Roles Of MiRNAs In Regulation Of Decidual Tissues With Early Pregnancy Loss

Human reproduction is regarded as being extremely complex and inefficient compared to that of other mammalian species.It has been estimated that approximately 50%-70%occurrence rate of early pregnancy loss during the first trimester.Even after a successful implantation(47.7%),approximately 15%-20%of clinical miscarriages were reported to be occurrence.Early pregnancy loss(EPL)is a clinical problem both puzzling clinicians and patients.The precise incidence of EPL has been more clearly defined with the history of menopause,transvaginal ultrasound and urine tests for human chorionic gonadotrophin(β-HCG).Through transvaginal ultrasound only the yolk sac has been documented on scan,without germ-free fetal heart,observed no signs of further growth.There is not double increasing every other day or falling down of blood β-HCG Excluded available reasons such as chromosomal abnormalities,abnormal anatomy of the maternal genital tract,endocrine disorders,autoimmune diseases,coagulation disorders,there is still a significant proportion about 41-70%of EPL are unknown.Doctors still could not give a definite cause of interpretation and effective preventive measures.Embryo implantation is the key process in reproduction of many species,and this process is a unique biological phenomenon.The whole process involves a series of key complex signal network system.It has been known that a successful pregnancy requires two important elements,a quality embryo and maternal endometrial receptivity.Precise dialogue between the maternal-fetal interface lead to a successful embryo implantation.Researches usually focus on villus on the causes of early pregnancy loss.But besides the embryo factors,as soil as seed breeding,maternal endometrial decidualization is also have an important guarantee for the maintenance of normal pregnancy.Earlier studies have pointed out that decidual tissue not only provides important nutritional support for embryo and protects embryo from maternal immune rejection,but also provide important guarantee for trophoblast cell invasion of endometrium.During the period of embryonic implantation,endometrium undergoes extensive cyclic biochemical and morphologic modifications,this process is regarded as decidualization of stromal cells,which is very important for the embryo to adhere and invasive into the uterine epithelium.Lots of steroids take part in endometrial regenerative and receptive processes.Accumulative evidence showed that endometrial expression of various autocrine/paracrine regulators,including many growth factors,cytokines,proteases,and extracellular matrix,serve as key components of these processes.Insufficient decidualization and defective placentation will lead to the department and development of early pregnancy loss,and,later complications in gestation such as fetal growth restriction and preeclampsia.Angiogenesis and vascular remodeling are regarded as key progress of pregnancy not only in the window of implantation but also placenta formation.Accumulating evidence suggests that different autocrine/paracrine regulation of expression in endometrium,including many kinds of growth factors,cytokines,protease and extracellular matrix,these factors regulated by estrogen and progesterone,through a variety of corresponding signal pathway to remodel decidual spiral arteries.Several investigations have discovered that uterine endometrium can be regulated by apoptosis,or programmed cell death.Even more,the increase of decidual cell apoptosis had been found in EPL.Over the past decade,the rapid development in molecular biology technologies with transcriptomic analysis give an opportunity for investigators to explore the genomics of human endomentrial development.During the whole gestation,embryo implantation,embryo development,placental formation and endometrial decidualization are also regulated by placenta-specific miRNA profile.More and more miRNAs have been discovered not only involving with the epithelial cell proliferation and x differentiation during the window of implantation,but also participate in the endometrial decidualization and the placenta development in the early stage of gestation.MicroRNAs(miRNAs)are kinds of small(-22nt),endogenous non-coding RNAs(small non-coding RNA,ncRNA),which is composed of 17-25 nucleotides,diversely regulate gene expression through their control of mRNA stability or translation.Until now,it has been evidenced that miRNAs participate in lots of cellular activities,such as proliferation,development,angiogenesis,differentiation,apoptosis,migration,and invasion.In recent years,people found that 30%of human genes regulated by miRNAs,and plays an important role in the embryonic development,growth and maturity,proliferation and differentiation.In the normal process of pregnancy,Sevarel investigations showed that a variety of miRNAs and their target genes have been discovered to play a crucial regulatory role in pregnancy.But their exact role in early stages of pregnancy and EPL remains unclear.In the present study,we expect to determine a potential underlying mechanism of early pregnancy loss(EPL)based on miRNA together with its target gene.Expect to find out the role of miRNAs in the regulation mechanism of EPL,and providing feasible direction of prevention and treatment of EPL.Part I:Microarray analysis of differentially expressed miRNAs and prediction of target genes in deciduas of early pregnancy lossObjective:The aim of this study is to identify differential expression of miRNAs in deciduas between EPL group and control group,and to predict the related-target genes of selected candidate miRNAs.Materials and Methods:1.Selected 3 pairs of of EPL decidual tissues and control group to be analyzed by miRNA microarray.2.Predicted the related-target of selected differentially expressed miRNAs with miRWalk including 10 databases(DIANA-mT,miRanda,miRDB,miRWalk,RNAhybrid,PICTAR4,PICTAR5,PITA,RNA22,TargetScan).3.According to the microarray results,we select 7 of differentially expressed miRNAs and 3 of differentially expressed target genes.Results:1.By the miRNA microarray analysis showed that total of 70 miRNAs were found to be differentially expressed in the decidual tissues of EPL patients,including 32 up-regulated expressed miRNAs,38 down-regulated expressed miRNAs.2.Three up-regulated expressioned miRNAs(miR-125a-5p,miR-29c-3p,miR-193a-5p)and four down-regulation expressed miRNAs(miR-378a-3p,miR-378a-5p,miR-422a,miR-378c)were selected.3.Through bioinformatics tools,we obtained some valuable target genes,which may be associated to the vascular growth(VEGFA and MMP2)and cell apoptosis(Caspase 10).Conclusions:1.Establish concomitant differential miRNA and mRNA expression profiles in deciduas of EPL.2.Predict that the selected differentially expressed miRNA may be involved in the occurrence of EPL by regulation of target genes through multiple signaling pathways.Part Ⅱ Real-time PCR verifying the expression of selected miRNAs and target genes in deciduas of early pregnancy lossObjective:Using RT-PCR to verify the expression of miR-125a-5p,miR-29c-3p,miR-193c-5p,miR-378a-3p,miR-422a,miR-378c and miR-378a-5p in deciduas of EPL and control group.Through dual-Luciferase Reporter Systerm to verify target-relationship of diffentially expression of selected miRNAs with predicted target genes.Methods:1.Verifying the results of miRNA chip and related target genes by real-time RT-PCR technology and western blotting technology.2.Verifing the relationship of selected candidate miRNAs with predicted target genes by 3’-UTR luciferase reporter assays.Results:1.RT-PCR validated that all of differential expression of miRNAs are well consistent with miRNA gene chip and are proved to be reliable.2.3’-UTR luciferase reporter assays confirmed that target genes of miR-125a-5p and miR-29c-3p are VEGFA,and MMP2 is the same target gene of miR-125a-5p,miR-29c-3p,miR-193c-5p.The target gene of miR-378a-3p and miR-378c are Caspase3.MiR-422a,miR-378a-3p,miR-378c and miR-378a-5p all can target to Caspase10.3.RT-PCR validated that there was no significant difference of VEGFA at the mRNA level in deciduas of two groups,but MMP2,CaspaselO and Caspase3 were increased dramatically in EPL.Western blotting showed that VEGFA and MMP2 were significantly decreased in protein levels.The expression of Caspase10 and Caspase3 in the deciduas were increased significantly in the EPL group(p<0.05).4.MiR-378a-3p significantly up-expressed in deciduas of EPL treated by progesterone than in deciduas of EPL group.Conclusions:1.MiR-29c-3p,miR-125a-5p,miR-193a-5p contribute to EPL through affecting angiogenesis and invasion of deciduas by regulating the expression of MMP2 and VEGFA.2.MiR-378a-3p family,miR-378a-5p contributed to EPL through affecting apoptosis of deciduas by regulating the expression of Caspase3 and Caspase10.Part Ⅲ MiR-378a-3p:induce apoptosis of decidual cells by regulating Caspase3 and Caspase10 expression in early pregnancy lossObjective:Further study on the differential expression of miR-378a-3p and target genes to determine their effects in the occurrence of EPL,through decidual cells cultured in vitro.Methods:1.Decidual cell in vitro culture.The expression of PRL in decidual cell was tested using immunofluorescence to confirm the decidual cell.2.Using Q-PCR,western blotting and immunohistochemistry method to determine the expression of Caspase3,Caspase10 in decidual cell.3.Overexpression and the interference of miR-378a-3p mimic and inhibitor,detection of target gene expression and decidual cell proliferation and apoptosis.Apoptosis was measured by flow cytometry through FL-1 filter(530 nm)and FL-2 filter(585 nm).4.Using Q-PCR to determine the expression of miR-378a-3p in decidual cell stimulated by progesterone through different pathway.Results:1.Decidual cells expressed high level of PRL which demonstrated that the culture of decidual cells in vitro was successful.2.Annexin V-FITC/PI double-labeled flow cytometry showed the apoptosis rate was the sum of early apoptosis and the late apoptosis.3.Trancfection with miR-378a-3p mimics increased miR-378a-3p expression dramatically(p<0.01).4.Overexpression of miR-378a-3p significantly inhibited the expression of Caspase3 and Caspase10 in the decidual cells(p<0.01).5.Q-PCR showed that the expression of miR-378a-3p is regulated by progesterone.MiR-378a-3p increased with raised concentrations and prolonged treatment of progesterone(p<0.05).Conclusions:1.The successful establishment of decidual cell model in vitro.2.Down expression of miR-378a-3p induced apoptosis of decidual cell through directly target Caspase3 and Caspase10 which may involve in the occurrence of EPL.3.The expression of miR-378a-3p in the decidual cell may be regulated by progesterone.