The Mechanism Of LncRNA-SNX17 Combining With MiR-517a To Regulage IGF-1 In The Placentation Of Diabetic Macrosomia

Background:Gestational diabetes mellitus（GDM）,as a common obstetric complication,has become a world-wide public health problem in recent years.GDM is defined as abnormal glucose tolerance first found in pregnancy period,which was normal before pregnancy.Most of patients with GDM will recover after delivery,but patients with GDM might be influenced by lots of pregnancy-associated adverse outcomes,such as abnormal glucose and lipid metabolism,hypoglycemia,ketoacidosis,abortion,premature delivery,polyhydramnios,fetal growth restriction,macrosomia,fetal malformations,neonatal respiratory distress syndrome and so on.Macrosomia,as one of adverse outcomes resulted by uncontrolled hyperglycemia of GDM,can not only result in short-time consequences,such as prolonged labor,postpartum hemorrhage,shoulder dystocia,neonatal hypoglycemia,neonatal respiratory distress syndrome and high risk of admission to newborn intensive care unit（NICU）,but also will lead to an increasing occurrence of adult metabolic syndrome,studies have already exhibited macrosomia is an independent risk factor of adult metabolic syndrome.Intrauteral programming hypothesis believed the enviorment in utero could effect the fetus and might be responsible for the the adult metabolic syndrome,including obesity,hypertension,diabetes mellitus and so on.It was proposed that both over-nutrition and under-nutrition could permanently alter the placenta and the fetal organ structure and function in response to environmental factors,which may lead to the abnormal metabolism in adult.The placenta,as the most important organ linking the mothers and the fetus in pregnancy,is the most important reaction place for the substance metabolism and energy exchange.The placenta is also one of the studied subjects by programming hypothesis.Studies have showed that the placenta make great effort in the pathogenesis of macrosomia with GDM.Epigenetics have received increasing attention in recent years.Epigenetics suggested post-transcriptional regulation of genes could modify the expression of genes and regulate the biological behavior.It was believed that the state of an individual’s DNA is both inherited and modifiable,which could be modified in response to the enviormental factors.The opinions of epigenetics and programming hypothesis are the same.which is fetal intrauterine exposure to enviornmental factors could regulate gene expression through epigenetic methods,the epigenetic regulation in turn played an important role in fetal growth and development and the pathogenesis of adult metabolic diseases.Long non-coding RNA is one of the most focused hot spots of epigenetics in recent years.Several studies have shown that long non-coding RNA can participate in the regulation of multiple biological processes,such as immunity,infection,and cancinogenesis in many different ways.However,there is still no study on the expression and mechanism of long non-coding RNA in placental tissues of macrosomia with gestational diabetes mellitus.Objective:This study is divided into three parts to explore the expression and mechanism of long non-coding RNAs in the placenta of macrosomia with diabetes mellitus and detect epigenetic regulation of long non-coding RNA on the biological behavior of trophoblast,and thus contributed to the study of possible mechanism of fetal growth regulation and adult metabolic disease pathogenesis through intrauterine programming.Methods:Part 1:The expression of lncRNAs in the placenta of macrosomia with diabetes mellitus.We collected the placenta of women deliveried in Shengjing Hospital by caesarean section without complications and detected the expression of long non-coding RNAs in fetal placenta tissues of both macrosomia group and normal birth weight newborn group was screened by microarray profile.According to the different birth weight,the newborns’placenta were divided into two groups,including macrosomia group（birth weight≥4000g）,normal birth weight group（control group,birth weight≥2500 and<4000g）（n=4,respectively）.Microarray profile was done in each group,and the microarray results were analysed by biological software（GO analysis and KEGG analysis）,and the proposed long non-coding RNA（lncRNA-SNX17）was selected.We collected 32 samples of placeta（macrosomia group,n=16;control group,n=16）to verify the microarray result by quantitative real-time PCR（qRT-PCR）.SPSS 19.0 software was used to analyse the results.HTR8/SVneo and JEG-3 cell lines were cultured with different glucose concentration,then we use qPCR to detect the expression of 4 candidate lncRNAs.Part 2:We use adenovirus infects HTR8/SVneo cell line,and constructed LncRNA-SNX17 overexpression cell line successfully,which was verified by PCR.Since part of the long-chain non-coding RNAs may regulate cell biological behavior by acting on its own gene protein expression,we first detected the effect of the overexpression of lncRNA-SNX17 on the expression of SNX17 protein through WB.Secondly,we tested the effect of LncRNA-SNX17 overexpression on proliferation of HTR8/SVneo trophoblast cells by CCK-8.The effect of LncRNA-SNX17overexpression on invasion of HTR8/SVneo trophoblast was detected by the Transwell.Effect of LncRNA-SNX17 overexpression on apoptosis of HTR8/S Vneo was detected by flow method.Part 3:The study on LncRNA-SNX17 regulating the regulation of HTR8/SVneo cell line by binding miR-517a to regulate IGF-1.1.The binding sites of miR-517a and LncRNA-SNX17 and IGF-1 were predicted by bioinformatics software TargetScan and miRanda.2.The expression of lncRNA-SNX17,miR-517a and IGF-1 in the placenta tissue of diabetic maocrosomia was detected by PCR and WB to partially detect the possible regulation mechanism of the three.HTR8/SVneo human trophoblast cell line was cultured in vitro.3.Luciferase report assay was used to validate the binding sites of miR-517a and lncRNA-SNX17 and IGF-1.4.siRNA technology was used inoverexpression or knock-out of miR-517a in HTR8/SVneo cell line,and the effects of miR-517a overexpression and knock-outon the expression of IGF-1 and were detected.The effects of over-expression and knock-out of miR-517a on proliferation and invasion of HTR8/SVneo cells were detected by CCK-8 and Transwell experiments.5.We used adenovirus transfection to induce over-expression of lncRNA-SNX17 in HTR8/SVneo cells,PCR and WB were done to detect the expression of miR-517a and IGF-1 in HTR8/SVneo trophoblast.6.We simultaneouslyupregulated lncRNA-SNX17 and miR-517ain HTR8/SVneo trophoblast cells,then WB was used to detecte the expression of IGF-1 after simultaneous over-expression of the two.CCK-8 and Transwell experiments were also done to test the proliferation and invasion ability of HTR8/SVneo cells.Results:Part 1:1.Clinical data analysis showed that there was no significant statistical difference between the two groups in age,pregnancy,birth time,pregnancy week,pre-pregnancy body mass index and pregnancy age（P>0.05）.Compared with the control group,the glycated hemoglobin,OGTT fasting blood glucose,1h blood glucose,2h blood glucose levels and newborn birth weight in the diabetic macrosomia group increased significantly（P<0.05）.2.Arrystar microarray results show that compared to the normal birth weight group,2,962 long non-coding RNAs and 4556 mRNAs were up-regulated,1921 long non-coding RNAs and 2527 mRNAs were down-regulated in the placenta of diabetic macrosomia（Fold change≥2.0,P<0.05）.3.GO analysis showed that the dysregulated mRNAs were mainly related to cellular process,cell part,binding,and biologic regulation.Pathway analysis showed 78 pathways were related to upward transcriptions and 27 pathways were related to downward transcriptions.4.qPCR results confirmed that NR₀49785,NR₀26709,NR₀24251,NR₀33967 expression in the placenta of diabetic macrosmia were up-regulated,which was consistent with the microarray results.5.In the HTR8/SVneo cell line treated with different glucose concentrations,the expression level of lncRNA-SNX17 increased significantly after 12h treatment,and the expression of lncRNA-SNX17 was positively related to the glucose concentration treated.Part 2:1.PCR results showed that the level of lncRNA-SNX17 was significantly higher than that of vector group and the control group.2.WB results showed that there was no significant difference in the expression level of SNX17 protein in the control group,vector group,and LncRNA-SNX17 overexpression group.3.CCK-8 results show that the proliferation activity of HTR8/SVneo trophoblast in lncRNA-SNX17 over-expression group increased.4.Transwell results show that the lncRNA-SNX17 over-expression increased HTR8/SVneo cells’invasiveness ability.5.Flow cell experiment showed an upregulation of apoptosis in HTR8/SVneo trophoblast in lncRNA-SNX17 over-expression group.Part 3:1.TargetScan and miRanda predicted that placental specific miR-517a has binding sites with lncRNA-SNX17 and IGF-1.2.The results of WB and qPCR showed that the expression level of miR-517a in placenta of diabetic macrosomia was significantly lower than that of control group.The expression level of lncRNA-SNX17 and IGF-1 in placenta of diabetic macrosomia was significantly higher than that of control group.3.The results of the luciferase report system showed that miR-517a had binding relationship with IGF-1 and LncRNA-SNX17.4.Over-expression of miR-517a in the HTR8/SVneo trophoblast line reduced the expression of IGF-1 significantly.CCK-8 and Transwell experiments suggested that over-expression of miR-517a inhibited the proliferation and invasion of HTR8/SVneo cells.5.Over-expression of lncRNA-SNX17 in the HTR8/SVneo cell line decreased the expression level of miR-517a significantly,and increased the expression of IGF-1.6.Simultaneous over-expression of lncRNA-SNX17 and miR-517a in the HTR8/SVneo cells reduced the expression of IGF-1,and HTR8/SVneo cell proliferation and invasion decreased.Conclusion:Part 1:Long non-coding RNAs participated in the placenta regulation of diabetic macrosomia.LncRNA-SNX17 was significantly higher in the placenta of diabetic macrosomia.The high expression of LncRNA-SNX17 may be related to high glucose stimulation.Part 2:LncRNA-SNX17 expression could not effect the expression of SNX17 protein.LncRNA-SNX17 over-expression could promote the proliferation and invasion activity of trophoblast HTR8/SVneo,and promote the apoptosis of HTR8/SVneo trophoblasts.Part 3:LncRNA-SNX17 over-expression could regulate the level of IGF-1 expression by binding miR-517a,and then regulate the proliferation and invasion of trophoblas.