MicroRNA-317a-5p Promotes Cell Apoptosis Via Regulation Of XIAP/caspase-3 Pathway Involving In The Pathogenesis Of Unexplained Recurrent Spontaneous Abortion

Chapter I Expression profiles of miRNAs and prediction of target gene in chorion villi of recurrent sponstanous abortionObjective To detect the expression profiles of miRNAs in chorion villi and target gene prediction for the differential miRNAs in unexplained recurrent sponstanous abortion and to predict whether there is a binding site between miRNAs and the target gene,which provides a theoretical basis for the next research.Methods 3 cases with unexplained recurrent sponstanous abortion and 3 cases with induced abortion were detected by high throughput sequencing,then the significantly differential miRNAs were used to annotate and predict target genes.The product results of sequencing were confirmed by agarose gelelectrophoresis and RT-q PCR.Results Comparing induced abortion with unexplained recurrent spontaneous,more than 2-fold multiplier and p<0.05 were regarded as the screening criterion of upregulation gene and downregulation gene.There was a significant difference in the expression levels of 111 mature miRNAs in unexplained recurrent spontaneous abortion and induced abortion,including upregulation expression of 78 miRNAs and downregulation expression of 33 miRNAs.The upregulated expression levels of miRNAs were more than the downregulated expression levels of miRNAs.Bioinformatics analysis were predicted to be target genes of miR-371a-5p,and there is a 3 ’-UTR binding region between miR-371a-5p and XIAP.The result of miR-371a-5p sequencing was in accordance with the products of agarose gelelectrophoresis and RTq PCR.Conclusions There was a significant difference in the expression levels of miRNAs in unexplained recurrent spontaneous abortion.The differential expression levels of miR-371a-5p and target gene XIAP in chorionic villi may be involved in the pathogenesis of unexplained recurrent spontaneous abortion.Chapter Ⅱ Expression profiles of differential miR-371a-5p and XIAP in chorion villiObjective To enlarge the number and analyze expression profiles of miR-371a-5p and XIAP gene in chorionic villi of unexplained recurrent spontaneous abortion and induced abortion.Methods Thirty samples in chorionic villi of unexplained recurrent spontaneous abortion and thirty samples in chorionic villi of induced abortion were detected the expression levels of miR-371a-5p and XIAP gene by RT-PCR.The correction of miR-371a-5p and XIAP gene was analyzed.Results The expression levels of miR-371a-5p and XIAP in unexplained recurrent spontaneous abortion were greatly decreased compared to that of induced abortion(P<0.05).There is a significant correlation between miR-371a-5p and XIAP(P<0.05).Conclusions There was a significant correlation between miR-371a-5p and XIAP in chorionic villi.The differential expression levels of miR-371a-5p and XIAP were related to the pathogenesis of unexplained recurrent spontaneous abortionChapter Ⅲ Detection of apoptosis rate of miR-371a-5p mimics and inhibitor transfected to JEG-3 cellsObjective To explore the role of miR-371a-5p in promoting cell apoptosis,mimics and inhibitor were transfected into JEG-3 cells to confirm the occurrence of apoptosis.Methods JEG-3 cells were transfected with miR-371a-5p mimics,inhibitor,and normal control using Lipofectamine 2000 for 48 hours,and the apoptosis rate was detected by flow cytometry.Results Mi R-371a-5p mimics transfected into JEG-3 cells significantly increased the rate of apoptosis after 48 hours.miR-371a-5p inhibitor transfected into JEG-3 cells significantly decreased the rate of apoptosis after 48 hours(P<0.05).Conclusions miR-371a-5p promoted the occurrence of apoptosis.Chapter ⅣConfirmation miR-371a-5p regulating XIAP to promote cell apoptosisObjective To confirm whether there is a binding site for miR-371a-5p in XIAP 3 ’-UTR and the apoptosis cascade is triggered by the XIAP/caspase-3 signaling pathway.Methods Dual luciferase genes of XIAP-miR-371a-5p-Wt and XIAP-miR-371a-Mut were transfected into JEG-3 cells and the luciferase activity were detected with dual luciferase reporter assayreporter assay.After 48 hours transfection of mimics and inhibitor,RT-PCR and Western blotting were used to respectively detect the expression levels of miR-371a-5p,XIAP,caspase-3 in m RNA and protein.Results There were significant differences in the luciferase activity of XIAP-miR-371a-5p-wt and miR-371a-5p mimics or inhibitor transfected into JEG-3 cells(P<0.05);There were no significant differences in the luciferase activity of XIAP-miR-371a-5pMut and miR-371a-5p mimics or inhibitor transfected into JEG-3 cells(P>0.05).There were significant differences in the expression levels of m RNA in miR-371a-5p,XIAP and caspase-3 before and after transfection(P<0.05);There were significant differences in the expression levels of protein in XIAP and caspase-3 before and after transfection(P<0.05).Conclusions The miR-371a-5p involved in unexplained recurrent spontaneous abortion was associated with XIAP gene of apoptosis.Mi R-371a-5p functions as an upstream regulator of XIAP/caspase-3 expression,cell apoptosis in trophoblasts.These findings provide strong and new evidence that miR-371a-5p regulates XIAP expression by reducing translation and is involved in the pathogenesis of unexplained recurrent spontaneous abortion.