| Research Background:Gestational Diabetes Mellitus(GDM)refers to the normal glucose metabolism,which occurs during the first time of pregnancy,and often occurs in the second half of pregnancy.It is one of the most easily occurring complications.In recent years,the incidence of GDM in China has shown a gradual increase,and it is currently as high as 14.8%.For the pregnant women,GDM increases the short-term risk of preeclampsia during pregnancy,the rate of cesarean delivery during childbirth,and the long-term risk of type-2 diabetes and cardiovascular disease after delivery.For the offspring,GDM causes high risks of macrosomia,shoulder dystocia,neonatal hypoglycemia,obesity in children and adolescents,and type-2 diabetes in a long term.The pathogenesis of GDM is complex and unclear.It is currently believed that a variety of factors have caused maternal pancreatic ? cells to fail to adapt to the increased insulin resistance during pregnancy,resulting in abnormal maternal glucose metabolism.Among them,leptin has been proved to play a role in the occurrence of GDM for interacting with insulin and sharing the downstream signal with it.In recent years,the results of epigenetic studies on the pathogenesis of GDM have shown that abnormally expressed microRNAs(miRNAs)in the placenta are involved in the occurrence of GDM.miRNAs are short,highly conserved non-coding RNA molecule,approximately 22 nucleotides in lenth.Mature miRNAs act on the post transcriptional mRNA sequence by incomplete pairing with the 3'-UTR of the target gene mRNA,and regulate the expression of nearly 60%of human genes by silencing the expression of RNA inhibitory protein,Based on their function,the miRNAs participate in various biological processes such as cell development,proliferation,differentiation,and apoptosis.At present,relevant studies have confirmed that the abnormal expression of miRNAs in placental tissues plays a role in GDM,but the studies are limited in number and the conclusions of the differentially expressed miRNAs are also inconsistent or even contradictory.Therefore,it is necessary to find and verify the differential expression profiles of miRNAs in placental tissues,explore its possible mechanism of causing GDM,and provide more insights to the research in this field.According to research that placental trophoblast cells can secrete exosomes to the periphery blood,and their concentration increases significantly with the growth of gestational age.This plays an important role in the maintenance of normal pregnancy and fetal development.Otherwise,the abnormal performance of concentration and biological activity is highly associated with pathologic pregnancy including GDM.Exosomes are extracellular vesicles that can be secreted by almost all body cells into the extracellular space.They play an important role in intercellular communication in the form of paracrine or remote secretion,and participate in a variety regulation including immune responses,tumor growth,and the metabolism of lipid and glucose.Exosomes in body fluids have been confirmed to have diagnostic value as a biological marker for a variety of diseases because they are enveloped by membranes and are rich in small fragments of mRNA,non-coding RNA,DNA and fragments of lipids.However,there is no research so far on the diagnostic value of dysregulated miRNAs in circulating exosomes for GDM.Based on the above research,this study intends to analyze the abnormal expression profile of miRNAs in placental tissues,confirm the existence of placenta derived-exosomes in the peripheral blood,and verify whether the dysregulated miRNAs in peripheral blood exosomes are consistent with the expression trends in placental tissues and its diagnostic value for GDM.In addition,we also seek to find the possible mechanism of how differentially expressed miRNAs causes GDM through bioinformatics analysis and confirm the mechanism through in vitro cell experiments.Part 1.The differential expression profiles of miRNAs in theplacenta in GDMObjective:To analyze the abnormal expression profile of miRNAs in GDM placenta,and to further verify its differential expression in a large sample of placental tissues,then analyze its possible mechanism of GDM.MethodsPregnant women with normal glucose tolerance(NGT)(n=3)and GDM(n=3)were recruited.Next-generation sequencing(NGS)was used to detect the abnormal expression profiles of miRNAs in the placenta tissues of the NGT group and GDM group.14 miRNAs with significant differences were selected from the differential expression profile,and the differential expression of the 14 miRNAs in these 6 placenta cases was verified by qRT-PCR test.The differentially expressed candidate miRNAs were validated on a larger cohort of placenta samples from 73 participants(37 NGT and 36 GDM).Clinical factors that may be related to glucose metabolism such as pre-pregnancy BMI,oral glucose tolerance test(oral Glucose Tolerance Test,OGTT)during pregnancy and weight gain during pregnancy were recorded.qRT-PCR test was used to verify the differential expression of selected miRNAs in the placenta,and Pearson correlation analysis was used to explore the correlation between the level of miRNAs and clinical factors in GDM group.Bioconductor(R)package miRNAtap,Cytoscape software(3.8.0)and KEGG were used to perform bioinformatics predictive analysis of prediction of target genes,Ming go and pathway enrichment analysis of candidate miRNAs.ResultsThe results of NGS in placental tissues of the two groups showed that that 764 differentially expressed miRNAs were totally detected,114 of which were significantly up-regulated and 43 were significantly down-regulated(p<0.05).14 miRNAs with significant dysregulation were selected(8 were up-regulated and 6 were down regulated),and verified using qRT-PCR in 6 placenta samples.The results showed that there were two miRNAs with inconsistent dysregulation trends(miRNA-21 and miRNA-409-3p)and 12 miRNAs with consistent trends.Five miRNAs are further selected from the 12 miRNAs with consistent trends of expression:miR-144,miR-451,miR-15b,miR-543,and miR-125b.A total of 73 cases were included in the NGT group(n=37)and GDM group(n=36).The results of qRT-PCR verification in the placental tissues showed that miRNA-125b and miRNA-543 were significantly down-regulated in the placental tissues of the GDM group than NGT group(p<0.001),whereas miRNA-144 was significantly up-regulated(p<0.001),however,the differential expression of other two miRNAs(miR-451,miR-15b)did not reach significance.The three results of OGTT during pregnancy in the GDM group were significantly higher than those in the NGT group(p<0.001).Neonatal weight was also(P=0.045).Other clinical data are meaningless.The level of mirna-144 in placenta was significantly increased in GDM group positively correlated with age and hemoglobin in term pregnancy(p=0.015,p=0.014),while miRNA-125b had no significant correlation with patient's characteristics.The results of bioinformatics analysis showed that miRNA-125b regulates 419 genes,including leptin(LEP),whereas miRNA-144 regulates 2076 genes.GO analysis results showed that the target genes of miRNA-125b were enriched in:maternal placental development(p=0.001612,),pathway regulation of TGF? receptor signal(p=0.00008325)and endocrine pancreatic development(p=0.005);miRNA-144 target genes are enriched in energy homeostasis(p=0.0017),pancreatic ?-cell development(p=0.01008),interleukin-17 production(p=0.009654)and interleukin-6-mediated signaling pathways(p=0.02004).The target genes of miRNA-125b were enriched in adipokines signaling pathway(p=0.001495),TGF?receptor signaling pathway regulation(p=0.001168)and insulin signaling pathway(p=0.02081),and miRNA-144 target genes are enriched in the TGF? receptor signaling pathway(p=0.02498).GO analysis was performed on both miRNA-125b and miRNA-144,and enrichment of two gene target cells such as metabolic processes and signal transduction.KEGG analysis showed the two miRNAs target genes were significantly enriched in the endocrine system,the immune system,the process of glucose and lipid metabolism,and the endocrine and metabolic diseases.Conclusion:?Compared with pregnant women with NGT,the placental tissues of pregnant women with GDM have significantly abnormal expression of miRNAs.?The expression of miRNA-125b and miRNA-543 in GDM was significantly lower than that in NGT,whereas miRNA-144 was significantly up-regulated,indicating that the abnormal expression of miRNAs in placental tissues is involved in the development of GDM.?The level of miRNA-144 in the term placenta of pregnant women with GDM was significantly positively correlated with the age and hemoglobin in term pregnancy.?Target genes regulated by miRNA-125b and miRNA-144 are enriched in glucose and lipid metabolismPart ?.Abnormal expression of miRNA-125b and miRNA-144 in plasma exosomes and its diagnostic value for GDMObjective:To study the differential expression of two miRNAs in peripheral blood of pregnant women in the third trimester of pregnancy,and to evaluate the value of them in the diagnosis of GDM.Methods:Pregnant women with NGT(n=61)and pregnant women with GDM(n=57)between 26-41 gestational weeks were recruited.Clinical data that might be related to glucose and lipid metabolism,such as pre-pregnancy BMI,pregnancy OGTT,weight gain during pregnancy,were recorded.Polymer precipitation was used to extract exosomes from the plasma.Western Blot was used to identify the markers of protein on the membrane of exosomes.Transmission electron microscopy was used to identify the extracted exosomes in morphology.Nanoparticle analysis methods were used to identify exosomes and their Diameter distribution.qRT-PCR method was used to detect the expression levels of miRNA-125b and miRNA-144 in the extracted exosomes.Factors influencing the pathogenesis of GDM were screened and analyzed using the univariate Logistic regression analysis.Stepwise Logistic regression analysis was used to screen for factors that increased blood glucose levels during pregnancy.Cross-validation were used to establish a Logistic diagnosis model using these factors.A receiver operating characteristic curve(ROC)was drawn.The prediction effect of the model was evaluated by area under curve(AUC).Results:The exosomes extracted from the plasma of the NGT group and the GDM group were identified:?Western Blot results showed that the extracted vesicles were enriched with exosomal marker molecules CD63,TSG101 and specific proteins of the placental-derived exosome,i.e.,placental alkalinity Phosphatase(Placental alkaline phosphatase,PLAP);? Transmission electron microscopy showed that the extracted vesicles showed a typical round cup-shaped structure with a diameter of 30-100nm;?Nanoparticle analysis showed that the both the particle size distribution curve of the vesicles extracted from the plasma of the two groups showed a single peak,with the peak size of 99.6 nm in GDM group and 98.2 nm in NGT group,and the proportion of vesicles at the peak size was 99.1%in GDM and 98.8%in NGT,respectively.The results of qRT-PCR test showed that the expression of miRNA-125b in plasma exosomes in the GDM group was significantly down-regulated than that in the NGT group(p<0.001),while miRNA-144 was significantly up-regulated than that in the NGT group(p<0.001).The pattern of difference in plasma exosomes is consistent with that in placenta.The levels of miRNA-125b and miRNA-144 in plasma exosomes were not significantly correlated with gestational weeks(p=0.3074,p=0.3167).Compared with NGT group,GDM group had higher OGTT,HbA1C level,age,weight,pre pregnancy body mass index(BMI),delivery,full-term pregnancy diastolic blood pressure and neonatal weight(P<0.05),while the number of platelets in term pregnancy were significantly lower than those in the NGT group(p=0.020).The level of miRNA-144 in plasma exosomes in the GDM group was significantly positively correlated with the value of 1-hour OGTT(p=0.044),significantly negatively correlated with BMI before pregnancy and before delivery(p=0.018,p=0.039),and positively correlated with the number of platelets in term pregnancy(p=0.028).Single factor Logistic regression statistics showed that the levels of miRNA-125b and miRNA-144 in circulating exosomes,age,number of childbirths,weight before pregnancy,BMI before pregnancy were significantly correlated with GDM(p<0.05).Among them,mirna-125b was negatively correlated with GDM(P<0.0001).and others were positively correlated.Multivariate Logistic regression analysis showed that the levels of miRNA-125b,miRNA-144 and BMI before pregnancy are significant risk factors of GDM(p=0.00030,p=00030,p=0339).The diagnostic model(model 1)established on the basis of these three factors has an AUC of 0.876,and the diagnostic model established on the basis of miRNA-125b and miRNA-144(model 2)has an AUC of 0.875.The diagnostic performance of Modell is significantly higher than that of miRNA-125b and miRNA-144 alone but is significantly lower than the value of 1-hour OGTT.Conclusion:?There are abundant placental-derived exosomes in the plasma of pregnant women with both NGT and GDM during the third trimester.?Compared with pregnant women with NGT in the third trimester,the expression of miRNA-125b in the plasma exosomes of pregnant women with GDM in the third trimester was significantly reduced,while the expression of miRNA-144 was significantly increased.The divergent change of these two miRNAs is the same in the placenta.It is suggested that the placenta may secrete dysregulated miRNAs into the peripheral circulation by secreting exosomes,and then act on target cells remotely and play a role in the pathogenesis of GDM.?The levels of miRNA-125b and miRNA-144 in plasma exosomes of pregnant women with NGT and GDM in the third trimester did not change across the gestational week.The level of miRNA-144 in plasma exosomes of the GDM group was significantly negatively correlated with BMI before pregnancy and before delivery.?The levels of miRNA-125b and miRNA-144 in plasma exosomes and the BMI before pregnancy are the risk factors of GDM,and the diagnostic model established based on these three factors has good diagnostic efficiency for GDM.The dysregulated miRNAs in plasma exosomes have a good diagnostic value for GDM.Part ?.Mechanism of miRNA-125b in the pathogenesis of GDMObjective:To investigate the effect of expression of miRNA-125b on regulation of expression of LEP,the activation of leptin receptor(LEPR)-JAK2-STAT3 pathway and the cell proliferation and apoptosis in JEG-3 cells by over-expression and knockout of miRNA-125b in vitro.Furthermore,to investigate the target regulation of miRNA-125b to LEP,so as to clarify the possible mechanism of miRNA-125b involved in the pathogenesis of GDM.Method:Placental tissues in the NGT group(n=17)and GDM group(n=19)in the full-term pregnancy were recruited.The clinical factors that may be related to energy metabolism,such as BMI before pregnancy,OGTT during pregnancy,and weight gain during pregnancy were recorded.JGE-3 trophoblast cells were used to construct miRNA-125b Mimics cells,mimics negative control cells,inhibitors cells and inhibitor negative control cells.The qRT-PCR test was used to detect the expression of LEP,and the Western Blot was used to detect the expression of LEPR,JAK2,pJAK2,STAT3,pSTAT3,cleaved caspase protein,Bax protein and Bcl2 protein.expression.CCK-8 test and clone formation test were used to detect the proliferation of trophoblasts in each group after transfection,and flow cytometry was used to detect the apoptosis of trophoblasts in each group after transfection.The dual luciferase test was used to verify the regulation of miRNA-125b on LEP.Result:(1)Compared with the NGT group,in the placenta of the GDM group:?The expression of LEP was significantly increased;?The expression of LEPR,p-JAK2,and p-STAT3 was significantly strengthened.(2)With overexpression of miRNA-125b,the expression of LEP in JEG-3 cells decreased,and the expression of LEPR,pJAK2 and pSTAT3 was significantly reduced.With miRNA-125b was knocked out,the expression of LEP in JEG-3 cells increased,and the expression of LEPR,pJAK2,and pSTAT3 was significantly enhanced.(3)The dual luciferase experiment showed that miRNA-125b directly regulates the expression of LEP.(4)With overexpression of miRNA-125b,the proliferation of JEG-3 trophoblasts was significantly reduced,and apoptosis was significantly increased;whereas the proliferation of JEG-3 cells was significantly increased and apoptosis was significantly reduced when miRNA-125b was knocked out.Conclusion:(1)The expression of LEP in full-term GDM placenta tissue was significantly increased,and the LEPR-JAK2-STAT3 pathway was significantly activated.(2)The over-expressed miRNA-125b in JEG-3 trophoblasts significantly inhibits the expression of LEP and the activity of the LEPR-JAK2-STAT3 pathway.Moreover,it inhibits cell proliferation while enhances its apoptosis.Conversely,the low-expressed miRNA-125b significantly enhances the expression of LEP,the activity of LEPR-JAK2-STAT3 pathway,and cell proliferation while attenuates cell apoptosis.(3)In JEG-3 trophoblasts,miRNA-125b directly combines with LEP mRNA to regulate the expression of LEP protein.(3)miRNA-125b may affect the expression of JAK2-STAT3 pathway by regulating the expression of LEP,leading to changes in proliferation and apoptosis of trophoblast,thereby playing a role in the pathogenesis of GDM. |
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