The Roles And Mechanism Of High Glucose-induced LncRNA MIR181A2HG In Regulating Proliferation And Migration Of Vascular Endothelial Cell

Vascular complication of diabetes is one of the common complications of diabetes,and is also the leading cause of death in diabetic patients.Previous studies have demonstrated that high glucose causes dysfunction of endothelial cells through the induction of oxidative stress and inflammatory responses.Vascular endothelial dysfunction plays an important role in the development of diabetic vascular disease.In recent years,although systematic studies have made on the molecular mechanism of oxidative stress induced by high glucose in vascular endothelial cells,most studies focused on the effects of longtime(continuous)high glucose on the function of vascular endothelial cells.The effects of transient high glucose stimulation on vascular endothelial cells have rarely been reported.Thus,it is important to study the roles and mechanism of transient high glucose stimulation on endothelial cells,which not only has a full understanding of the effects of high glucose on endothelial cells,but also has important implications for the experimental conditions of cell culture.Studies have found that only less than 2% of the gene sequences in the human genome can encode protein,whereas 98% of the remaining gene sequences are transcribed into lots of non-coding transcripts,including microRNAs(mi RNAs)and long non-coding RNA(lncRNA).miRNAs are a class of non-coding small RNAs with length of ~20nt,which regulates gene expression by binding to its target gene 3’UTR.Studies have found that miRNAs are widely involved in a variety of physiological and pathological processes,including cardiovascular disease.For example,miR-155 is involved in vascular remodeling.MiR-29 a is involved not only in the formation of atherosclerosis,but also in myocardial fibrosis.LncRNAs are a class of non-coding RNAs which length exceeds 200 nt.Many studies have shown that lncRNAs play an important role in various of cellular processes and regulate gene expression in epigenetic regulation,transcription,RNA processing and translation level.For example,lncRNA-MALAT1 is overexpressed in endothelial cells under hypoxia.Knockdown of MALAT1 can inhibit the proliferation of endothelial cells.It has been shown that lncRNA regulates the expression of gene at post-transcriptional level by inhibiting the binding of miRNA to its target gene competitively.Muscle-specific lncRNA(linc-MD)1 regulates the expression of MAML1 and MEF2 C by adsorption of mi R-133.Myocardial apoptosis-related lncRNA-CARL regulates the expression of PHB2 by adsorption of miR-539 and regulates apoptosis by regulating mitochondrial division.However,it remains unknown whether and how high glucose changes the expression of lncRNA and regulates endothelial cell function.Therefore,this study screened and verified the differential expression of lncRNA in HUVEC treated with different concentrations of glucose and investigated the roles and molecular mechanism of the newly identified lncRNA on biological behavior in endothelial cells.Part one MIR181A2 HG regulates AKT2 expression by competing with miRNAs.Objective: To explore the differential expression of lncRNAs induced by high glucose,verify the miRNAs directly binding with this lncRNA and identify the miRNAs’ possible target genes.Method:1.MTS method was used to detect the effect of cell viability on glucose.2.LncRNA array combined with bioinformatics methods were used to detect and predict lncRNA expressed in HUVEC treated with different concentrations of glucose.3.CeRNA prediction analysis was used to predict the mi RNAs which could bind with MIR181A2 HG and the target gene of these miRNAs.4.Real-time quantitative PCR was used to verify the expression of MIR181A2 HG in HUVEC treated with different concentrations of glucose.5.Real-time quantitative PCR was used to verify the expression of miR-6832-5p,miR-6842-5p and miR-8056 in HUVEC treated with glucose(5.5 mM and 30 mM).6.Luciferase reporter assay and RNA pulldown experiments were used to verify the direct binding of miR-6832-5p,miR-6842-5p and miR-8056 to MIR181A2 HG.7.Construction of the luciferase reporter plasmids containing AKT2 3’UTR and AKT2 3’UTR mut.8.Real-time quantitative PCR,Western blotting and Immunofluorescence were used to detect the mRNA and protein expression of AKT2.9.Immunofluorescence assay was used to detect the expression and localization of AKT2 when MIR181A2 HG was overexpreesed or knocked down.Results:1.Screen and vertify the lncRNA expressed differently in high glucose-induced HUVEC.Compared with the control group(5.5 mM D-glucose),the cell viability was increased significantly in HUVEC induced by high glucose treated 24 h.Cell viability began to decrease after 5 days.Compared with the control group,MIR181A2 HG was increased after 12,24 and 36 h treatment with high glucose.NCBI’s ORF Finder predicted that MIR181A2 HG has no protein coding ability and no Kozak sequence was found in its sequence.2.MIR181A2 HG can specifically bind miR-6832-5p,miR-6842-5p and miR-8056.Bioinformatics and ceRNA predictive analysis revealed that MIR181A2 HG might bind miR-6832-5p,miR-6842-5p and miR-8056,and these miRNAs might interact with AKT2 3’ UTR.Compared with the control group,expression of miR-6832-5p,miR-6842-5p and mi R-8056 in high glucose-induced HUVEC decreased significantly.Luciferase reporter assay showed no significant difference in luciferase activity after co-transfection with miR-6832-5p,miR-6842-5p and miR-8056 with pmirGLO compared with control group,respectively.After co-transfection of these three miRNAs with pmirGLO-MIR181A2 HG,the luciferase activity was 58.52%,48.52% and 83.03% of the control group,respectively.RNA pulldown assay showed that MIR181A2 HG could specifically bind to miR-6832-5p,miR-6842-5p and miR-8056.3.miR-6832-5p,mi R-6842-5p and miR-8056 inhibit the expression of AKT2 by targeting its 3’UTR.Luciferase reporter assay showed that when co-transfection of miR-6832-5p,miR-6842-5p or miR-8056 with pmirGLO-AKT2 3’UTR,luciferase activity was 32.41%,41.73% and 54.42% of the control group.Luciferase activity was significantly increased when MIR181A2 HG was co-transfected with pmirGLO-AKT2 3’UTR,but luciferase activity was significantly decreased after co-transfection of shR-MIR181A2 HG with pmirGLO-AKT2 3’UTR.The expression of AKT2 was decreased by 51.92%,47.13% and 27.39% when transfected with these three mi RNAs,respectively.Overexpression of them reduced by 54.30%,52.70% and 27.67% in AKT2 protein expression.Immunofluorescence assay showed that over-expression of these three miRNAs markedly reduced the fluorescence intensity.4.Glucose-induced MIR181A2 HG upregulates AKT2 expression.Overexpression of MIR181A2 HG significantly promoted the mRNA level of AKT2 significantly,whereas knockdown of MIR181A2 HG decreased it.Real-time quantitative PCR showed that,compared with the control group,high glucose promoted AKT2 mRNA expression significantly.Western blotting showed that high glucose promoted protein expression of AKT2 significantly.Real-time quantitative PCR and Western blotting showed that overexpression of AKT2 could rescue the reduction of AKT2 mRNA level induced by shR-MIR181A2 HG.Conclusion:1.High glucose promotes the expression of MIR181A2 HG.2.MIR181A2 HG can specifically bind to mi R-6832-5p,miR-6842-5p and miR-8056.3.miR-6832-5p,mi R-6842-5p and miR-8056 inhibit the expression of AKT2 by targeting its 3’UTR.4.MIR181A2 HG induced by high glucose promotes AKT2 expression.Part two MIR181A2 HG promotes the proliferation,migration and angiogenesis of HUVECObjective: To explore the effects of MIR181A2HG/mi RNA/AKT2 on the proliferation,migration and angiogenesis of HUVEC.Methods:1.MTS assay was used to detect the effect of MIR181A2 HG on the viability of HUVEC.2.Scratch assay was used to detect the effects of MIR181A2 HG on the proliferation and migration of HUVEC.3.Transwell migration assay was used to detect the effect of MIR181A2 HG on the migration of HUVEC.4.3D-culture was used to detect the effect of MIR181A2 HG on the angiogenesis of HUVEC.5.Western blotting was used to detect the expression of PCNA,MMP2 and VE-cadherin when MIR181A2 HG was overexpressed or knocked down.Results:1.MIR181A2 HG promotes the proliferation,migration and angiogenesis of HUVEC.Overexpression of MIR181A2 HG promoted the cell viability,whereas knockdown of MIR181A2 HG inhibited the cell viability of HUVEC.Scratch assay showed that compared with the control group,overexpression of MIR181A2 HG promoted the migration of HUVEC significantly,whereas knockdown of MIR181A2 HG decreased it.Transwell migration assay showed that the number of migrated cells increased after overexpressing of MIR181A2 HG significantly,whereas the number of migrated cells was reduced after knocking down of MIR181A2 HG.3D-culture showed that overexpression of MIR181A2 HG facilitated the angiogenesis of HUVEC,whereas knockdown of MIR181A2 HG reduced it.Compared with the control group,high glucose promoted HUVEC proliferation.The promotion of cell proliferation made by high glucose was reduced after knocking down of MIR181A2 HG.Transwell migration assay and 3D-culture revealed that knockdown of MIR181A2 HG could counteract the high glucose-induced promotion of and angiogenesis in HUVEC.Overexpression of MIR181A2 HG could promote the expression of MMP2 and VE-Cadherin,whereas knockdown of MIR181A2 HG reduced the expression of MMP2 and VE-cadherin.2.miR-6832-5p,mi R-6842-5p and mi R-8056 inhibit proliferation,migration and angiogenesis in HUVEC.Compared with the control group,overexpression of miR-6832-5p,miR-6842-5p and miR-8056 significantly decreased the proliferation and migration of HUVEC.3D-culture showed that overexpression of miR-6832-5p,miR-6842-5p or miR-8056 decreased the formation of the ring structures significantly.Western blotting showed that overexpression of miR-6832-5p,miR-6842-5p and miR-8056 could inhibit the expression of PCNA,MMP2 and VE-Cadherin.3.Knockdown of AKT2 can abolish proliferation,migration and angiogenesis induced by high glucose/MIR181A2HG/mi RNA axis.Overexpression of MIR181A2 HG promoted proliferation,whereas knockdown of AKT2 could counteract the promotion of proliferation and migration made by MIR181A2 HG in HUVEC.3D-culture showed that overexpression of MIR181A2 HG promoted the formation of the ring structures of HUVEC,whereas knockdown of AKT2 in MIR181A2HG-overexpressed cells reduce it significantly.Conclusion:1.MIR181A2 HG promotes the proliferation,migration and angiogenesis of HUVEC.2.miR-6832-5p,miR-6842-5p and miR-8056 inhibit the proliferation,migration and angiogenesis of HUVEC.3.Knockdown of AKT2 can abolish the proliferation,migration and angiogenesis induced by high glucose/MIR181A2HG/miRNA axis.Part three That high glucose/MIR181A2HG/miRNA/AKT2 promotes proliferation and migration is coupled with glucose metabolismObjective: To explore the relationship between the promotion of proliferation and migration made by high glucose/MIR181A2HG/miRNA/ AKT2 and glucose metabolism of HUVEC.Methods:1.Chemiluminescence was used to detect the formation of ATP.2.Glucose oxidase method was used to detect the glucose uptake.3.Glycogen content kit was used to detect the glycogen synthesis;4.Western blotting was used to detect AMPK,GSK3β phosphorylation and GLUT1 expression.Result:1.MIR181A2 HG promotes ATP production,glucose uptake and glycogen synthesis in HUVEC.Overexpression of MIR181A2 HG increased the production of ATP in HUVEC significantly,while knockdown of MIR181A2 HG decreased it.Glucose uptake increased significantly after overexpressing of MIR181A2 HG and decreased after knocking down of MIR181A2 HG in HUVEC.Overexpression or knocked down of MIR181A2 HG could increased or decreased the glycogen synthesis,respectively.2.miR-6832-5p,miR-6842-5p and mi R-8056 inhibit ATP production,glucose uptake and glycogen synthesis in HUVEC.Compared with the control group,the ATP production,glucose uptake and glycogen synthesis decreased after overexpression of miR-6832-5p,miR-6842-5p and miR-8056 significantly.3.AKT2 promotes ATP production,glucose uptake and glycogen synthesis in HUVEC.After overexpressing of AKT2,the production of ATP increased significantly,while knocking down of AKT2 decreased it.Overexpression or knockdown of AKT2 increased or decreased glucose uptake in HUVEC respectively.The ability of glycogen synthesis increased after overexpressing of AKT2 and decreased after knocking down AKT2 in HUVEC.4.MIR181A2HG/mi RNA/AKT2 promotes AMPK,GSK3β phosphorylation and GLUT1 expression.Overexpression of MIR181A2 HG did not affect the level of AMPK and GSK3β,but increased phosphorylation of AMPK and GSK3β.In contrast,knockdown of MIR181A2 HG reduced phosphorylation of AMPK and GSK3β.Furthermore,mi R-6832-5p,mi R-6842-5p and miR-8056 did not affect the total protein of AMPK and GSK3β,but decreased AMPK and GSK3β phosphorylation.Similarly,AKT2 promoted the level of phosphorylation of AMPK and GSK3β was increased.MIR181A2 HG increased the expression of GLUT1.In addition,miR-6832-5p,mi R-6842-5p and mi R-8056 mimics reduced the expression of GLUT1.Overexpression or knocked down of AKT2 promoted or inhibited the expression of GLUT1 respectively.Conclusion:1.MIR181A2 HG promotes ATP production,glucose uptake and glycogen synthesis in HUVEC.2.miR-6832-5p,miR-6842-5p and mi R-8056 inhibit ATP production,glucose uptake and glycogen synthesis in HUVEC.3.AKT2 promotes ATP production,glucose uptake and glycogen synthesis in HUVEC.4.Glucose/MIR181A2HG/miRNA/AKT2 promotes AMPK and GSK3β phosphorylation and GLUT1 expression.