The Roles And Mechanisms Of Placental MiRNAs In Selective Intrauterine Growth Restriction

Selective intrauterine growth restriction(sIUGR)is a special complication of monochorionic and double amniotic(MCDA)twins.The sIUGR occurs in 10%~15% of the monochorionic twins.At present,the commonly accepted diagnostic criteria are that the weight of the small fetus is less than the 10 th percentile of the growth curve and the difference between the estimated weight of the twins is more than 25%.sIUGR is often accompanied by fetal nervous system damage,which is prone to fetal intrauterine death and poses a serious threat to the prognosis of perinatal infants.With the gradual deepening of basic research,the focus of basic research on sIUGR also goes deep into the molecular mechanism.Hypotheses such as unequal placental sharing,abnormal umbilical cord insertion,abnormal placental vascular anastomosis,oxidative stress,and epigenetic modification have been derived from the pathogenesis of sIUGR.Although some theories and hypotheses have been put forward and demonstrated,the exact pathogenesis of sIUGR is still unclear.Micro RNA is a kind of endogenous small molecule single-stranded RNA in eukaryotic organisms.It can induce the degradation of target RNA or inhibit its translation by pairing with specific base pairs of target RNA,thus regulating the post-transcriptional expression of genes.About 30% of human genes expression may be regulated by micro RNAs and participate in all life processes including cell proliferation,differentiation,metabolism,and apoptosis.Functional studies have shown that micro RNAs play an important role in regulating placental development and function.In recent years,many studies have shown that abnormal expression of micro RNAs has been found in many pregnancy-related diseases,such as preeclampsia,fetal growth restriction,premature delivery,etc.These abnormal expressions of micro RNAs are related to the occurrence,development,and prognosis of diseases.Inconsistent placental development is the main cause of selective intrauterine growth restriction.It is well-recognized that unequal placental sharing is the major placental determinants of selective birth weight discordance in monochorionic twins.Placental development is closely related to the invasion of trophoblasts to decidua.The biological behavior of trophoblasts,such as proliferation,invasion,and migration,is similar to that of malignant tumors.We found that 14 placental-derived micro RNAs were differentially expressed in sIUGR fetuses compared with normal monochorionic twins,suggesting that these micro RNAs may be involved in the development of sIUGR,but the specific pathogenesis of sIUGR remains unclear.In recent years,micro RNA-338-5p has been found to regulate cell growth,differentiation,and tumorigenesis.It has been confirmed that micro RNAs are abnormally expressed in many malignant tumors,which affects the biological behavior of tumor cells in proliferation,invasion,and metastasis.We found that the expression of micro RNA-338-5p in larger fetuses was significantly lower than that of small fetuses.It is speculated that the abnormal expression of micro RNA-338-5p may affect the growth of trophoblasts and thus participate in the pathogenesis of sIUGR.This study aimed to detect the expression of micro RNAs in the placenta of sIUGR and normal monochorionic twins and to establish the expression profile of placentalderived micro RNAs.At the same time,we studied the effects of abnormal expression of micro RNA-338-5p on the proliferation,invasion,and migration of trophoblasts in HTR-8/SVneo cells,and further explored the role of micro RNA-338-5p in regulating the development of sIUGR.Part ? The establishment of placental-derived micro RNAs expression profile in fetuses with selective growth restrictionObjective: To explore the expression profiles of placenta-derived micro RNAs in sIUGR and normal MCDA twins,by comparing the differences of sIUGR and normal MCDA twins.Methods: The placental tissues of sIUGR fetuses and normal monochorionic twins with different body mass were detected by microarray.The differences of micro RNAs expression in placental tissues of sIUGR fetuses and normal MCDA fetuses were compared,and the expression profile of placental-derived micro RNAs of sIUGR was established.Results: 1.The results of the cluster analysis of micro RNAs showed that 45 kinds of micro RNAs expressed significantly in placenta tissues of sIUGR fetuses with different body mass.2.Comparing the placental differential miRNA profiles of sIUGR fetuses with different body weights with those of normal MCDA fetuses,there were 7 specifical upregulated miRNAs and 7 down-regulated miRNAs in sIUGR placental differential miRNAs.Conclusion: There are differences in the expression of micro RNAs in placental tissues of sIUGR fetuses of different body weight.These placental-derived micro RNAs may be related to the occurrence and development of sIUGR.Part ? The bioinformatics analysis and validation of differentially expressed micro RNAs derived from placenta with selective growth restrictionObjective: To construct the regulatory network of placental-derived and target genes of sIUGR via the results of gene chip,and to verify the expression profile of micro RNAs in placental tissues of sIUGR by RT-q PCR.Methods: Based on the results of the micro RNA chip,target gene prediction was carried out by Gene Spring software,target gene function enrichment analysis and signal transduction pathway enrichment analysis were carried out by the DAVID database after the screening,and micro RNA pathway network and signal pathway network were constructed by Cytoscape software.Collect 15 cases of sIUGR placental tissues,15 cases of normal MCDA twins placental tissues,extracting RNA for RT-q PCR assay to detect the expression of miR-5189-5p? miRNA-1? miR-370-3p? miR-373-3p?miR-338-5p? miR-590-5p.Results: 1.In the sIUGR group,there are 712 target genes of up-regulated placental-derived micro RNAs,and 929 target genes of down-regulated micro RNAs were found in large body mass fetuses.2.Further GO and Pathway analysis of 14 target genes of micro RNAs revealed that 7 up-regulated micro RNAs involved 101 signaling pathways,while 7 down-regulated micro RNAs involved 49 signaling pathways.3.The key signaling pathways involved in key micro RNAs are MAPK signaling pathway,TGF-beta signaling pathway,Wnt signaling pathway,and HTLV infection pathway.4.The RT-q PCR expression files of miR-5189-5p,miRNA-1,and miR-370-3p were up-regulated by in large-body mass fetal placenta of sIUGR,while the expression files of miR-373-3p,miR-338-5p and miR-590-5p were down-regulated,with significant difference(p<0.05),which were consistent with the microarray detection results.5.The top five micro RNAs involved in more signaling pathways were down-regulated in the placenta of large-body mass fetuses.Conclusion: 1.Placental-derived micro RNAs of sIUGR involves multiple signaling pathways,including MAPK signaling pathway,TGF-beta signaling pathway,Wnt signaling pathway,and HTLV infection pathway.2.miR-338-5p may be associated with the pathogenesis of sIUGR.Part ? The effects of miR-338-5p on proliferation,invasion,and migration of HTR-8/SVneo trophoblastObjective: To investigate the effects of miR-338-5p on proliferation,invasion,and migration of HTR-8/SVneo trophoblasts.Methods: HTR-8/SVneo cells were cultured and transfected with miRNA-338-5p inhibitor to inhibit the expression of miR-338-5p.The interference titer was determined by RT-q PCR.CCK-8 method was used to detect the proliferation of trophoblasts after inhibited by miR-338-5p,Transwell method was used to detect the invasive ability of trophoblasts,and the scratch test was used to detect the changes of cell migration ability.Results: 1.miR-338-5p inhibitor was transfected into HTR-8/SVneo trophoblast cells,and the expression of miR-338-5p was significantly down-regulated by RT-q PCR after 48 hours(p<0.0001).2.CCK-8 assay showed that HTR-8/SVneo trophoblast proliferation increased significantly 72 hours after miR-338-5p inhibitor transfection(p<0.01).3.Transwell results showed that the invasion of HTR-8/SVneo trophoblasts was enhanced 48 hours after the transfection of miR-338-5p inhibitor(p<0.01).4.Scratch test showed that the migration of HTR-8/SVneo trophoblast cells was enhanced 48 hours after miR-338-5p inhibitor transfection(p<0.0001).Conclusion: 1.The proliferation,invasion,and migration of HTR8/SVneo cells were enhanced after down-expression of miR-338-5p.Part ? The miR-338-5p targeting inhibits EFEMP1 expression in sIUGRObjective: To predict and identify target genes regulated by miR-338-5p in sIUGR.To study the regulation of EFEMP1 by miR-338-5p in HTR-8/SVneo trophoblasts.Methods: The expression of miR-338-5p and EFEMP1 in placenta of sIUGR was detected by RT-q PCR?immunohistochemical method and Western-blot.Using luciferase reporter gene assay to detect whether EFEMP1 is a target gene of miR-338-5p.The expression of EFEMP1 in HTR-8/SVneo cells after 48 hrs transfected with miR-338-5p inhibitor was analyzed by RT-PCR and western-blot.Results: 1.The expression of miR-338-5p was negatively correlated with EFEMP1 expression.in sIUGR placenta tissues.2.miRNA-338-5p could significantly inhibit the luciferase activity of wild-type reporter gene vector EFEMP1-3'UTR-psi-CHECK 2,did not affect the luciferase activity of mutant reporter gene vector Mut-EFEMP1-3'UTR-psi-CHECK 2(p<0.01).3.Compared with the negative control,the expression of EFEMP1 in trophoblast cells increased 48 hours after the transfection of miR-338-5p inhibitor(p<0.001).Conclusion: 1.Compared with the placenta of small-body mass fetuses,the expression of miR-338-5p was down-regulated in the placenta of large-body mass fetuses with sIUGR,while the expression of EFEMP1 was up-regulated.2.The expression of EFEMP1 in HTR-8/SVneo cells was up-regulated after the down-expression of miR-338-5p.3.EFEMP1 is one of the target genes of miR-338-5p.