The Mechanism Of NDRG1 Gene In The Migration And Invasion Of Trophoblast Cells In Sheep

N-myc downstream regulated gene 1(NDRG1)is one of the earliest tumor suppressor genes in the NDRG family,which plays a role in regulating cell differentiation,proliferation,apoptosis,angiogenesis,tumor progression and metastasis,as well as heavy metal and hypoxia sensing.NDRG1 has attracted attention because of its ability to inhibit tumor growth and metastasis.After been identified as a metastasis factor in colon cancer,it has been proved that NDRG1 can play an anti-tumor and anti-metastasis ability in lots of tissues.Embryo implantation is a crucial step in mammalian pregnancy.The migration and invasion of trophoblast cells ensure the smooth completion of embryo implantation.At the same time,trophoblast cells can produce a large number of hormones and factors necessary for embryo development and pregnancy maintenance.Therefore,trophoblast cells play an extremely important role in the process of pregnancy.Many studies have shown that NDRG1 is closely related to human or mouse pregnancy,and the erosion behavior of the trophoblast is similar to that of tumor cells.This study explored whether the differential expression of NDRG1 in sheep trophoblast cells can change the migration and invasion of trophoblast cells,so as to affect the pregnancy process of sheep,so as to provide a scientific basis for the study of sheep pregnancy process.Objective :(1)Objective to explore the localization of NDRG1 gene in trophoblast cells,reveal the relationship between NDRG1 and trophoblast cells,and prove the expression of NDRG1 gene and migration-related genes in abortion and pregnancy tissues;(2)To constructed the expression vector and interference vector of NDRG1 lentivirus and establish the differential expression cell model of NDRG1 in trophoblast tissue of sheep;(3)To explore the effect of differential expression of NDRG1 on the migration and invasion of sheep trophoblast cells.Methods :(1)Collected and isolated sheep uterus,judged whether the fetus is normal pregnancy,according to the fetal pregnancy schedule,located the trophoblast cells and NDRG1 in the trophoblast of different tissues by HE and IHC,and detected the expression of NDRG1 and migration related genes(E-cadherin,vimentin,MMP2 and MMP9)in different tissues by q RT-PCR;(2)Constructed NDRG1 overexpression and interference lentivirus vector,transient transfected to293 T,verified its function by q RT-PCR and Western blot。Used the three-plasmid system to package lentivirus,determined the titer of the virus,tested its infection efficiency,and constructed sheep trophoblast cell model with differential expression of NDRG1;(3)According to q RT-PCR explored the effection of differential expression of NDRG1 gene on cell cycle,migration and invasion.CCK-8 and scratch test were used to detect the effects of differential expression of NDRG1 gene on cell biological behavior.Results :(1)It was proved that the expression of NDRG1 was different in the trophoblast tissue of stillbirth and normal pregnancy,and the expression of NDRG1 in trophoblast tissues of stillbirth was significantly increased(P<0.01).The expression of migration and invasion related genes Vimentin and MMP2 in stillborn tissue were significantly decreased(P<0.01).What show that abnormal expression of NDRG1 in stillborn trophoblast tissue affects the migration and invasion ability of trophoblast cells,and increased expression of NDRG1 may be the cause of pregnancy failure;(2)Enzyme digestion identification and sequencing result show that the overexpression vector of Lentivirus NDRG1(CD513B-1-NDRG1)was constructed successfully.The overexpression vector and the lentivirus interference vector were transfected into 293 T cells simultaneously,and an optimal interference vector(NDRG1-SHRNA3)was selected for subsequent experiments.Lentiviral vectors were introduced into 293 T cells and packaged.The concentration titer of CD513B-1-NDRG1 virus was 2E+8TU/m L,and the concentration titer of ndrg1-shrna3 virus was 6E+7TU/ml;The virus concentrates successfully infected sheep trophoblast cells,and the cell model of differential expression of NDRG1 gene in sheep trophoblast cells was constructed;(3)NDRG1 overexpression significantly enhanced the expression of E-cadherin and β-catenin(P<0.01),and significantly inhibited the transcription levels of Vimentin and MMP2(P<0.05),MMP9 and Slug(P<0.01).The expression level of CCND1 was significantly inhibited(P<0.05),which hindered the normal cell cycle.CCK8 and scratch test showed that it inhibited cell proliferation and migration in vitro.NDRG1 interference significantly enhanced the expression of Vimentin and MMP9(P<0.01),MMP2 and Slug(P<0.05),and significantly inhibited the expresion of E-cadherin(P<0.01)and β-catenin(P<0.05).CCK8 and scratch test showed that it promoted cell proliferation and migration in vitro.Conclusion : The study proved that the expression of NDRG1 in stillborn trophoblast tissue was increased and the expression of migration-invastion-related genes was decreased,suggesting that the increased expression of NDRG1 was associated with pregnancy failure.In vitro cell experiments showed that NDRG1 could regulate cell cycle,cell proliferation,cell migration and invasion.These results indicated that the increased expression of NDRG1 in sheep trophoblast cells hindered the occurrence of pregnancy,providing clues for the study of the molecular mechanism of early pregnancy failure,and expanding the field of understanding the gene function of NDRG1.