The Expression Of CF6 In Preeclamptic Plactal Tissues And The Effect Of It On The Biological Behaviour Of Trophoblast Cells

Preeclampsia(PE)is a pregnancy disorder in which hypertension coupled with proteinuria or other symptoms of organ damage are present after the 20 th week of gestation.The increased morbidity and mortality in pregnant women occurs due to the serious damage to body systems.Since the first patient was recognized to have eclampsia in 1840,more researchers have been devoted to clarification of the mechanism of this disease to find proper prevention and treatment measures.However,the only effective way to treat this condition is to detach the placenta,which is based on the hypothesis that in the complex process of pregnancy,some factors are released from the placenta into the mother's bloodstream that trigger the spasm of systemic small vessels,ischaemia of organs and ultimately damage to entire organ systemsAs the main component of human placenta,trophoblast cells play an irreplaceable role in the process of normal pregnancy.Trophoblast cells are derived from the the trophoblast which stay at the surface of blastocyst.At the time of the late blastocyst adhering to the endometrium,trophoblast cells start to differentiate,and to invade into the endometrium,the 1/3 of uterine muscle layer and the vascular,which guarantee the placenta receiving enough blood supply,maintaining the normal development of embryos.Studies have confirmed that invasive insufficiency of the trophoblast cells can lead to the occurrence of preeclampsia.Coupling factor 6(CF6),also named ATP5 J,is a part of ATP synthase,which is expressed on the inner membrane of mitochondria and the cell membrane of various cells.ATP synthase is an energy converter and includes two subunits,namely,F0 and F1.This enzyme is responsible for conducting the synthesis and hydrolysis of ATP to supply energy for cellular metabolism.As a component of F0,CF6 is released into the extracellular fluid as a type of circulating peptide as well as being located on the membrane.After binding with particular ligands,CF6 interrupts vascular homeostasis via activating the signal factors downstream.Some studies have proven that when comparing normal pregnant women and normal non-pregnant women of childbearing age,the level of CF6 was increased in the peripheral blood of pregnant women with preeclampsia,which was particularly evident in early-onset preeclampsia and severe preeclampsia.Thus,we hypothesized that a portion of the CF6 detected in the peripheral blood circulation was secreted from the placenta;however,there have been no reports in the scientific literature about the expression of CF6 in the placenta.In the current study,we aimed to determine whether any difference existed in the CF6 expression between the normal placenta and the severe preeclamptic placenta via the detection of CF6 protein on placental tissue microarrays(TMAs)and detection of CF6 mRNA and protein in fresh placental tissues.The results could help to explain the clinical significance of the CF6 alteration in preeclampsia.The human choriocarcinoma cell lines JAR and JEG-3 cells with similar molecules and biological charactoristics with trophoblast cells can be used as the cellular models to research the invasiveness of trhophoblast.Based on the results of placental tissues study,the two cell lines were cultrued in the low oxygen to detect the CF6 secretion level,next then the cells were incubated in the normal medium with recombinant human CF6 to analyze the cells' invasiveness and expression of MMPs for exploring whether CF6 could influence on trophoblast invasion and the role of it during the mechanism of preeclampsia.ObjectThe aims of this study were to clarify the location of CF6 and to analyze the changes in mRNA and protein levels of CF6 in normal and preeclamptic placentas.In addition,the detection of CF6 of JAR and JEG-3 cells following the culture in low oxygen by ELISA,Real-time PCR(polymerase chain reaction)and Western Blot assays to demonstrate the low oxygen influence on CF6 secretion and expression.Furthermore,JAR and JEG-3 cells were incubation with exogenous recombinant human CF6 and detect the possible function of CF6 on trophoblast invasiveness using Transwell and MTT assays,combined with detection of MMPs by Real-time PCR and Western Blot assays in order to explore whether CF6 induce the invasion of trophoblast cells.Materials and methods 1 MaterialsThe placental TMAs,including placental villous cytotrophoblast(VCT)microarray and extravillous cytotrophphoblast(EVCT)microarray,were successfully constructed and validated by the Maternal and Children's Translational Medical Engineering Laboratory of Henan province.The placental tissues collected for constructing TMAs were delivered from the pregnant women who were recruited at the Third Affiliated Hospital of Zhengzhou University,China,from December 2007 to December 2010.The placental TMAs were used for detection of CF6 location and expression in placenta.Other 60 fresh placenta delivered from 30 severe preeclamptic patients and 30 normal pregnant women hospitalized in the third affiliated hospital of Zhengzhou university from January 2015 to January 2016 to further detect CF6 mRNA and protein expression level in the severe preeclamptic placentas.JAR and JEG-3 cell lines purchased from the American Type Culture Collection(ATCC)were maintained with complete medium containing 90% RPMI-1640(without FBS),10% FBS(Fetal Bovine Serum),100U/ml penicillin,100U/ml streptomycin at 37°C under the moist atmosphere of air with 5% CO2.The two cells in exponential growth stage were used for all exposure experiments.2 MethodsImmunohistochemistry was used for detection of location and expression of CF6 in the placental TMAs.Real-time PCR and Western-blot were used for detection of CF6 mRNA and protein expression level in the placental tissues,respectively.JAR and JEG-3 cell lines were resuspended in complete RPMI-1640 medium and maintained in humidified incubator with 5% CO2 at 37°C constant temperature chamber.The exponential phase cells were seeded into 6-well cell culture plate containing 2 ml of complete medium at the density of 1.5×106 cells/well.After incubation for 24 h,JAR and JEG-3 cells were made into three groups respectively according their different batches of cell cryopreservation: the control group,the low oxygen coltured group and the CF6(0.1?M)treatment group.Different groups of the two cells were cultured for another 24 h and then used to obtain the culture medium,total RNA and proteins.Western Blot,Real-time PCR,Transwell assay and MTT assay were used to investigate the changes in the expression of CF6 and MMPs,invasion and proliferation of JAR and JEG-3 choriocarcinoma cells.Based on the paired design,all the data from these assays were used to analyze the CF6 expresion in placentas and effect of CF6 on JAR and JEG-3 cells biological behaviors.3 Statistics analysisStatistical analysis was undertaken using SPSS 21.0 software.Data were presented as the mean ± SD.For comparisons between the two groups,we used an unpaired Students' s t test,Mann-Whitney U test,Kruskal-Wallis test or Chi square(?2)test as appropriate.P < 0.05 value was considered as statistically significant.Results 1 The clinical characteristics of pregnant women of TMAs and the fresh placentasTo analyse the differences between the preeclampsia and normal control groups of pregnant women from which placental tissues were collected and delivered,we kept statistics related to the clinical characteristics of the pregnant women from both groups of TMAs and the fresh placentas.These data are shown in Table 3.1 and Table 3.2.2 Location of CF6 protein in the VCT and EVCT tissue microarraysTo verify whether CF6 was expressed in the placenta,IHC was first conducted in VCT and EVCT TMAs,which included both preeclamptic blocks and normal blocks.The results showed that in the VCT TMA,CF6 was expressed in the plasma and on the membrane of cytotrophoblasts.CF6 displayed stronger staining intensity in the preeclamptic blocks compared to the normal control blocks.The same results were observed in the EVCT TMAs of the two groups.The immunostaining intensities of CF6 were then categorized and are shown in Table 3.3 and Table 3.4.The data indicated that positive percentages of immunostaining for CF6 in the preeclamptic VCT TMA reached 47/56(83.9%),which was significantly higher than in the normal VCT TMA(P<0.05).Among the preeclamptic VCT blocks,the positive percentages of severe preeclampsia was 42/48(87.5%).The difference between the severe preeclampsia group and the normal group was statistically significant(P<0.05).Similar results were found in the EVCT TMA.The percentages of CF6 expression in preeclampsia and severe preeclampsia were 39/47(83.0%),36/44(81.8%),respectively.Compared to the normal group(30/42,71.4%),these differences were statistically significant(P<0.05).3 Increased CF6 expression level in the severe preeclamptic placentasAs demonstrated previously in the immunostaining of VCT and EVCT TMAs,compared with normal placentas,a higher expression of CF6 was observed in preeclampsia,especially the severe ones.Nevertheless,CF6 expression in the placenta has not been discussed previously,we intended to further investigate if any change occurred at the post-transcriptional or transcriptional levels in our study.The CF6 mRNA and protein expression level of the 60 fresh placentas were measured.The results showed that a statistically significant increase occurred in both of them of the severe preeclamptic placentas compared to the normal control specimens(P<0.05).4 Establishment of hypoxic cellular modelAccording to some relevant researches before,hypoxia that also existed in the placental microenvironment,might have influence on the expression and release of CF6.As the first step,the hypoxic cellular model was established.However,on account of lacking hypoxic incubator,CoCl2(200?M)was used for making the hypoxic environment and increased levels of hypoxia induced factor-1?(HIF-1?)expression in cells could indicate that the cells have suffered hypoxia.By Western-blot assay,we found that the HIF-1? protein expressed higher significantly in the cells incubated with CoCl2 for 24 h than in the normal cells(JAR: 1.170±0.295 VS 0.311±0.193;JEG-3: 0.795±0.304 VS 0.210±0.101;P<0.05).The result showed that the hypoxic cellular model was established successfully which could provide the appropriate experimental condition for the following research.5 Effect of low oxygen on secretion and expression of CF6 in JAR and JEG-3 cellsIn order to analyse the induction of CF6 expression by low oxygen,CoCl2 was used to establish a cell model of hypoxia.Incubation with CoCl2(200?M)for 24 h led to increased levels of hypoxia induced factor-1?(HIF-1?)which indicated that the cells have suffered hypoxia.In the presence of CoCl2(200?M),ELISA demonstrated the secretion of CF6 increased in the medium of the cells.Western-blot suggest that the protein expression of CF6 increased significantly compared with control levels(P<0.05).However,Real-time PCR assay didn't show any significant change of the CF6 mRNA expression levels in the cells between the hypoxia group and the normal group(P>0.05).6 Effect of exogenous CF6 on invasion and proliferation of JAR and JEG-3 cells in vitroAfter incubation with 0.1?M CF6 for 24 h,MTT assay showed that there was no statistical difference of the cell proliferation between CF6 stimulation group and the corresponding control group(P>0.05).Nevertheless Transwell assay indicated that the two cells treated with exogenous CF6(0.1?M)showed decreased invasion compared with control group(P<0.05).MMP-2 and MMP-9 were generally regarded as the molecular biomarker standing for the ability of cell invasion.Therefore,we chose these two factors to detect to explore the changes of JAR and JEG-3 invasion on the molecular level.The results demonstrated that both MMP-2 mRNA and protein level were increased after CF6 stimulation(P<0.05).However,neither MMP-9 mRNA or protein level changed in the CF6 stimulation group compared with the control group(P>0.05).ConclusionsThis study suggested that CF6 expressed in placental cytotrophoblast cells and may be connected with the hypoxia signaling.Moreover,CF6 play an important role in reducing invasion of JAR and JEG-3 cells and might take part in the mechanism of preeclampsia.