The Role And Mechanism Of Long Non-coding RNA HCG27 Regulating MiR-378a-3p/MAPK1 Axis In Gestational Diabetes Mellitus

Objective: Gestational diabetes mellitus(GDM)is the most common metabolic disorder concerning hyperglycemia,characterized by glucose intolerance that first noticed peculiar to pregnancy.The ceRNA(competitive endogenousRNAs)network is a delicate and complex regulation of gene expression,which has great biological significance.The phenomenon of poor glycemic control at early times leading to long-term adverse effects is referred to as ‘metabolic memory’.Many studies have confirmed that ‘metabolic memory’may lead to persistent dysfunction of vascular endothelial cells.Therefore,it is important to analyze and construct ceRNA networks in GDM vascular endothelial cells and find out the molecular biomarkers contributing to the correction of ‘metabolic memory’.The study aims at constructing the lncRNA-mediated ceRNA networks in GDM vascular endothelial cells.The lncRNA possibly related to the change of ‘metabolic memory’ in GDM was screened out and its ceRNA regulatory network was verified to explore its possible mechanism in GDM vascular endothelial cells.Methods: By integrating gene expression profiles of GDM patients and pregnant women in control group from Gene Expression Omnibus(GEO)database,differential expression lncRNAs(DELncs),differential expression miRNAs(DEMis)and differential expression mRNA(DEMs)were screened out,and their biological functions were analyzed.By integrating the interaction network of DEMis targeted DEMs and DELncs,the ceRNA regulatory network was constructed.LncRNA HCG27 was screened out as the research target and then its miRNA-378a-3p/MAPK1 interaction regulation axis in umbilical vein endothelial cells was verified.The placenta tissues and primary umbilical vein endothelial cells from GDM patients and normal pregnant women were collected.The expression of lncRNA HCG27,miR-378a-3p and the target gene MAPK1 were detected by q RT-PCR and Western Blot to determine the expression difference in GDM.After knocking down the expression of lncRNA HCG27 in human umbilical vein endothelial cell line(HUVEC),the differential expression of MAPK1 was detected by q RT-PCR and western blotting and the change of glucose uptake ability was detected by glucose consumption test.Then the rescue test was performed by transfecting the overexpression plasmid of MAPK1.After knocking down the expression of lncRNA HCG27 in HUVEC,the rescue test was performed by transfecting the miR-378a-3p inhibitor to inhibit the expression of miR-378a-3p.The expression change of target gene MAPK1 was detected by q RT-PCR and western blotting,and the change of glucose uptake ability was detected by glucose consumption test.To verify the targeting regulatory relation between miR-378a-3p and lncRNA HCG27,and the targeting regulatory relation between miR-378a-3p and MAPK1,the luciferase reporter gene assay was performed.The palmitic acid was used to construct the insulin resistance model in HUVEC.The actuation duration and effective concentration of palmitic acid were selected through CCK8 experiment and glucose consumption experiment.After transfecting the overexpression plasmid of MAPK1 in HUVEC insulin resistance model,glucose consumption experiment was performed to detect its effect on glucose uptake function of HUVEC with insulin resistance.Results: The lncRNA-mediated ceRNA network was constructed in GDM vascular endothelial cells through bioinformatics analysis.Compared with the control group,the expression of lncRNA HCG27 and MAPK1 in placenta tissue of GDM patients were decreased,while the expression of miR-378a-3p was increased.In the primary umbilical vein endothelial cells from GDM patients,the expression of lncRNA HCG27 and MAPK1 were decreased,while the expression of miR-378a-3p was increased.The knockdown of lncRNA HCG27 in HUVEC showed its positive regulation on MAPK1 and could reduce the glucose uptake function of HUVEC.The overexpression of MAPK1 could rescued the inhibition of glucose uptake function caused by lncRNA HCG27.Mi R-378a-3p negatively regulates MAPK1.The inhibition of miR-378a-3p expression in HUVEC can rescued the inhibitory effect of lncRNA HCG27 on glucose uptake.The luciferase reporter gene assay confirmed that there were targeting sites between miR-378a-3p and lncRNA HCG27,and targeting sites between miR-378a-3p and MAPK1.The actuation duration of 48 hours and effective concentration of 250μM were chosen to be the most appropriate palmitic acid insulin resistance model in HUVEC.In this model,overexpression of lncRNA HCG27 can improved the glucose uptake function.Conclusions: LncRNA HCG27 can promote the glucose uptake function of insulin resistant HUVEC by regulating miR-378a-3p/MAPK1 axis,providing new biomolecular target on controlling the long-term complications of GDM.