| Objective: The prevalence of placenta accreta has increased in recent years along with the increased incidence of cesarean section.Patients complicated with palcenta accreta can easily lead to intrapartum and postpartum hemorrhage and placenta accreta is a major cause of adverse outcomes on the mother and newborn in modern obstertics.However,the specific pathogenesis of placental accreta is still unclear.Futher elucidation of these mechanisms would provide theory for early diagnosis and treatment of high-risk populations.Micro RNAs(mi RNAs)are small non-coding RNA molecules(~22 nucleotides long)that suppress gene expression by binding to the 3' end of the untranslated region(3'-UTR)of target m RNAs.Numerous of mi RNAs are expressed in the placenta,which play critical roles in placental development,and their abnormal expression is closely related to complications during pregnancy,such as severe preeclampsia,abortion,fetal growth restriction,gestational diabetes and so on.With the development of gene chips and deep sequencing technologies,transcriptome sequencing can be used to study specific genes that are critical for development and function to help understand and identify the mechanisms of normal pregnancy and pathological pregnancy.Mi RNA expression profiling and transcriptome combined analysis can systematically study the molecular mechanism of mi RNA regulation in gene expression under two levels.There is no report on the combined analysis of mi RNA expression profiling and transcriptome in placenta accreta.In the physiological environment,mi R-193a-3p can limit the proliferation and cell cycle of normal cells.However,as a member of the mi R-193 family,mechanism of mi R-193a-3p in different diseases is still controversial.Mi R-193a-3p is down-expressed in various tumors,such as hepatocellular carcinoma,gastric cancer,colorectal cancer,non-small cell lung cancer,and so on.Conversely,mi R-193a-3p is significantly up-expressed in renal cell carcinoma and esophageal squamous cell carcinoma.It can play a tumor suppressing or promoting role in cancer by targeting different genes related to proliferation,apoptosis,migration,invasion and metastasis.What's more,mi R-193a-3p can regulate cartilage matrix degradation in human osteoarthritis,attenuate neurotoxicity in patients with Alzheimer's disease,and protect umbilical vein endothelial cells from hypoxic damage,but its expression and role in placental accreta have not been reported.Ephrin and its receptors are the largest family of tyrosine kinase receptors,which includes 8 ligands and 14 receptors.Previous studies have confirmed that ephrin and its receptors play an important role in the development of placenta,and EFNB2 are expressed spatially and developmentally during human placentation,which can inhibit endothelial progenitor cell-mediated angiogenesis in vitro.However,only a few studies have investigated the role of EFNB2 in trophoblast invasion.Therefore,this study explored the role of mi RNAs in pernicious placenta previa patients by small RNA sequencing and transcriptome sequencing in placenta tissues between placenta accreta patients and control patients and focused on the effects of mi R-193a-3p by regulating EFNB2 on placenta accreta and its underlying mechanism.Methods: 1.Ten patients with pernicious placenta previa diagnosed and delivered at Shengjing Hospital affiliated to China Medical University from June 2016 to October 2017 were selected.Prenatal ultrasound examinations(two-dimensional + 3D color Doppler + 3D energy Doppler)were performed at 72 hours before operation and placental tissue samples were collected for small RNA sequencing to screen differentially expressed mi RNAs and perform bioinformatics analysis.2.Placental tissue samples from 10 patients with pernicious placenta previa were collected for transcriptome sequencing to screen differentially expressed messenger RNA(m RNAs)and bioinformatics analysis were used to combined analyze mi RNAs expression profiles and transcriptome data.3.The expression of mi R-193a-3p and EFNB2 were detected by Real-time PCR,western blot in placental tissue samples of 40 pernicious placenta previa patients and their correlation were anlysed.Bioinformatics software was used to predict the binding site of mi R-193a-3p to EFNB2,and luciferase assay was used to verify that EFNB2 was the target gene of mi R-193a-3p on the basic of contruction of EFNB2 wild-type and mutant vectors.mi R-193a-3p mimics,inhibitors and their corresponding negative control were transfected into the HTR-8/SVneo human extravillous trophoblasts cell which was cultured in primary human endometrial stromal cell(h ESC)-conditioned medium.Transwell migration and invasion experiments were performed to verify the effect of mi R-193a-3p on the migration and invasion ability of HTR-8/SVneo cells.The expression of EFNB2 affected by mi R-193a-3p were examined by Real-time PCR and western blot after transfection.EFNB2 was silenced to mimic the effect of mi R-193a-3p and transwell migration and invasion experiments were used to detect the effect of EFNB2 on the migration and invasion ability of HTR-8/SVneo cells.The expression of EFNB2 were examined by Real-time PCR and western blot after transfection.To explore the underlying mechanism that affects the migration and invasion of HTR-8/SVneo cells,the expression of E-cad,N-cad,vimentin,MMP2 and MMP9 was detected by western blot after the above transfection.Results: 1.There were 128 differentially expressed miRNAs in placenta accreta patients compared with control patients,in which 77 were up-regulated and 51 were downregulated by analysis of micro RNA expression profiles.KEGG enrichment analysis showed that target genes of these differentially expressed mi RNAs were enriched in focal adhesion,extracellular matrix((ECM)receptor interaction and immune-related pathways.What's more,GO enrichment analysis suggested that biological adhesion,cell aggregation,cell killing were enriched in biological processes and they were mainly enriched in cell junction and extracellular matrix in cellular component.What's more,enrichment of binding-related molecules is evident in molecular functions.2.There were 37743 differentially expressed m RNAs in placenta accreta patients compared with control patients by placental transcriptome sequencing.KEGG enrichment analysis showed that these m RNAs are mainly involved in Notch pathway,PI3K/Akt pathway,TNF pathway,MAPK and m TOR pathways.The GO enrichment analysis found that in biological processes,it was mainly enriched in biological adhesion,cell aggregation,cell proliferation and response to stimulues;while in molecular functions,binding-related to molecules were obviously enriched.3.Combined analysis of placenta mi RNAs expression profiles and transcriptome in placenta accreta patients,we screened 1044 target genes of differentially expressed mi RNAs in differentially expressed m RNAs.KEGG enrichment analysis showed that these significantly differentially expressed m RNAs were enriched in cell adhesion and immune-related pathways.The GO enrichment analysis suggested that in the biological process,it is enriched in angiogenesis-related pathways,cell adhesion, and cell communication.GSEA and GSVA analysis of transcriptome data confirmed that placenta accreta has poor angiogenesis and abnormal epithelial-mesenchymal transition(EMT)pathway.4.mi R-193a-3p was upregulated but EFNB2 downregulated in the placenta tissues of placenta accreta group and the expression of the two is negatively correlated.Luciferase assay confirmed that EFNB2 was a direct target of mi R-193a-3p.The extracted primary endometrial stromal cells have a typical fibroblastic morphology with spindle-shaped and expressed the vimentin protein in the cytoplasm.mi R-193a-3p promoted the migration and invasion of HTR-8/SVneo cells,while EFNB2 inhibited the migration and invasion of HTR-8/SVneo cells in human endometrial stromal cell(h ESC)-conditioned medium,which might be achieved through the EMT pathway.We found that N-cad,vimentin,MMP2,and MMP9 increased and E-cad decreased in the placenta accreta group and in HTR-8/SVneo cells transfected with mi R-193a-3p mimics or si-EFNB2.Reverse experiments confirmed that mi R-193a-3p promotes the migration and invasion of HTR-8/SVneo cells by inhibiting the expression of EFNB2.Conlusions: 1.The funtions of differentially expressed miRNAs and their target genes in placenta accreta placental tissues mainly include cell adhesion,cell aggregation,cell killing,immune system response and so on.2.The functions of differentially expressed m RNAs in placenta accreta placental tissues include cell adhesion,cell aggregation,cell proliferation,PI3K/Akt pathway and so on.Target genes of differentially expressed mi RNAs intersect with differentially expressed transcriptomes and these target genes participate in angiogenesis-related pathways and cell adhesion processes in placental accreta placenta.There are insufficient angiogenesis and abnormal EMT in placenta accreta placental tissues.3.mi R-193a-3p increases HTR-8/SVneo cell migration and invasion by targeting EFNB2 via the EMT pathway under decidua defect conditions. |
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