The Importance Of P53-tristetraprolin-stathmin1 Molecular Pathways On Trophoblast

Objective: During normal pregnancy,cell proliferation and apoptosis are wellbalanced in placental villi and decidual cell.Abnormal apoptosis may lead to pathological pregnancy and spontaneous abortion.Recently,it was found that p53 as a kind of apoptosis pathway is also involved in spontaneous abortion.Stathmin1(STMN1)is a major cytosolic phosphoprotein that regulates microtubule catastrophe.STMN1 is involved in tumor cell proliferation,invasion,differentiation,and motility.The study found that in breast cancer,gastric cancer and prostate cancer STMN1 involved in regulating the AKT signaling pathway,beta catenin signaling pathways,such as promoting the migration and proliferation of tumor cells.In comparison,trophoblastic cells in the maternal-fetal interface STMN1 biological function is still poorly understood.This topic by collecting recurrent miscarriage samples and setting up trophoblast cell model in vitro to explore STMN1 differentially expressed in maternalfetal interface and its importance,and further study of biological mechanisms affecting STMN1 expression and interaction,so as to better illustrate its important role in maternal-fetal interface provides the basis.Methods: By collecting recurrent abortion and normal abortion villus samples immunohistochemistry and Western blot assay were performed to investigate the expression level STMN1 and p53 on maternal-fetal interface.Stimulated HTR8-S/V neo cells with doxorubicin(doxo)to build a trophoblast cell model of recurrent spontaneous abortion in vitro,qRT-PCR,western blot and immunofluorescence assay was performed to investigate the expression level of STMN1 and Tristetraprolin(TTP)on mRNA and protein respects.Constructing TTP gene pairs of luciferase reporter gene plasmid,that with the mutation sequence of STMN1 transcription factor expression plasmid co-transfection HTR8 cell line,by detecting the expression of STMN situation,explore STMN1 relations with TTP.Nuclear/Cytosol Fractionation Kit was used to the cell lysis solution which have been stimulated with doxorubicin in order to determine TTP and STMN location and changes in the cell.To study the relationship between STMN1 and TTP,Western blot,gene knockdown and overexpression experimental methods were used to confirm the influence of TTP on expression of STMN1.Results: The immunohistochemistry and Western blot performed with human villus showed that STMN1 expressed in the villus syncytiotrophoblast and lower in recurrent spontaneous abortion,but the expression of p53 is higher than normal pregnancy.Cells in vitro experimental results showed that when using the doxorubicin to activate p53,STMN1 expression is reduced,but increased expression of TTP.The time of doxorubicin produces difference is at about 24 h and the optimal concentration is 1.0 nmol/ml.Western blot found that TTP is mainly located in nuclei,and after doxorubicin activated it appeared the phenomenon of nucleus expression increased.Moreover,the simultaneous addition of MUT1 and MUT2 had an obvious effect on STMN1.In a rescue experiment using TTP overexpression,WT,MUT1,MUT2,and MUT1+MUT2 transfection significantly decreased STMN1 expression as compared with transfection in the vector control cells After overexpressing TTP,STMN1 expression decreased and after knockdown TTP,the expression of STMN1 rise.Conclusions: P53 apoptosis pathway in patients with recurrent miscarriage expression increase,may cause tristetraprolin expression increased resulting in STMN1 expression is insufficient.Then because of the lack of trophoblast cell invasion,embryonic development and placental implantation are impacted which then lead to pregnancy failure.