A Study About The Effect Of MicroRNA-125a And MicroRNA-29a/b/c On The Pathologic Mechanism Of Placenta Accreta

Chapter I THE EXPRESSION OF microRNA-125a microRNA-29a/b/c AND MCL1 IN PLACENTA ACRETA AND THEIR CLINICAL SIGNIFICANCEObjective1.To investigate the expression of microRNA-125a(miR-125a)and microRNA-29a/b/c(miR-29a/b/c)in creta sites tissue and their corresponding adjacent noncreta sites tissues.2.To detect the MCL1 expression,and to analyse its correlation with miR-125a and miR-29a/b/c level in placenta acreta.3.To determine whether MCL1 is a target gene of miR-125a and miR-29a/b/c.Methods1.Maternal placental tissues containing villous and extravillous trophoblasts,the fibrinoid layer,and the basal plate layer were separately isolated from both creta(also included basal plate myometrial fibers)and noncreta sites2.Real-time RT-PCR was used to detect the expression of miR-125a miR-29a/b/c and MCL1 expression in creta sites tissue and their corresponding adjacent noncreta sites tissues.3.The 3'UTR sequence of MCL1,which contains the putative miR-125a or miR-29a/b/c-binding site,was subcloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector.The construct containing the wild type 3'UTR was designated Wt 3'UTR.Mutagenesis of the miR-125a or miR-29a/b/c-binding site was performed on the Wt 3'UTR plasmid using the QuikChange Lightning Site-Directed Mutagenesis Kit.The resulting plasmid contained a mutated 3'UTR and was designated Mut 3'UTR.microRNA mimics or a negative control were cotransfected into the 293T cells using Lipofectamine 3000 together with Wt 3'UTR plasmid or Mut 3'UTR plasmid.4.Luciferase activity,to determine whether MCL1 is a target gene of miR-125a and miR-29a/b/c,was measured using the Dual-Luciferase Reporter Assay System.Results1.Relative expression of miR-125a and miR-29a/b/c were lower in creta sites,compared to noncreta sites(p =0.005,0.018,0.041,and 0.022,respectively).2.Expression of the MCL1 mRNA was significantly increased in the creta sites of placenta accreta compared to noncreta sites(p = 0.039).3.Pearson correlation analysis showed a significantly inverse correlation between miR-125a miR-29a/b/c and MCL1(Pearson correlation =-0.701,-0.543,-0.876,-0.767,p<0.05).4.Luciferase activity measured using the Dual-Luciferase Reporter Assay System revealed that miR-125a and miR-29a/b/c directly targets the 3'UTR of MCL1.ConclusionExpression of miR-125a and miR-29a/b/c were decreased in creta sites,compared to noncreta sites,while expression of the MCL1 mRNA was significantly increased in the creta sites.Luciferase activity revealed that miR-125a and miR-29a/b/c directly targets the 3'UTR of MCL1.Our findings suggest that miR-125a and miR-29a/b/c play a critical role in placenta accreta.Chapter II microRNA-125a and microRNA-29a/b/c CONTRIBUTE TO BIOLOGICAL BEHAVIOR OF TROPHOBLAST BY REGULATING MCL1 IN PLACENTA ACCRETAObjective1.In vitro,To investigate whether Mcll could be modulated by miR-125a and miR-29a/b/c in HTR-8/SVneo cell line.To further confirme the negative regulation of target genes MCL1 by miR-125a and miR-29a/b/c.2.To explore the influence of abnormal expression of miR-125a and miR-29 a/b/c on the apoptosis of the trophoblast cell.3.To determine the apoptosis cell types,and preliminarily explore the pathological mechanismof placenta accreta.Methods1.In HTR-8/SVneo cell line,gain-and loss-function of miR-125a or miR-29a/b/c was achieved by transfection with chemical synthetic miRNA mimic and miRNA inhibitor oligonucleotides with Lipofectamine 3000 Reagent.2.The influence of miR-125a and miR-29a/b/c on mRNA and protein level of MCL1 was evaluated by qRT-PCR and western blot assay,respectively.3.The functional effect of miR-125a and miR-29a/b/c on cell apoptosis was evaluated using a FACScan flow cytometer,after cell was performed using the Annexin V-FITC/PI Apoptosis Detection Kit.Results1.Compared with control group,transfection of miR-125a and miR-29a/b/c mimic resulted in down-regulation of MCL1 expression(fold,0.60,0.42,0.58 and 0.43.p =0.0497,0.002,0.008,0.013),and decrease of MCL1 protein expression.2.After transfected miR-125a and miR-29a/b/c mimics and scramble control(NC),the levels of apoptosis of HTR-8/Svneo cells increased.3.The number of ISIT cells was increased at the implantation site in cases of placenta accreta.Importantly,these ISIT cells were positive for MCL1 expression.In creta site,the artery and vein is invaded and remodeled by intermediate trophoblast cells.ConclusionThe over-expression of miR-125a and miR-29a/b/c could significantly inhibit the expression of MCL1,and promote apoptosis of HTR-8/Svneo cell line.The number of intermediate trophoblast cells,which were positive for MCL1 stain,was increased at the implantation site in cases of placenta accreta.All those suggest that the increased number of ISIT cells in placenta accreta may be triggered,in part,inhibition of trophoblast apoptosis,driven by upregulation of MCL1.