The Study On The Mechanism Of Ndrg1 Regulating Preeclampsia By Weakening Invasion Of Trophoblast Cells

Introduction:Preeclampsia(PE)is a special type of hypertensive disorders complicating pregnancy(HDCP).PE's major characteristic is emerging high blood pressure,proteinuria and edema after 20 weeks of gestation,however,blood pressure returns to normal after delivery.Because of the differences of geography,society,economy,race,culture and so on,its incidence is different in various regions.Worldwide,the incidence of PE is 2%-8%,however,the incidence of PE is higher(9.4%)in China.Although more and more countries continuously improve maternal and child health,but over half a million women die from pregnancy-related diseases each year.Among them 10%~15% direct maternal deaths are associated with PE and eclampsia.The etiology and pathogenesis of PE have not been fully elucidated,it is generally believed that restrictive trophoblast invasion in uterine spiral artery is the key link of PE.Trophoblast cells are the main components of placenta,which play an important role in the formation of placenta and the normal growth of the fetus.In the early stage of blastocyst implantation,trophectoderm cells differentiate into two types of trophoblast cells,namely cytotrophoblasts and syncytiotrophoblasts.The former has two differentiation pathways:fusion with syncytiotrophoblast and conversion to extravillous trophoblasts(EVTs).The later has invasion ability.A portion of EVTs invade into the deep layer of endometrium and a third of myometrium and are named interstitial trophoblast cells.A portion of EVTs intrude into maternal uterine spiral artery(SPA)and are named intravascular trophoblast cells.They gradually replace the vascular endothelial and vascular smooth muscle cells and cause the degradation of vascular basement membrane.This process completes the reconstruction of SPA,which forms high blood flow and low resistance of SPA to supply enough blood into fetus and placenta.If invasion disorder occurs in placentation and blood volume into placenta decreases,severe impariment will appear.On one hand,it can result in fetal intrauterine growth restriction.On the other hand,trophoblast cells will secrete some cytokines or hormones due to hypoxia and they cause systemic vascular endothelial function damage and excessive inflammatory reaction which leads to a series of clinicalsymptoms in PE patients,such as hypertension and proteinuria.Trophoblast cells have the similar invasion to tumor cells.However,their invasion progress is regulated strictly and complex cellular signal transduction pathways are involved.Nowadays the specific molecular mechanism has not yet been fully elucidated.N-myc downstream regulated gene 1(NDRG1),also known as differentiation related gene 1(DRG1),response induced by stress 42(Rit42),protein regulated by oxygen-1(PROXY1),etc.,is discovered in colon cancer cell line in 1997 for the first time.NDRG1 is a relatively conservative sequence in the process of evolution,which exists in multiple tissues and participates in various physiological functions,such as proliferation,differentiation,hypoxia and stress reaction.In recent years it is found that NDRGl plays an important role in the process of tumor invasion.NDRGl can downregulate the expression of matrix metalloproteinases(MMPs)and reduce the invasion capacity of many tumors,such as prostate carcinoma,rectal carcinoma,breast carcinoma,pancreatic carcinoma and gastric carcinoma.In vivo and in vitro studies show that overexpression of NDRG1 could inhibit invasion and metastatic potential of tumor cells.The erosion behavior of trophoblast cells is similar to that of tumor cells.Previous studies show that NDRG1 mainly lies in trophoblast cells of placenta and its expression is enhanced in placenta of PE patients compared with that of normal pregnant women,which indicates NDRG1 expression is related with the occurrence of PE.According to the onset time,PE can be divided into early-onset and late-onset PE.The earlier the onset,the more serious the illness.Is NDRG1 differentially expressed in the two types of PE? There is no report until now.The invasion dysfunction of trophoblast cells is the core of pathophysiological mechanisms of PE,which causes remodeling disorder of SPA and the decrease of blood volume in placenta.However,NDRG1 can inhibit the invasion of tumor cells.Thus,we speculate NDRG1 may regulate trophoblast invasion process.In the study,we will investigate the change of invasion in JEG-3 cells in vitro after silencing NDRG1 expression.It is well known that the invasive progress of cells is completed through hydrolysis of protease in tissue.MMPs,zinc dependent proteolytic enzymes,are capable of degrading most of extracellular matrix with the generation of their active forms.Previous studies show that the remodeling process of uterine SPA is closely related to the expression of MMPs.Among them,MMP-2 and MMP-9 have been known as rate-limiting proteases during invasive process of trophoblasts.Extracellular regulated protein kinases(ERK)belong to the family of mitogen activated protein kinase(MAPK).Its abnormal activation can affect the invasion and metastasis of malignant tumor.Meanwhile it can also improve MMP-9 expression.However,whether ERK/MMPs signal pathway is involved in regulatory effect of NDRG1 has not been reported.Therefore,in the study,we also investigate the molecular mechanism of NDRG1 regulation of trophoblast invasion by silencing the expression of NDRG1 in JEG-3 cells.Objective:First of all,this study is to confirm that NDRG1 is closely related to the occurrence and development of PE through detecting the location and expression of NDRG1 on placenta of early and late PE patients.After that,to explicit NDRG1's effect on the invasion of trophoblast cells and PE occurrence and explore the underlying molecule mechanism,we silenced NDRG1 expression and observed the difference of cell viability and invasion capacity of trophoblast cells and the proteins expression in ERK/MMP signal pathway.The aim of our study is to provide valuable research information for pathogenic mechanism of PE.Methods:1.NDRG1's expression on placenta of PE patients(The related content has been published in PLACENTA.)The pregnant women involved were all from Affiliated Hospital of Qingdao University.Their production inspection and delivery were finished from September 2014 to March 2015 in Department of Gynaecology and Obstetrics of this hospital.All participants were divided into 5 groups: normal group,early gestation group,premature delivery group,early-onset PE group and late-onset PE group.Normal group(n=20)included normal singleton term pregnancies.Early gestation group(n= 20)included healthy women undergoing legal abortion for nonmedical reasons.Premature delivery group(n=20)included 24~37 years old women with gestational age less than 37 weeks without other complications.PE group included 40 individuals with PE between the ages of 25~44 years and the diagnosis of PE was based on clinical evidence.According to onset time,they were divided into two subgroups,early-onset PE(n=20),and late-onset PE(n=20).Their clinical data(times of gravidity,gestational age,proteinuria,blood pressure and birth weight)were collected.After a vaginal or abdominal delivery,the tissue was frozen around fetal surface and umbilical root of placenta.As for artificial abortion patients,chorionic villi of early pregnancy was frozen.Then the location and expression of NDRG1 on placenta of these groups were compared by immunohistochemistry,real time PCR and western blot technique.2.The effect of silencing NDRG1 on invasion capacity in JEG-3 cellsIn cell experiments,human choriocarcinoma cell line JEG-3 was used and cells were divided into 2 groups,namely,control group and siRNA group.In siRNA group NDRG1 expression was silenced by transfection.Cell proliferation was tested by MTT.Cell apoptosis was determined by flow analysis technology.Cell invasion capacity was detected by transwell experiment.3.The underlying mechanism of NDRG1 regulating invasion capacity of JEG-3 cellsThe normal and NDRG1-silenced JEG-3 cells were cultured and the supernatant of the two groups of cells were collected.MMP-2 and MMP-9 levels in the medium were determined by ELISA.Meanwhile,cell protein was extracted.Western Blot analysis was applied to test the expressions of ERK,phosphorylated ERK(p-ERK),MMP-2 and MMP-9.Results:1.Clinical characteristicsOur results showed there were no significant differences in maternal age and times of gravidity among all groups.All the PE patients suffered from hypertension and proteinuria.The systolic blood pressures were respectively increased by 26% and 40% in early-onset PE group and late-onset PE group,and the diastolic pressures were increased by 52% and39%,respectively.Compared with that of control group,gestational age of early-onset PE patients were shortened by 14%(P< 0.05)and all the patients in this group belonged to premature delivery.More importantly,birth weight in early-onset and late-onset PE group were significantly lower by 54% and 41% correspondingly than that in control group and neonates from early-onset PE group had lower birth weight than those from late-onset PE(P < 0.01).2.NDRG1 expression in placentaImmunohistochemistry staining of placenta showed NDRG1 was located in the cytotrophoblasts,syncytiotrophoblasts and intercellular substance.NDRG1 staining was found in cell membrane and cytoplasm.There was a marked difference in the intensity of NDRG1 staining among different groups.Compared with that of control group,NDRG1 expression was decreased in early gestation group(P< 0.05)and was significantly increased in premature delivery group(P< 0.01).Meanwhile,NDRG1 expression in PEgroups was highest in all groups and the expression of NDRG1 in early-onset PE group was higher than that in late-onset PE group(P< 0.01).Results from RT-PCR and western blot were consistent with immunohistochemistry.It was noteworthy that the expression of NDRG1 in early-onset PE group was higher than that in premature delivery group with the same gestational age and the expression of NDRG1 in late-onset PE group was higher than that in control group with the same gestational age,which indicated NDRG1 expression was upregulated in placentas of PE patients at the same stage of placenta development.3.The effect of NDRG1 expression on invasion capacity in JEG-3 cellsWestern blot analysis showed the expression of NDRG1 was efficiently decreased by91.14% in NDRG1-silenced JEG-3 cells compared with control cells,which indicated the success of gene silencing.MTT testing and flow analysis showed silencing NDRG1 did not change cell proliferation ability and apoptosis percentage.However,the number of invasive cells in siRNA group was significantly enhanced compared with that in control group(P<0.01)4.The effect of NDRG1 on ERK/MMPs expression in JEG-3 cellsIn the study,MMP-2 and MMP-9 levels in the cell culture supernatant were determined by ELISA and the data indicated the secretions of MMP-2 and MMP-9 from JEG-3 cells was raised by 101.2% and 65.01% correspondingly after silencing NDRG1.Western blot analysis revealed the relative expression level of MMP-2 was increased by 53.7% after silencing NDRG1 in JEG-3 cells and MMP-9 expression was elevated by48.83%.The total content of ERK1 and ERK2 had no significant difference between the two groups,however,the total protein level of p-ERK1 and p-ERK2 in siRNA group was upregulated by 61% compared with that in control group.Conclusions:1.NDRG1 expression on human placenta is low in the first trimester of pregnancy,however,it is raised with increasing of gestational age.2.NDRG1 expression is significantly upregulated in placentas of PE patients,especially in early-onset PE patients.3.Silencing NDRG1 enhances cell invasion capacity in JEG-3 cells.4.NDRG1 regulates cell invasive via ERK/MMP 2/9 pathway which is probably one of molecular mechanisms involved in PE's pathogenesis.