| BackgroundPreeclampsia(PE)is a systemic multisystem syndrome in pregnancy,which has a series of clinical characteristicses,such as hypertension,proteinuria,and edema,appearing after 20 weeks of pregnancy.It is one of the common perinatal complications for pregnant women,in view of a global incidence of about 2%-8%.Moreover,PE accounts for 10%-15%of maternal deaths and is one of the four major causes of death in obstetrics.MicroRNA(miRNA)are kind of small and non-coding RNA,with a length of 22-25 nt,which regulate gene expression at the post-transcriptional level by degrading or inhibiting the mRNA of target gene.miRNA involve and play an important role in all biological processes,e.g,cell growth and differentiation.MicroRNA 155(miR-155)is located at the third exon of BIC gene on human chromosome 21.It is considered being an inflammation-related miRNA and is related to the maturation and differentiation of immune cells,immune regulation Angiogenesis,and vasomotor function.The expression changes of miR-155 are highly correlated with many diseases,such as cancer,hypertension,atherosclerosis,viral infection,etc.Many studies have found that it plays a critical role in the occurrence and development of PE.Specifically,miR-155 can guide the proliferation,migration,differentiation and apoptosis of trophoblast cells and participate in many mechanisms,including inflammation,immunity,and vascular remodeling.Therefore,miR-155 is an important regulatory factor and may become a molecular target for the diagnosis and treatment of PE.However,the mechanism of miR-155 in the occurrence and development of PE remains unclear.The presented studies are inspired by this research gap.The expression of miRNA is affected by a variety of epigenetic mechanisms,among which DNA methylation is one of the important contents of epigenetic modification while DNA methylation mainly occurs in CpG island.Approximately,70%of the gene promoter regions contains CpG island.With bioinformatics analysis,we found that there is a CpG island in the miR-155 promoter region.Furthermore,the analysis for the target genes of miR-155 using the online biological prediction software indicated that 1)the 3’UTR of forkhead box transcription factor O3(FOXO3)has a site that can bind to miR-155;2)FOXO3 is a member of the FOXO transcription factor family which contains four members working together as transcription factors to control oxidative stress and other biological functions;and 3)FOXOs express in a variety of organs and tissues,including placenta,heart,vascular endothelium,and adipose tissue,as well as involve in cell development,metabolism,apoptosis,autophagy,and anti-oxidative stress.However,the FOXO3’ function in preeclampsia is still inconclusive.On the one hand,FOXO3 has a potential inhibitory effect on inflammatory response and oxidative stress.On the other hand,some studies have found that FOXO3 can also promote oxidative stress and inflammatory response.This divergence also exists in the study of preeclampsia.In order to explore the effect of miR-155 in preeclampsia,the study is organized as follows.First,the inflammatory factors and expression of miR-155 in the blood and placental tissues from PE patients and healthy pregnant were explored individually.The methylation level of miR-155 promoter was investigated.The relationship between miR-155 expression and methylation levels was analyzed.Secondly,the effects of miR-155 expression on trophoblast proliferation,apoptosis,migration,invasion,and the expression of MMP2,SOD1,CytC were studied.The effect of miR-155 on the regulation of trophoblast function after treatment with the demethylating drug 5-Aza was investigated.Subsequently,we will verify whether FOXO3 has a targeting relationship with miR-155 at the cellular level and explore the impact of this relationship between miR-155 and FOXO3 on the biological function of trophoblast cells.Finally,a rat model of preeclampsia was constructed to verify the effect of miR-155 in PE model.Part Ⅰ Concentration of inflammatory factors in blood and expression characteristics of miR-155 in placental tissue of patients with preeclampsiaObjectiveTo explore the concentration of inflammatory cytokines in blood of patients with PE and the expression of miR-155 in placenta in PE.Method1.20 pregnant women,who were diagnosed as PE and cesarean section in the third affiliated Hospital of Zhengzhou University from August 2017 to November 2019,were selected as PE group.Another 20 normal pregnant women,who were delivered by cesarean section in the same period,were randomly selected as control group(N-PE group).The concentrations of IL-6,IL-8,and TNF-α in serum were detected by ELISA.2.qRT-PCR was used to detect the difference of miR-155 expression in placental tissue;BSP was used to detect the methylation level of miR-155 promoter region.3.The statistical analysis of data was analyzed by SPSS Ver.22.0.Result1.There was no significant difference in age between PE group and control group(N-PE,P>0.05).The systolic blood pressure and diastolic blood pressure in PE group were significantly higher than those in N-PE group(P<0.001),the gestational age,newborn birth weight,Apgar score at 1 minute and Apgar score at 5 minutes in PE group were significantly lower than those in N-PE group(P<0.05).2.The serum levels of IL-6,IL-8,and TNF-α in PE group were significantly higher than those in N-PE group(P<0.01).3.The relative expression of miR-155 in placenta of PE group was significantly higher than that of N-PE group,which was approximately 3 times higher than that of N-PE group(P<0.01),and the expression level of miR-155 showed a significant positive correlation with the serum levels of IL-6,IL-8 and TNF-α(P<0.05).4.The higher the level of DNA methylation in the miR-155 promoter region,the lower the expression of miR-155,the expression level of miR-155 has a negative correlation with the level of methylation in the promoter region(P<0.05).SummaryThe serum inflammatory reaction in patients with PE was significantly higher than that in normal pregnant women.In addition,miR-155 was highly expressed in placenta of patients with PE and the expression level of miR-155 was negatively correlated with the level of methylation in its promoter region,suggesting that inflammation and miR-155 methylation may play an important role in the occurrence and development of PE.Part Ⅱ Effect of miR-155 on biological function of trophoblasts and its preliminary mechanismTo study the effect of miR-155 expression on trophoblast proliferation,apoptosis,migration and invasion;the expression of MMP2,CytC,and SOD1;and the effect of demethylation drug 5-Aza on miR-155 expression and trophoblast function.MethodTrophoblast JEG-3 and HTR-8/Svneo were selected for in vitro experiment.1.Cells were divided into three groups:NC group(transfection of mimic NC),mimics group(transfection of miR-155 mimic),and inhibitor group(transfection of miR-155 inhibitor).The effects of miR-155 on the proliferation,apoptosis,migration,invasion,and the expression of MMP2,CytC,and SOD1 in trophoblasts were detected.2.Cells were divided into three groups:NC group(transfection inhibitor NC),inhibitor group(transfection of miR-155 inhibitor),and inhibitor+5-Aza group(transfection of miR-155 inhibitor after treatment with demethylation drug 5-Aza).The combined effect on proliferation,apoptosis,migration,invasion,and the expression of MMP2,CytC,and SOD1 in trophoblasts were explored.3.The statistical analysis of sample data was analyzed by SPSS Ver.22.0.Result1.The relative expression of miR-155 in mimics group was significantly higher than that in NC group in JEG-3 and HTR-8/Svneo.The relative expression of miR-155 in inhibitor group was significantly decreased(P<0.05),indicating that the transfection was successful.2.The cell viability of mimics group at 24 h,48 h,and 72 h was significantly lower than that of NC group(P<0.05).The apoptosis(green fluorescence)in mimics group was significantly higher than that of NC group(P<0.05).The ability of cell migration and invasion significantly decreased in mimics group than that of NC group(P<0.05).While the inhibitor group showed the opposite effect in JEG-3 and HTR-8/Svneo.3.The concentration and expression of MMP2 in the supernatant of the mimics group was significantly decreased compared with the NC group(P<0.05).The expression of MMP2 at the mRNA and protein levels was significantly decreased in JEG-3 and HTR-8/Svneo(P<0.001).The expression of SOD 1 was significantly decreased(P<0.05),and the expression of CytC was significantly increased(P<0.05).While the inhibitor group showed the opposite effect in JEG-3 and HTR-8/Svneo.4.In both trophoblastic cells,the results showed that the expression of miR-155 in inhibitor+5-Aza group was significantly enhanced than that in inhibitor group(P<0.01).Cell viability in inhibitor+5-Aza group was significantly lower than inhibitor group at 24 h,48 h,and 72 h(P<0.05).The apoptosis(green fluorescence)in inhibitor+5-Aza group was significantly higher than that of inhibitor group(P<0.01).The ability of cell migration and invasion significantly decreased in inhibitor+5-Aza group than that of inhibitor group(P<0.05).Compared with inhibitor group,the expression of MMP2 and SOD1 were significantly weakened in inhibitor+5-Aza group(P<0.05),a significantly weakened expression on CytC was found in inhibitor group than that of inhibitor+5-Aza group(P<0.01).SummaryMiR-155 inhibitor can effectively promote the proliferation,migration,and invasion of HTR-8/S Vneo and JEG-3 cells by inhibiting miR-155.It is related to the enhanced expression of MMP2 and SOD1 and the weakened expression of CytC.While the effect of miR-155 mimic is opposite to that of miR-155 inhibitor.Based on the miR-155 inhibitor treatment,demethylation drug 5-Aza can reverse the effects of miR-155 inhibitor on trophoblast proliferation,apoptosis,migration,and invasion,as well as the expression of MMP2,CytC,and SOD1.Part Ⅲ Effects of miR-155 targeting FOXO3 on the biological function of trophoblasts and the placental function of PE rat modelObjectiveFirst,verify whether the target gene of miR-155 is FOXO3.Then,explore the effect of the relationship between miR-155 and FOXO3 on the biological function of trophoblasts through the rescue experiment.Finally,verify the effect of miR-155 on PE model from the animal level.Method1.Verification of miR-155 targeting FOXO3 3’UTR by double-luciferase assay.2.JEG-3 and HTR-8/SVneo were divided into five groups:NC group,inhibitor group,si-NC group(transfected with siRNA negative control),si-FOXO3 group(transfected with FOXO3 siRNA sequences),and inhibitor+si-FOXO3 group.The expression levels of FOXO3,MMP23 CytC,and SOD1,along with cell proliferation,apoptosis,migration,and invasion were measured.3.The PE model of rats with preeclampsia was established and divided into three groups:Sham,PE and PE with miR-155 inhibitor injection.MMP2,IL-6,IL-8,and TNF-α in serum were detected in serum.The expression of miR-155,methylation of miR-155 promoter,and the expression of MMP2,CK7,and FOXO3 in placenta were detected in placenta.4.The statistical analysis of sample data was analyzed by SPSS Ver.22.0.Result1.MiR-155 can directly target FOXO3 3’UTR and inhibit FOXO3’ expression.2.In both trophoblastic cells,compared with si-NC group,the ability of cell proliferation,migration,and invasion decreased in si-FOXO3 group(P<0.05),the apoptosis level of cell was up-regulated(P<0.01).The concentrations of IL-6,IL-8,and TNF-α in the cell supernatant were all increased(P<0.05).The concentration of MMP2 in the supernatant of cells in the si-FOXO3 group was significantly lower than that in the si-and protein levels was significantly lower(P<0.05).The expression of SOD1 was NC group(P<0.05)and the expression of MMP2 in cells at both mRNA weakened(P<0.05),while the protein expression of CytC was enhanced(P<0.05).3.In trophoblastic cells,compared with si-FOXO3 group,the cell viability of inhibitor+si-FOXO3 group was significantly increased(P<0.05),the migration and invasion ability of the cells were significantly increased(P<0.05),the apoptosis signal was significantly decreased(P<0.01),and the concentrations of IL-6,IL-8,and TNF-αin the supernatant were significantly decreased(P<0.05).The concentration of MMP2 in the supernatant and the expression level of MMP2 mRNA in the cells of inhibitor+si-FOXO3 group were significantly higher than those in si-FOXO3 groups(P<0.05),the expressions of FOXO3,MMP2,and SOD1 were significantly up-regulated(P<0.05),while the expression of CytC was significantly down-regulated(P<0.05).4.Compared with control group,urinary protein and systolic pressure in PE group were significantly increased,indicating that the model was successfully established.The concentrations of IL-6,IL-8 and TNF-α in serum in PE group were significantly increased(P<0.01),while the concentration of MMP2 and expression of FOXO3,MMP2 and CK7 was significantly decreased(P<0.01),together with significantly increase expression of miR-155(P<0.01).5.In preeclampsia rats injected with miR-155 inhibitor,that is,miR-155 inhibitor group,urinary protein and systolic blood pressure decreased significantly compared with PE group.The concentrations of IL-6,IL-8,and TNF-α in serum were significantly decreased(P<0.05),while the concentration of MMP2 was significantly increased(P<0.05).The expression levels of FOXO3,MMP2 and CK7 in placental tissue were significantly enhanced(P<0.05).The expression of miR-155 was significantly weakened(P<0.05),while the methylation level of miR-155 promoter region was not changed significan5tly(P>0.05).SummaryMiR-155 can target the FOXO3 3’UTR region,inhibit the expression of FOXO3,and may play an important role in trophoblasts by promoting apoptosis and inhibiting migration,invasion,and proliferation.In the animal model of PE,miR-155inhibitor treatment can significantly attenuate the inflammatory response,promote the expression of MMP2 and CK7 and significantly enhance the expression of FOXO3,indicating that the inhibition of miR-155 has a potential therapeutic effect in PE disease.Therefore,miR-155 may be a potential therapeutic target for PE.Full text conclusions1.MiR-155 is highly expressed in placental tissue in patients with preeclampsia and is inversely correlated with the level of DNA methylation in its promoter region.2.MiR-155 has the effect of inhibiting trophoblastic proliferation,migration,invasion,and promoting apoptosis and inflammatory response.3.MiR-155 regulates trophoblast proliferation,migration,invasion,apoptosis,inflammatory response,and oxidative stress levels by targeting FOXO3.4.In the PE model of rats,inhibition of miR-155 expression can significantly improve inflammatory response,promote the expression of MMP2 and CK7 in rat placental tissue,and significantly increase the expression of FOXO3. |
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