level of 5mC in the placenta of preeclampsia patients was higher than that of the normal control group.The level of 5hmC in the placenta of patients with preeclampsia was lower than that in the normal control group.The expression of TET2 mRNA and protein in the placenta of patients with preeclampsia was lower than that in the normal control group.The expression of MMP9 mRNA and protein in the placenta of patients with preeclampsia was lower than that of the normal control group.The methylation level of the-233 bp site in the promoter region of MMP9 in patients with preeclampsia was higher than that in the normal group.In vitro cell experiments found:After knockdown of TET2 in HTR8 cells,the migration and invasion of HTR8 cells was decreased,the secretion of MMP9 in HTR8 cell culture medium was reduced,the expression of MMP9 mRNA and protein in HTR8 cells was decreased,the methylation level of MMP9 promoter region in HTR8 cells was increased.TET2 can bind to the promoter region of MMP9.The degree of methylation of the-233 bp CpG site in the MMP9 promoter region had a significant effect on promoter activity.Vitamin C can partially restore the phenotype was caused by knockdown of TET,and MMP9 mRNA and protein expression.In summary,(1)TET2 is a DNA demethylase that oxidizes 5mC to 5hmC.In HTR8 cells,TET2 regulates the promoter activity of MMP9 and control the expression of MMP9 by determining the methylation level in the MMP9 promoter region.Then it affect the trophoblast cell migration and invasion.(2)Vitamin C can increase the enzyme activity of TET2 and restore the abnormal phenotype caused by knockdown of TET2.Therefore,this article provides a certain basis for the pathogenesis of preeclampsia by TET2,and lays a foundation for clinical prevention and treatment of preeclampsia.