Study On The Effect Of Dual Specificity Phosphate1( DUSP1) In Severe Pre-eclampsia

Background and ObjectivePre-eclampsia is best described as a pregnancy-specific syndrome of reduced organ perfusion secondary to vasospasm and endothelial activation. It is severe and clinically important manifestations of placental insufficiency. PE is known as a placenta-dependence disease, which is a leading cause of maternal and fetal death. Usually, PE occurs after 20 weeks gestation. High blood pressure, proteinuria and edema are the main symptoms of PE. The clinical symptoms would disappear after the pregnancy termination. Recently, the study about the etiology and pathogenesis of PE has become one of the popular topics in the obstetric field. Many theories, of which the Two Stages Theory is the most accepted one, have been formed about PE. What is the real and most important reason of PE? How can we successfully predict PE? Problems such as these still remain unknown. Many biologic factors such as HIF-1α, PLGF, PAPP, VEGF and IL-2 are related with PE, but which one can be the marker of PE? More studies are needed.Dual specificity phosphates(DUSPs) are a superfamily of phosphates including DUSP1, DUSP2, DUSP5, DUSP9 etc. DUSPs play an important role in signal transduction pathways in inactivating mitogen-activated protein kinases(MAPK). Researches have shown that down regulation of DUSP1 induces changes in the expression levels of genes involved in specific biological pathways, including angiogenesis. DUSP1 is not only an important negative regulator of p38 MAPK activity but also a potent inhibitor of MAPK activity through dephosphorylation of MAPK. Researchers have investigated and found DUSP1 to be an important negative regulator of acute inflammatory responses and also the function of DUSP1 in protecting over-activation of hypoxia-inducible factor 1(HIF-1) through inactivating ERK/MAPK. Another study showed that the inflammatory response and HIF-1α played an important role in the pre-eclamptic patients, where HIF-1 was one of transcription factors, and played an important role in controlling oxygen delivery and metabolic adaptation in hypoxic conditions. HIFs and vascular endothelial growth factor(VEGF) may play an important role in the establishment and development of pregnancy. The HIFs were tested to regulate placentation and vascularization by stimulating VEGF gene expression.The purpose of this study was to test DUSP1 expression in normal human pregnancy and severe pre-eclampsia(sPE) and to analyse what was the relationship between DUSP1 and HIF-1α in s PE. DUSP1 and HIF-1α protein and mRNA expression in villi of the first trimester of pregnancy and placenta of normal human gestation and in s PE were tested. We also used Be Wo cell culture in the normoxia( the O2 concentration 20%) and hypoxia circumstance(the O2 concentration 1%) at 37oC, then DUSP1 and HIF-1α protein and m RNA expression were tested, at the same time, we tested the function of proliferation and invasion of the Be Wo cell. We analysed the relationship between DUSP1 and HIF-1α in Be Wo cell.Materials and methods1. Patient selection and placental collectionThe study was approved by the Ethics Committees of Daping Hospital, Third Military Medical University, and all the patients signed consents for their sample donation. Between January 2013 and May 2014, 94 Villi was gained from those women who terminated their pregnancy during the first trimester(5-10weeks), 90 terminal gestations and 70 s PE admitted to the Department of Obstetrics and Gynecology, Daping Hospital and Xi’nan Hospital, Third Military Medical University, Chongqing, were included in the study. All the patients included had no history of chronic hypertension, diabetes mellitus, nephropathy etc. All the placental tissues including both normal terminal pregnancy and sPE were obtained by cesarean sections. Placental collection was performed as per the protocol. Once the collection was complete, samples were sent to the seventh department of Daping Hospital and Research Institute of Surgery for analysis.2. Be Wo cell cultureThe human choriocarcinoma cell line Be Wo(passages 10e) was kept in frozen nitrogen canister. After thawing, the cells were maintained in DMEM/high glucose media(Gibco, United States), supplemented with 100 units/ml penicillin and streptomycin(North China pharmaceutical Group Corporation, China) and 10% fetal bovine serum(Gibco, United States), in normoxia(20% O2) and hypoxia atmospheric(1% O2) at 37oC in a sealed chamber. In order to further proceed with the observations, after 24 h, 36 h, 48 h, 60 h and 72 h incubation, cells were extracted by using RIPA lysis buffer(Beyotime Institute of Biotechnology, PR China) which included both protease and phosphatase inhibitors.3. Protein and mRNA level of DUSP1 and HIF-1α detectionELISA and Western blotting were used to characterize DUSP1 and HIF-1α protein and Q-PCR for gene expression levels in villi of different ages of the first trimester pregnancy, placentas of terminal gestations pregnancy and in s PE. DUSP1 and HIF-1α protein and gene expression levels were detected in Be Wo cell in different circumstance culture.4. To change the expression of DUSP1 in BeWo cellWe used DUSP1 overexpression and siRNA to transfect Be Wo cells, after 24 h, 36 h, 48 h, 60 h and 72 h incubation, then we tested the expression of HIF-1 protein and m RNA level in Be Wo cells. And the ability of invasion and proliferation of BeWo cells were also tested in different circumstance.Results1. In the clinical experiment part, DUSP1 and HIF-1a expression was different in villous trophoblast of the first trimester and placentas of normal terminal pregnancy and sPE. DUSP1 protein level by ELISA were respectively 824.22±236.17,1058.43±266.23 and 774.12±144.76pg/ml, DUSP1 mRNA relative level by Q-PCR were 0.0263±0.0082, 0.3535±0.0142, 0.0062±0.0018 in villous of first trimester, placentas of normal terminal pregnancy and sPE. HIF-1α protein level by ELISA were 264.42±66.23, 155.25±36.60 and 314.42±44.76 pg/ml; HIF-1α m RNA relative level by Q-PCR were 0.326±0.036,0.239±0.038, 0.445±0.052 in villous of first trimester, placentas of normal terminal pregnancy and sPE.2. DUSP1 and HIF-1α protein and mRNA level ranged in normoxia and hypoxia circumstance of Be Wo cells. DUSP1 protein and m RNA level decreased in BeWo cells cultured in hypoxia circumstance. On the contrary, the level of HIF-1α increased in BeWo cells cultured in hypoxia circumstance when the time proceeded. The level of DUSP1 and HIF-1α protein and m RNA was significantly different especially in 48 h, 60 h and 72 h culture in the two groups(normoxia and hypoxia group). The expression levels of DUSP1 in trophoblast would have a big impact on cell invasion and proliferation.3. In the BeWo cells with DUSP1 overexpression,P-ERK and HIF-1α protein and m RNA level was decreased. The ability of invasion improved by the test of Transwell, and the ability of proliferation was decreased by CCK8. On the contrary, In the Be Wo cells with DUSP1 si RNA,P-ERK and HIF-1α protein and m RNA level was increased. And the ability of invasion was decreased but proliferation was increased.Conclusions1. The DUSP1 protein and mRNA level was significantly lower in sPE placentas compared to normal terminal pregnancy(p<0.05). The HIF-1α protein and m RNA level was significantly higher in sPE placentas compared to normal terminal pregnancy. So there was some relationship between the level of DUSP1 and HIF-1α and the placental abnormality development of s PE.2. DUSP1 protein and mRNA level decreased but HIF-1α protein and mRNA level increased in hypoxia circumstance of Be Wo cells. The ability of invasion and proliferation of Be Wo cells was different in normoxia and hypoxia circumstance, the decreased DUSP1 protein expression could decrease the ability of cell invasion and improve the proliferation function. The oxygen concentration could be one of the key links in PE, We could prevent the occurrence of PE by adjustment of the oxygen concentration.3. DUSP1 overexpression Be Wo cells, P-ERK and HIF-1α protein and mRNA level was decreased. The ability of invasion improved and the ability of proliferation was decreased. DUSP1 might protect over-activation of HIF-1α through inactivating ERK/MAPK in PE.