MiR-889-3p Regulates Glucose Metabolism By Targeting Smad7 In Gestational Diabetes Mellitus

Objective: Gestational diabetes mellitus(GDM),characterized by glucose intolerance with onset or first recognition during pregnancy,is one of the most common pregnancy complications and could result in short-term and long-term adverse outcomes for both the mother and fetus.The pathogenesis of GDM is similar to type 2 diabetes mellitus,including insulin resistance(IR)and relative insufficiency of insulin secretion.As a hormone lowering blood glucose,insulin plays a biological role through target organs,and the liver is one of the most important target organs,which regulates and maintains the glucose and lipid homeostasis.Hepatic insulin resistance is a decrease in the ability of insulin inhibiting the hepatic glucose production which lead to increased hepatic glucose production;furthermore,insulin resistance affects lipid metabolism,which result to liver lipid deposition and reduce insulin sensitivity.The main pathophysiological features are the abnormal insulin signaling pathway,and the increase of gluconeogenesis and glycogen decomposition.Initially,non-coding RNAs(ncRNAs)were regarded as "transcriptional noises",but accumulating evidences have demonstrated that these ncRNAs have a series of crucial regulatory potentials both in transcription and post-transcription.MicroRNA(mi RNA),a class of single-stranded RNA of 19-24 nucleotides in length,is recognized as post-transcriptional regulators of gene expression by binding to the target sites to influence mRNA degradation or translational inhibition.Numerous studies have indicated that mi RNA was involved in various biological processes,and dysregulated expression of miRNA have been found to be associated with many human diseases.The Smad family is an intracellular signal transduction factor.Smad family member 3(Smad3)is a receptor-regulated Smad,while Smad family member 7(Smad7)is an inhibitory Smad.Smad7 could inhibit Smad3 and act as a negative regulator.Studies have shown that Smad participated in many metabolic pathways such as adipogenesis,lipid accumulation,fatty acid beta oxidation,hepatic gluconeogenesis,and pancreatic β-cell.As a temporary organ formed during pregnancy,the placenta is not just the only interface connecting the mother and fetus,participating in the process of nutrient transport,gas exchange and blood circulation,but also has important endocrine function.Placenta-derived hormones antagonize insulin and increase insulin resistance,which in turn lead to GDM.Therefore,the aim of this study was to screen out differentially expressed nc RNAs in GDM placentas,and to explore the molecular mechanism of the differentially expressed miR-889-3p in GDM.Methods: 1.Six placenta samples from healthy pregnant women(n=3)and GDM(n=3)were collected to analyze the whole transcriptome profiles by high-throughput sequencing.Differentially expressed ncRNAs were further validated by quantitative real-time polymerase chain reaction(qRT-PCR)on an independent set of normal(n=20)and GDM(n=20)placenta samples.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to describe genes annotations of differentially expressed ncRNAs in biological process,cellular component and molecular function and analyze the functions and related biological pathways.Competitive endogenous RNA(ceRNA)was used to predict the interactions between the possible targeted miRNA of differentially expressed ncRNA and mRNA.2.8-week-old C57BL/6J female mice was used to establish an animal model of GDM through a high-fat diet.Blood glucose levels of intraperitoneal glucose tolerance test were measured,and the area under the curve and the relevant indexes of glucose metabolism were calculated.The expression of miR-889-3p was detected.HE staining,oil red staining and the content of triglyceride and glycogen were performed in liver tissues.The expression of key enzyme of gluconeogenesis,including phosphoenolpyruvate carboxykinase(PEPCK)and glucose-6-phosphatase(G-6-Pase),and the expression of key molecules of the insulin signaling pathway,such as insulin receptor substrate-2(IRS-2),phosphatidyl inositol 3-kinase(PI3K),protein kinase B(Akt)and glycogen synthase kinase-3β(GSK-3β),were detected.3.The bioinformatics software was used to predict the binding sites of miR-889-3p and Smad7,and the luciferase reporter assay was used for validation.qRT-PCR and Western Blot were used to detect the expression of Smad7,Smad3,forkhead box O1(FoxO1)and peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1α)in liver tissues.The insulin resistance cell model was established by PA treatment with human normal hepatocyte L02.After treatment,miR-889-3p minic,miR-889-3p inhibitor and miR-889-3p NC were transfected to IR cell model.The glucose residue in the medium,the expression of key enzyme of gluconeogenesis,key molecules of the insulin signaling pathway,Smad7,Smad3,FoxO1 and PGC-1α were determined.Smad7 agonist was used to treat transfected IR cell model to demonstrate the effect of miR-889-3p on hepatic insulin sensitivity and glucose metabolism.Results: 1.A total of 2,817 miRNAs,23,339 lncRNAs and 9,513 circRNAs were identified.There were 290 differentially expressed ncRNAs in GDM placentas compared with healthy pregnant women.2 miRNAs,86 lncRNAs and 55 circRNAs were upregulated while 2 miRNAs,86 lncRNAs and 59 circRNAs were downregulated in GDM.The expression of the selected ncRNAs was consistent with the sequencing results.GO and KEGG pathway analysis demonstrated that the major targets of these ncRNAs were associated with insulin resistance and abnormal glucose and lipid metabolism.2.Pregnant mice had normal blood glucose in gestational day 0.5(GD0.5).Blood glucose levels of GD11.5 and GD16.5 increased gradually,and insulin resistance of mice in GD16.5 was heavier than GD11.5.Meanwhile,their weight gradually increased during pregnancy.These phenotypes indicated that the animal model of GDM was successfully established.The expression of miR-889-3p in placenta and liver tissues was significantly higher in the GDM group than the control group(P<0.05).Histopathological examination revealed that the liver of GDM group showed steatosis and lipid drip infiltration.Compared with the control group,the TG content of liver was significantly increased,and glycogen content was reduced in the GDM group.The mRNA expression of glycogenic PEPCK and G-6-Pase in liver tissues of the GDM group was significantly increased compared with the control group(P<0.05).Furthermore,the expression of IRS-2,PI3 K,Akt and GSK-3β in the GDM group were significantly lower than the control group(P<0.05).3.The luciferase reporter assay confirmed that Smad7 was the target gene of mi R-889-3p.Compared with the control group,the expression of Smad7 in the liver of GDM mice decreased,while the expression of FoxO1 and PGC-1α,and the phosphorylation level of Smad3 was increased(P<0.05).IR L02 cell model was established by PA intervention.After transfected with miR-889-3p minic,miR-889-3p inhibitor and miR-889-3p NC,the amount of glucose residual in the PA+miR-minic group was higher than that in the PA and PA+mi R-inhibitor group(P<0.05).The mRNA expression of PEPCK and G-6-Pase in the PA+mi R-minic group were significantly higher than those in the PA and PA+miR-inhibitor group(P<0.05).Compared with the PA and PA+mi R-inhibitor group,the phosphorylation levels of IRS-2,PI3 K,Akt and GSK-3β in the PA+mi R-minic group were significantly lower(P<0.05).Furthermore,the expression of Smad7 was lower and the expression of p-Smad3,FoxO1 and PGC-1α were higher than those in the PA and PA+mi R-inhibitor group(P<0.05).After treatment with Smad7 agonist,the effects of mi R-889-3p on hepatic gluconeogenesis and insulin signaling pathway were weakened.Conclusions: 1.The whole transcriptome profiles significantly differed in GDM placentas compared with healthy pregnant women,and may be a potential biomarker for GDM.2.miR-889-3p could regulate Smad3 and FoxO1 by targeting Smad7 to affect hepatic gluconeogenesis and insulin signal pathway,resulting in abnormal liver glucose and lipid metabolism and exacerbation of insulin resistance,and then exert in GDM.