| Background:Preeclampsia(PE)is a common pregnancy-specific syndrome characterized by newly occurred hypertension and proteinuria at>20 weeks of gestation,which affects approximately 2-8%of pregnancies and is a leading cause of maternal and fetal mortality and morbidity all over the world.According to gestational age of preeclampsia onset,PE is classified early onset PE(EOPE)with an onset before 34 weeks of gestation and late onset PE(LOPE)with an onset at or after 34 weeks of gestation.Although the incidence of EOPE is lower compared to the LOPE,the early onset PE is more severe,with more serious complications,and it is more likely to cause maternal and perinatal death.In addition,the early onset PE has a higher recurrence rate in the second pregnancy and her offspring are also at greater risk of cardiovascular disease and preeclampsia.Studies suggestd that EOPE was mainly a placental disease,while late onset preeclampsia is mostly a maternal disease,which might have different pathogenesis,but the specific pathogenesis of PE is still unknown.Until now,termination of pregnancy and delivery of the placenta is the most effective way to treat preeclampsia,indicating that the abnormal functions of the placenta trophoblast cell behaviors are indispensable to the onset and development of PE.The placenta trophoblast cells and tumor cells have many similarities,but regulated by many factors in the human body,the trophoblast cells function present strict time and space control.Its insufficient invasive ability may be related to fetal intrauterine growth restriction(FGR),the onset of preeclampsia and so on.If the ability was too aggressive,it was more likely to get hydatidiform mole,choriocarcinoma and other diseases of gestational trophoblastic cells.Although preeclampsia is diagnosed after 20 gestational weeks,abnormalities in placental trophoblast function may begin at the beginning of pregnancy and continue until the end of pregnancy and delivery of the placenta.To study the factors that regulate the placental trophoblast cells behaviors may increase our understanding of the onset and development of PE and provide potential novel treatment methods for PE.Placenta expresses a singular microRNA(miRNA or miR)profile which varies during different stages of pregnancy and whose abnormal alteration may be associated with PE.MicroRNAs are endogenous small non-coding RNAs,usually 19-22 nucleotides long,which regulate target gene expression by binding to the 3'untranslated region(UTR),suppress the translation or accelerate the degeneration of target mRNA,resulting in decreasing the protein production.In recent years,the roles of miRNAs in modulating diverse cell processes such as embryo development,differentiation,proliferation,apoptosis,metabolic homeostasis,tumorigenesis and immune response have been widely demonstrated.MiR-21 highly expressed in human placenta,cancers and so on.In the tumor microenvironment,the expression of miR-21 is significantly up-regulated,and the highly expressed miR-21 can promote the proliferation,invasion,migration and growth of tumor cells,which is widely recognized as an oncomir.Low expression of miR-21 was associated with fetal growth restriction suggesting that the dysregulation of miR-21 might inhibit trophoblast cells proliferation and invasion abilities.Recent studies reveal that miR-31 is involved in fertility,embryonic development,cancers and antoimmune diseases formation.Kresowik at al found that in early pregnancy compared to estrous cycle,miR-31 expression was significantly elevated and the observed increased miR-31 expression during pregnancy suggested that it may participate in the creation of an immune-tolerant maternal environment.Reduced miR-31 expressions perhaps disturb the immune-tolerant environment and could contribute to pregnancy complications such as maternal abortion and preeclampsia.Although increasing numbers of studies about miRNAs in preeclampsia are becoming available in the literature,few studies have evaluated miR-21,miR-31 in regulating trophoblast cell behaviors for preeclampsia.The two miRNAs regulate diverse cellular and developmental processes by targeting genes involved in proliferation,apoptosis,invasion,angiogenesis and immune tolerance.Using bioinformatic analysis TargetSCAN,miRanda and miRDB database,we found RAS p21 GTPase activating protein 1(RASA1)appeared to be a promising candidate target of miR-21 and miR-31.RASA1 is a member of the Ras GTPase activating protein(RAS-GAP)family and can stimulates the intrinsic GTPase activity of RAS p21,leading to hydrolysis of RAS-GTP to RAS-GDP and then inactivating the RAS p21 protein.As a result.the RAS-MAPK signaling pathways which have anti-apoptotic and pro-survival effects are turned off.However,to date,it has not been elucidated whether miR-21 and miR-31 are important in onset and development of PE through targeting RASA1.Our research mainly includes the following three parts:1.MiR-21 and miR-31 contribute to the onset of preeclampsia through targeting RASA1.2.The regulation roles of miR-21 and miR-31 on the trophoblast cell biological functions.3.The predictive value of circulating miR-21 and miR-31 for preeclampsiaPart ?:MiR-21 and miR-31 contribute to the onset of preeclampsia through targeting RASA1.Objective:To analyze the miR-21,miR-31 and RASA1 different expression levels between early onset PE,late onset PE and their respective gestational age matched normal placenta tissues.To investigate the possible targeted bindings between miR-21 and RASA1,miR-31 and RASA1.Methods:1.Seventy-seven women who underwent caesarean section form Qilu Hospital and Zibo Central Hospital were recruited in this study during the time period January 2017 and March 2018.The study was comprised the following four groups:early onset preeclampsia(EOPE,n=20),EOPE gestational age-matched normal pregnancy(Control 1,n=17),late onset preeclampsia(LOPE,n=20),LOPE gestational age-matched normal pregnancy(Control 2,n=20).2.Stem-loop real-time quantitative polymerase chain reaction was performed to investigate the different expression of miR-21 and miR-31 in normal and PE placenta tissues.3.Using bioinformatics analysis,we found RASA1 appeared to be a promising candidate target of miR-21 and miR-31.Firstly,the expression of RASA 1 in placental tissues was confirmed by IHC.Real-time quantitative polymerase chain reaction and Western blot were used to test the RASA1 different expressions in preeclampsia and normal placental tissues.Pearson correlation analysis was used to analyze the correlation between miR-21 and RASA1,miR-31 and RASA1.4.In human trophoblasts HTR-8/SVneo cells,miR-21 and miR-31 were treated by transfection of mimics and inhibitors,following verifying the transfection efficiency.The expression of RASA1 was detected by Real-time quantitative polymerase chain reaction and Western blot.The dual-luciferase reporter assay was used to test whether miR-21 and miR-31 directly regulate RASA1.Results:1.There were no significant differences in maternal age,gestational weeks at delivery,nulliparity and newborn sex between early onset PE,late onset PE and their respective gestational week matched control groups.The blood pressure,BMI and 24h urine protein of preeclampsia women were significantly higher than that in gestational week matched normal pregnancy.In the LOPE group,the birth and placenta weight were significantly lower than that in control 2,however no significant difference was observed between EOPE and control 1.2.Both miR-21 and miR-31 expression levels were significantly decreased in early and late onset PE when compared with their respective normal gestational age-matched control 1 and 2 groups3.Bioinformatics analysis showed that both miR-21 and miR-31 have binding sites at the RASA 1 mRNA 3'-UTR.IHC revealed that RASA 1 was confined to both cytoplasm and nucleus of trophoblasts in human placenta and most abundantly expressed in syncytiotrophoblasts.The mRNA and protein expressions of RASA 1 in placenta were increased both in EOPE when compared to control 1 and in LOPE when compared to control 2.Using Pearson's correlation analysis,the miR-21 and the protein expressions of RASA1,the miR-31 and RASAlprotein were both highly correlated and negatively associated4.In HTR-8/SVneo cells,the mRNA and protein expression levels of RASA1 were significantly repressed transfected with miR-21 or miR-31 mimics,whereas the levels were increased transfected with miR-21 or miR-31 inhibitors.Dual-luciferase reporter assays showed that the luciferase activities in RASA1 wild-type group were significantly reduced by miR-21 or miR-31,while that in RASA1 mutate group this reduced effect was abolished.These results demonstrated that both miR-21 and miR-31 restrained RASA1 expression by binding to RASA1 3'-UTR in human HTR-8/SVneo cells.Conclusions:1.The decreased expressions of miR-21 and miR-31 in placental tissues were related to the pathogenesis of preeclampsia.2.The RASA1 expressions in the placentas of PE were increased compared to normal pregnancy.Both miR-21 and miR-31 in placental tissues were negatively correlated with RASA1.3.In human HTR-8/SVneo cells,the expression of RASA1 could.be negatively regulated by miR-21 or miR-31.Both miR-21 and miR-31 restrained RASA1 expression by directly binding to RASA1 mRNA 3'-UTR in HTR-8/SVneo cells4.Both miR-21 and mir-31 could contribute to the onset of preeclampsia through targeting RASA1.Part ?:The regulation roles of miR-21 and miR-31 on the trophoblast cell biological functionsObjective:In human placental trophoblasts HTR-8/SVneo cells,miR-21 or miR-31 was overexpressed and down-regulated respectively,then to study the changes in the trophoblasts proliferation,migration,invasion and apoptosis,and to investigate the regulatory roles of miR-21 and miR-31 on trophoblast biological functions.Methods:1.The human HTR-8/SVneo cells were transfected by miR-21 or miR-31 mimic,mimic NC,inhibitor and inhibitor NC respectively and verified the transfection efficiency.The CCK-8 assays were performed to test the trophoblasts proliferation after transfection at 0,24.48 and 72 h.2.In HTR-8/SVneo cells,miR-21 or miR-31 was overexpressed and down-regulated respectively.Firstly,the effects of miR-21 or miR-31 on trophoblast cell migration were detected by scratching experiment.and the effects of miR-21 and miR-31 on trophoblast cell migration and invasion were further verified by Transwell migration and invasion experiment.3.In HTR-8/SVneo cells,miR-21 or miR-31 was overexpressed and down-regulated respectively.The changes in trophoblast cell apoptosis were analyzed by flow cytometry,and the expression changes of apoptosis-related proteins Caspase-3,Bcl-2 and Bax were detected by Western Blot.Results:1.The CCK-8 experiments showed that the survival rate of trophoblast cells did not change significantly when miR-21 or miR-31 were overexpressed or down-regulated for 24h,respectively;after miR-21 overexpression for 48 or 72h,the cell survival rate was significantly increased;when miR-21 down-regulated for 48 or 72h,the cell survival rate was significantly reduced;after miR-31 overexpression for 48 or 72h,the cell survival rate was significantly increased;when miR-31 down-regulated for 48 h,the cell survival rate was significantly decreased;after miR-31 down-regulated for 72 h,the change in cell survival rate was not statistically significant.2.The Scratch and Transwell migration experiments showed that overexpression of miR-21 and miR-31 significantly increased the migration abilities of trophoblast cells;the cell migration abilities were markedly suppressed in the miR-21 or miR-31 inhibitor groups.The Transwell invasion experiments showed that overexpression of miR-21 or miR-31 significantly enhanced the invasion abilities of trophoblast cells;the cell migration abilities were markedly decreased in the miR-21 or miR-31 inhibitor groups.3.Flow cytometry and Western Blot showed that overexpression of miR-21 or miR-31,the apoptosis rates of trophoblast cell were significantly reduced,the expressions of Caspase-3 were significantly inhibited-the expressions of Bcl-2 were significantly increased,and the expressions of Bax were significantly inhibited;when miR-21 or miR-31 down-regulated,the apoptosis rates of trophoblast cell were increased and the expressions of Caspase-3,Bcl-2 and Bax were opposite to that of overexpression.Conclusions:MiR-21 or miR-31 could promote the proliferation,migration and invasion of trophoblast cells,and inhibit the apoptosis of trophoblast cells through regulating apoptosis-related proteins Caspase-3,Bcl-2 and Bax.Part ?:The predictive value of circulating miR-21 and miR-31 for preeclampsiaObjective:The purpose of this part was to compare the circulating miR-21 and miR-31 levels in plasma from non-pregnancy,preeclampsia and gestational age matched normal pregnancy women,in order to identify the circulating miR-21 and miR-31 predictive value for preeclampsia.Methods:Ninety women who went to Qilu Hospital and Zibo Central Hospital were recruited in this study between January 2017 and March 2018.The study was comprised the following five groups:non-pregnancy(n=10),20-33+6 gestational weeks normal pregnancy(n=20),early onset preeclampsia(n=20),34-41 gestational weeks normal pregnancy(n=20)and late onset preeclampsia(n=20).Using miRcute Plasma miRNA Isolation Kit and stem-loop real-time quantitative polymerase chain reaction to detect plasma miR-21 and miR-31 expressions,we analysis the miR-21 and miR-31 difference between preeclampsia and gestational age matched normal pregnancy women with the non-pregnancy as the calibrator.In addition,plasma was sampled again from the early and late onset preeclampsia patients at the time of delivery to evaluate miR-21 and miR-31 changes comparing to the expression levels at diagnosed preeclampsia.Receiver operating characteristic(ROC)curve was used to analysis whether miR-21 and miR-31 had predictive value for preeclampsiaResults:1.All the study groups were similar in maternal age and body mass index(BMI).There were no significant differences between 20-33+6 gestational weeks normal pregnancy and 34-41 gestational weeks normal pregnancy groups in blood pressure,the gestational week at delivery,mode of delivery,newborn sex,birth and placental weight.In the early onset PE group,24h urine protein was significantly greater than that in LOPE,gestational week at delivery,birth and placental weight were significantly lower than that in late onset PE.however no significant difference was observed in the blood pressure,mode of delivery and newborn sex between early onset PE and late onset PE groups2.The miR-21 and miR-31 were both expressed in the plasma of non-pregnancy,20-33+6 gestational weeks normal pregnancy,early onset PE,34-41 gestational.weeks normal pregnancy and late onset PE.With the non-pregnancy group as the calibrator,the relative expression in early onset PE group was lower than gestational weeks matched 20-33+6 normal pregnancy group,however there was no significant difference.Maternal plasma miR-21 was significantly under-expressed in late onset PE when compared to 34-41 gestational weeks normal pregnancy group.We found significantly higher expression of miR-21 in 34-41 gestational weeks normal pregnancy group compared to non-pregnancy and 20-33+6 gestational weeks normal pregnancy groups.With the non-pregnancy group as the calibrator,plasma miR-31 levels were significantly reduced in early onset PE group compared with 20-33+6 gestational weeks normal pregnancy group,however there was found no evident difference between 34-41 gestational weeks normal pregnancy group and late onset PE group.Meanwhile,the miR-31 expressions in normal pregnancy revealed up-regulated at 20-33+6 gestational weeks normal pregnancy and 34-41 gestational weeks normal pregnancy,when compared to non-pregnancy.Above all,measurements of miR-21 and miR-31 both showed that there were remarkably lower in women with early onset PE compared with late onset PE3.There were no significant changes in the miR-21 and miR-31 plasma expressions between sampled at diagnosed and sampled at delivery in both early and late onset preeclampsia patients.4.The ROC curves analysis showed that the plasma expression levels of miR-21 discriminated the late onset PE patients from the gestational weeks matched healthy controls,with 65.1%sensitivity and 90.3%specificity,and the cut-off value was 2.309;the plasma expression levels of miR-31 differentiated early onset PE patients from the gestational weeks matched healthy controls and the cut-off value was 0.947,which was associated with sensitivity of 95.0%and specificity of 70.0%.Conclusions:1.Compared with the gestational weeks matched normal pregnancy,miR-21 plasma expression was significantly lower in patients with late onset preeclampsia,and miR-31 plasma expression was markedly lower in early onset preeclampsia patients.2.Measurements of miR-21 and miR-31 both showed that there were remarkablv lower in women with early onset PE compared with late onset PE3.There were no significant changes in the miR-21 and miR-31 plasma expressions between sampled at diagnosed and sampled at delivery in both early and late onset preeclampsia patients.4.MiR-21 and miR-31 expression levels changed throughout different stages of pregnancy.When studying the pathophysiological differences between preeclampsia and normal pregnancy,it is necessary to be corrected by gestational age.Only by comparing preeclampsia with the gestational weeks matched normal pregnancy,we can get a more accurate and scientific conclusion5.The ROC curve analysis showed that miR-21 had high predictive value for late onset PE and miR-31 had high predictive value for early onset PE. |
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