The Histopathological Changes Of Embryonic Villi During Early Pregnancy And The Role Of MiRNA In Abnormal Placental Development

Early embryo growth arrest,known as missed abortion(MA),is a common and frequent disease in obstetrics.It refers to embryo growth arrest in early pregnancy,absence of gestational sac and cardiovascular pulsation in embryo.The clinical incidence of MA in China is 13%,and it shows an upward trend in recent years.It has become a major health problem to the women of childbearing age.But the mechanism of this disease is still inadequately definite.Placenta is a temporary organ that ensuresthe growth and development of the fetus duringpregnancy.It is the only channel to exchange the nutrition and gas between the fetus and the mother.The degree of formation and development of placental vessels is closely related to the success of pregnancy during embryonic development.Abnormal placental development is an important cause for missed abortion.The pathophysiological changes of placenta in missed abortion are not very clear.The molecular mechanism of placental abnormality also needs further study.MicroRNAs(miRNA)are a class of endogenous small non-coding RNA which usually have 18-25 nucleotides.They are widely existing in eukaryotes.They have the characteristics of highly conservative,tissuespecificity and timedependence.They can regulate human gene expression,and generally participate in cell proliferation,apoptosis,differentiation and other life activities.Previous studies in our laboratory showed that abnormal expression of placental miRNAs was involved in the occurrence of gestational diabetes mellitus.However,the role of miRNAs in the abnormal development of placental tissue in missed abortion is not clear.In order to observe the pathological changes of villus tissues in MA,immunohistochemistry was used to detect the difference of vascular endothelial growth factor(VEGF),which is the markers of placental angiogenesis and trophoblast invasion,cytokeratin(CK19),which reflects the distribution of trophoblast cells,and Vimentin,which reflects the distribution of mesenchymal cells in decidua,in echorionic villus in patients with MA(case)and normal early pregnancy(control).Apoptosis of chorionic villus in case and control was determined by Tunel assay.Research resultshow thatVEGF,CK19 and Vimentin are all expressed in chorionic villus of early embryo growth arrest group and the control group.VEGF expression sites in chorionic trophoblasts(including cytotrophoblasts and syncytiotrophoblasts)and stromal cells.CK19 is mainly expressed in trophoblast cells,and Vimentin is abundant in stromal cells and vascular endothelial cells.Differences in expression of VEGF,CK19 and Vimentin between case group and control group by H-score.The expression level of VEGF in the control group was significantly higher than that in the case group(P<0.005).This result is consistent with the results of several studies showing that VEGF expression levels in spontaneous abortion are low.There was no significant difference in the expression of CK19 and Vimentin between the control group and the case group.Apoptotic cells were found in the placenta villi of MA patients and normal early pregnant women.The apoptotic index in the case group was significantly higher than that in the control group(P<0.001).These results suggest that placental angiogenesis and invasion of villous trophoblastic cells has been hindered in MA.Trophoblast cell apoptosis,which may cause chorionic growth restriction,inadequate invasion,hinder the development of fertilized egg,and thus MA occurrence.To investigate the molecular mechanisms of abnormal placental development in MA patients,we first filtered differentially expressed miRNAs in placental tissues of MA patients.MiRNA probe in situ hybridization and RT-PCR were used to detect the expression of miR-98,miR-16,miR-503 and miR-125a in placenta of MA patients.In situ hybridization results showed that miR-98,miR-16,miR-503 and miR-125a were widely expressed in MA patients and normal early pregnancy villus tissues,mainly distributed in villus trophoblast cells,syncytiotrophoblast and interstitial cells.Statistical analysis for the mean optical density of miRNAs positive signals revealed that the expression of miR-98,miR-16,miR-503 and miR-125a was significantly increased in the villus tissues of MA patients compared with the control group(P<0.05).Real-time PCR results showed that the expression differences of miR-98,miR-16 and miR-125a in the control group and the case group were consistent with the in-situ hybridization results(P<0.05,P<0.01,P<0.001).These results suggest that up-regulation of miR-98,miR-16 and miR-125a may be risk factors for embryonic cessation.Our previous results show that miR-98 and miR-125a play important roles in embryo implantation and recurrent abortion.Therefore,miR-98 and miR-125a were selected for further study.To further analyze the relationship between these miRNAs and MA,we investigated the function and mechanism of miRNAs in trophoblast cells.The Edu,MTT,and transwell chamber assays were used to analyze the effects of miRNAs on human trophoblastic function;the miRWalk and targetscan databases were used to predict miRNA target genes;the dual luciferase reporter assay system and qRT-PCR were used to identify and to verify the target gene of miRNA;gene compensation experiments were used to further confirm the relationship between miRNAs and target genes.The results showed that after overexpression of miR-98,the migration ability and activity of HTR8 trophoblast cells were significantly decreased(P<0.01,P<0.05),when knocking down miR-98,HTR8 cells proliferative capacity and activity were significantly increased(P<0.05,P<0.05),suggesting that miR-98 inhibits cell proliferation and migration.Bioinformatics predictions indicate that GDF6 and PLEKHA8 are target genes for miR-98.Verification by dual luciferase system,after co-transfection of recombinant plasmid which including GDF6 3'UTR seed sequence and miR-98 mimics,luciferase activity was significantly reduced(P<0.01),and co-transfection with miR-98 inhibitor could significantly increased luciferase activity(P<0.05),suggesting that miR-98 could bind to the GDF63'UTR.Site-directed mutagenesis of GDF6 3 'UTR region that recognized with miR-98,luciferase activity increased significantly(P<0.05),revealing that this mutation could inhibit the binding of miR-98 to GDF 3'UTR.Overexpression of miR-98 could reduce GDF6 mRNA expression level in HTR8 cells(P<0.001),and knockdown of miR-98,GDF6 expression was significantly increased(P<0.05).These results suggest that GDF6 is a target gene of miR-98.Similarly,dual luciferase assay showed that miR-98 overexpression significantly inhibited PLEKHA8 activity(P<0.05),knockdown significantly promoted PLEKHA8 activity(P<0.05),Mutation of the 3'UTR site of PLEKHA8 recognized by miR-98 significantly inhibited the binding of miR-98 to PLEKHA8 3'UTR(P<0.001).qRT-PCR results showed that miR-98 overexpression could decrease the expression of PLEKHA8 mRNA in cells(P<0.001),and knock-down expression of miR-98 could significantly increase the level of PLEKHA8 mRNA(P<0.05).These results suggest that PLEKHA8 is a miR-98 target gene.Target gene RESCUE showed that knocking down microRNA-98 and inhibiting the expression of PLEKHA8 could restore cell proliferation and migration to normal level.Overexpression of miR-125a,migration and proliferation ability of HTR8 cells significantly decreased(P<0.05,P<0.05),knockdown of miR-125a,HTR8 cells proliferative capacity and activity were significantly increased(P<0.05,P<0.05).This indicates that miR-125a inhibits cell proliferation and migration.Overexpression of miR-125a can inhibit the expression of CBX7,suggesting that CBX7 may be the target gene of miR-125a.Conclusion:There is no significant change in the distribution of trophoblast cells and interstitial cells in embryonic cessation patients,but the invasive ability of villus trophoblast cells is decreased,and cells are hyperapoptosis,indicating that low expression of VEGF and abnormal apoptosis are related with MA;miR overexpression of-98,miR-16 and miR-125a is involved in the development of MA;miR-98 and miR-125a inhibit cell proliferation and migration,miR-98 acts by down-regulating GDF6 and PLEKHA8,miR-125a works by regulating CBX7.